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CN121464348A - T cell activation markers and methods for assessing T cell activation - Google Patents

T cell activation markers and methods for assessing T cell activation

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Publication number
CN121464348A
CN121464348A CN202480045753.6A CN202480045753A CN121464348A CN 121464348 A CN121464348 A CN 121464348A CN 202480045753 A CN202480045753 A CN 202480045753A CN 121464348 A CN121464348 A CN 121464348A
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cells
markers
group
cell
notch
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G·M·科莱特
M·V·古特肖
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Juno Therapeutics Inc
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Juno Therapeutics Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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Abstract

The present disclosure relates to methods of assessing the activation state of T cells within a cell composition, which methods may be used in conjunction with cell therapies or cells that produce for cell therapies. The cells of the cell composition may express recombinant receptors, such as chimeric receptors, e.g., chimeric Antigen Receptors (CARs), or other transgenic receptors, such as T Cell Receptors (TCRs). The methods provide markers and how to use the markers to assess activation of T cells within a cell composition.

Description

T cell activation markers and methods for assessing T cell activation
Cross Reference to Related Applications
The present application claims priority from U.S. provisional application No. 63/468,524 entitled "T cell activation marker and method FOR assessing T cell activation (ACTIVATION MARKERS OF T CELLS AND METHODS FOR ASSESSING T CELL ACTIVATION)" filed on month 23 of 2023, the contents of which are incorporated by reference in their entirety.
Reference to electronic sequence Listing
The present application is presented in conjunction with a sequence listing in electronic format. The sequence listing is provided in a file created at 2024, 5, 21, under the name 7350420272 540seqlist. Xml, which is 99,949 bytes in size. The information in electronic format of the sequence listing is incorporated by reference in its entirety.
Technical Field
The present invention relates to methods for assessing activation of T cells in a cell composition for use in conjunction with cell therapy or cell transduction. T cells of the cell composition may express recombinant receptors such as chimeric receptors, e.g., chimeric Antigen Receptors (CARs), or other transgenic receptors such as T Cell Receptors (TCRs). 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 markers to assess activation.
Background
There are a variety of methods for determining the activation state 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. Activation status was assessed by examining these defined markers and their surface expression levels or the percentage of T cells within the cell composition that were positive for the marker. However, markers may be present on only some subset of T cells, so a combination of markers is used to assess T cell activation, resulting in the benefit of having multiple markers available for selection for assessing T cell activation. These methods of assessing T cell activation using markers rely on characterization results that demonstrate which surface markers have varying levels of expression during T cell activation and benefit from a larger pool of available markers. Methods to address such needs are provided herein.
Disclosure of Invention
Provided herein is a method for assessing activation of T cells within a cell composition, the method comprising (a) detecting the level of surface expression of or the percentage of cells positive for one or more markers in cells of the 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 chains, CD105 (endostatin), CD262 (DR 5), 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、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD196 (CCR 6), CD230 (prion), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR 6), GARP (LRRC 32), CD116, CD334 (FGFR 4), CD66a/c/e and TSLPR (TSLP-R), and one or more markers of group (ii) is 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 receptors, Comparing the surface expression level or percentage of positive cells 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 percentage of positive cells of the marker in (i) is indicative of the T cells being activated compared to the reference and a lower level or percentage of positive cells of the marker in (ii) is indicative of the T cells being activated compared to the reference.
In some of any of the embodiments, the reference consists of a control cell composition that is not stimulated.
In some of any of the embodiments, 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 of the embodiments, the reference is an average expression level or average percent positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor. In some of any of the embodiments, 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.
Provided herein is a method for assessing surface expression of a T cell activation marker of a T cell, the method comprising detecting the level of surface expression of or the percentage of cells positive for one or more markers in cells of a composition comprising the T cell, 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 the group consisting 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 strand), CD105 (endostatin), CD262 (DR 5, TRAIL-R2), CD170 (Siglec-5), CD73 (extracellular-5' -nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g-strand ）、CD148、CD150（SLAM）、CD151（PETA-3）、CD154、CD165、CD166、CD200 R、CD217、CD218a（IL-18Ra）、CD227（MUC-1）、CD245（p220/240）、CD30、CD317（BST2、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG 3k, 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-2 Rb), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD196 (CCR 6), CD230 (prion), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR 6), GARP (LRRC 32), CD116, CD334 (FGFR 4), CD66a/c/e and TSLPR (TSLP-R), and 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 receptors, CD229 (Ly-9), CD84 and EGFR.
In some of any of the embodiments, the surface expression level or positive cell percentage of the one or more markers in (i) is positively correlated with T cell activation. In some of any of the embodiments, the surface expression level or positive cell percentage of the one or more markers in (ii) is inversely related to T cell activation.
Provided herein is a method of comparing activation of T cells in a donor, the method comprising (a) detecting the surface expression level of or the 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 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 strand, CD105 (endostatin), CD262 (DR 5, 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、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD196 (CCR 6), 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, CD230 (prion), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR 6), GARP (LRRC 32), CD116, CD334 (FGFR 4), 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 receptors, Comparing the surface expression level or the percentage of positive cells to the level or percentage of positive cells of each of the one or more markers in an unstimulated cell composition, wherein a higher level or percentage of positive cells of the marker in (i) indicates that the T cells are activated and a lower level or percentage of positive cells of the marker in (ii) indicates that the T cells are activated as compared to the unstimulated cell composition.
In some of any of the embodiments, prior to the detecting, the composition comprising T cells is incubated with a T cell stimulator under conditions that induce T cell activation.
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.
In some of any of the embodiments, the incubation with the T cell stimulating agent is performed in the subject. In some of any of the embodiments, the incubation with the T cell stimulating agent is performed in vitro or ex vivo.
In some of any of the embodiments, the incubation with the T cell stimulating agent is performed for 12-72 hours. In some of any of the embodiments, the incubation with the T cell stimulating agent is performed for about 24 hours.
In some of any of the embodiments, the one or more markers of group (i) are selected from the group consisting of CD20, CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (FGFR 4), CD66a/c/e, and TSLPR (TSLP-R), and the one or more markers of group (ii) are selected from the group consisting 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.
In some of any of the embodiments, the incubation with the T cell stimulating agent is performed for about 48 hours.
In some of any of the embodiments, the one or more markers of group (i) are selected from the group consisting of CD20, CD105 (endostatin), CD107a (LAMP-1), CD109, CD120b, CD132 (co-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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD230 (prion), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, CD73 (extracellular-5' -nucleotidase), CD186 (lrp 3) and one or more markers selected from the group consisting of CD32 and (lrf) thereof.
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-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b and CX3CR1.
In some of any of the embodiments, the one or more markers are (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.
Provided herein is a method of identifying activated T cells, the method comprising detecting cell surface expression of one or more markers in cells of a composition comprising T cells, wherein the one or more markers 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、CD165、CD355（CRTAM） and GPR56, and wherein the cells expressing high levels of the one or more markers are activated T cells.
In some of any of the embodiments, the one or more markers is selected from group (i) and consists of CD200 (OX 2), CD357 (GITR), CD120b, CD155 (PVR), CD107b (LAMP-2).
In some of any of the embodiments, the one or more labels are of (ii) and are selected from CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b and CX3CR1.
Provided herein is a method of identifying activated T cells, the method comprising detecting cell surface expression of one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from the group consisting of 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.
In some of any of the embodiments, the detecting is detecting cd4+ or cd8+ T cells in the composition comprising T cells.
In some of any of the embodiments, 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, and the one or more markers from group (ii) are selected from CD49f, CD124 (IL-4rα), CCRL2, CD217, CD192 (CCR 2), CD96 (TACTILE), and CD195 (CCR 5).
In some of any of the embodiments, the detecting is detecting cd4+ T cells in the composition comprising T cells.
In some of any of the embodiments, 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 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7rα).
Provided herein is a method for assessing surface expression of a T cell activation marker of a cd4+ T cell, the method comprising detecting the level of surface expression of one or more markers or the percentage of cells positive for one or more markers in a cd4+ T cell 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-4 ra), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7 ra).
In some of any of the embodiments, the surface expression level or positive cell percentage of the one or more markers in (i) is positively correlated with cd4+ T cell activation. In some of any of the embodiments, the surface expression level or positive cell percentage of the one or more markers in (ii) is inversely correlated with cd4+ T cell activation.
Provided herein is a method for assessing activation of cd4+ T cells comprising (a) detecting the surface expression level of 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) 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-4 ra), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7 ra), and (b) comparing the surface expression level or the percentage of positive cells to the level or percentage of positive cells of each of the one or more markers in cells of an unstimulated control cell composition, wherein a higher level or percentage of positive cells in (i) than the unstimulated control cell composition indicates that a higher level or percentage of positive cells in the control cell composition is activated by the CD4+ cells than the level of the non-stimulated control cell.
In some of any of the embodiments, the detecting is detecting cd8+ T cells in the composition comprising T cells.
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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1, and CD127 (IL-7rα).
Provided herein is a method for assessing surface expression of a T cell activation marker of a cd8+ T cell, the method comprising detecting the surface expression level of one or more markers in a cd8+ T cell of a composition comprising T cells or the percentage of cells positive for the one or more markers, 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1, and CD127 (IL-7 ra).
In some of any of the embodiments, the surface expression level or positive cell percentage of the one or more markers in (i) is positively correlated with cd8+ 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 in (ii) is inversely correlated with cd8+ T cell activation.
Provided herein is a method for assessing activation of cd8+ T cells comprising (a) detecting the surface expression level of or the 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1, and CD127 (IL-7rα), and (b) comparing the surface expression level or the percentage of positive cells to the level or percentage of positive cells of each of the one or more markers in cells of a non-stimulated control cell composition, wherein a higher level or percentage of positive markers in (i) is activated by a higher level of the marker than the non-stimulated control cell composition and a lower percentage of positive cells in the cells of the non-stimulated control cell composition is indicative of the level of activation of the T8+ cells.
In some of any of the embodiments, the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor.
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-4rα), CD217, CD192 (CCR 2), CD195 (CCR 5) and CD96 (TACTILE).
Provided herein is a method for assessing activation of T cells comprising (a) detecting the surface expression level of or the 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、CD83、CD357（GITR）、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD355（CTRAM）、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（CTRAM）、CD155（PVR）、CD74、CD170（Siglec-5） and GPR56.
In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5), and the one or more markers selected from group (ii) are selected from the group consisting of CD49f, CCRL2, CD124 (IL-4 ra), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE).
In some of any of the embodiments, the detecting is detecting a recombinant receptor expressing cd4+ T cell in the composition comprising a T cell.
In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR) and CD74, and/or the one or more markers selected from group (ii) are selected from the group consisting of CD49f, CCRL2 and CD124 (IL-4 ra).
In some of any of the embodiments, the detecting is detecting a recombinant receptor expressing cd8+ T cell in the composition comprising a T cell.
In some of any of the embodiments, the one or more markers from group (i) are selected from CD200 (OX 2), CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM) and GPR56, and/or the one or more markers from group (ii) are selected from CCRL2, CD217, CD96 (TACTILE).
In some of any of the embodiments, the one or more markers of group (i) are selected from the group consisting of CD36L (SCARB 1, SR-BI), CD262 (DR 5, trail-R2), CD105 (endostatin), CD73 (extracellular-5' -nucleotidase), CD83, CD119 (IFN-g ra chain), CD154, CD170 (Siglec-5), notch 1, and CD360 (IL-21R), and the one or more markers of group (i) are selected from the group consisting of CD96 (TACTILE).
Provided herein is a method for assessing activation of T cells comprising (a) detecting the surface expression level of or the 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 (SCARB 1, SR-BI), CD262 (DR 5, trail-R2), CD105 (endoglin), CD73 (extracellular-5' -nucleotidase), CD83, CD119 (IFN-g ra chain), CD154, CD170 (Siglec-5), notch 1 and CD360 (IL-21R), and group (ii) consists of CD96 (TACTILE).
In some embodiments, the composition comprising T cells comprises cells that express a recombinant receptor.
In some of any of the embodiments, the detecting is detecting a recombinant expression cell of the T cell composition.
In some of any of the embodiments, the surface expression of the one or more markers of group (i) is increased on cells expressing the recombinant receptor as compared to cells not expressing the recombinant receptor.
In some of any of the embodiments, the surface expression of the one or more markers of group (ii) is reduced on cells expressing the recombinant receptor as compared to cells not expressing the recombinant receptor.
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.
In some of any embodiment, the recombinant receptor is a Chimeric Antigen Receptor (CAR).
In some of any of the embodiments, the recombinant receptor stimulant comprises a recombinant target antigen recognized by the recombinant receptor. In some of any of the embodiments, the recombinant receptor stimulant is an antibody specific for an extracellular antigen-binding domain of the recombinant receptor. In some of any of the embodiments, the recombinant receptor stimulant is an anti-idiotype antibody specific for the extracellular antigen-binding domain of the recombinant receptor.
In some of any of the embodiments, the recombinant receptor stimulant 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 or flask of a microplate. In some of any of the embodiments, the solid support is a bead.
In some of any of the embodiments, 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 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.
In some of any of the embodiments, the detecting is detecting a recombinant receptor expressing T cell in the composition comprising a T cell.
In some of any of the embodiments, 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 (OX 40), and/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD192 (CCR 2), CD11b, and CX3CR1.
In some of any of the embodiments, the one or more markers from group (i) are selected from CD71, notch 1, CD107a (LAMP-1), CD166, CD245 (p 220/240), CD154, notch 2, CD165, and CD83, and/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD11b, and CX3CR1.
In some of any of the embodiments, the detecting is detecting cd4+ T cells in the composition comprising T cells.
In some of any of the embodiments, the one or more markers from group (i) are selected from CD71, notch 1, CD107a (LAMP-1), CD166, CD245 (p 220/240), CD154, notch 2, and CD165, and/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), and CD127 (IL-7 Ra).
In some of any of the embodiments, the detecting is detecting cd8+ T cells in the composition comprising T cells.
In some of any of the embodiments, the one or more markers from group (i) are selected from CD71, notch 1, CD107a (LAMP-1), CD166, notch 2, CD165, CD83, and/or the one or more markers from group (ii) are selected from CD11b, CX3CR1, and CD127 (IL-7 Ra).
In some of any of the embodiments, the T cell stimulating agent is a pan T cell activator. In some of any of the embodiments, the pan-T cell activating agent comprises an anti-CD 3 antibody and an anti-CD 28 antibody, optionally wherein the pan-T cell activating agent comprises an anti-CD 3 Fab and an anti-CD 28 Fab. In some of any of the embodiments, the pan-T cell activating agent comprises anti-CD 3/anti-CD 28 beads. In some of any of the embodiments, the pan-T cell activating reagent comprises a soluble anti-CD 3/anti-CD 28 streptavidin oligomerization reagent.
In some of any of the embodiments, 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 labels.
In some of any of the embodiments, 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 comprising a substance for binding the one or more labels.
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 are detectably labeled. In some of any of the embodiments, the one or more binding agents are fluorescently labeled.
In some of any of the embodiments, the detecting is by flow cytometry. In some of any of the embodiments, the detecting of step (a) is performed in combination with a CITE-Seq or REAP-Seq. In some of any of the embodiments, the detecting of step (a) is performed by immunohistochemistry, optionally by immunohistochemical fluorescence.
In some of any of the embodiments, 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 labels are used for the detection in step (a).
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers in some of the methods of any of the embodiments.
Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding one or more markers in the methods of some of any of the embodiments.
In some embodiments, the substance for detecting each of the one or more markers is an antibody.
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from group (i) and/or group (ii), wherein group (i) consists of CD20, CD105 (endostatin), 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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, CD73 (extracellular-5' -nucleotidase), DR3 (mp), CD186 (CXCR 6), GARP 32, CD334, tsrp (tsrp) and CD 34 b.sup.3284, and the receptor (tsr) consists of EGFR cells (tsr) and of group (lp-84).
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from group (i) and/or group (ii), wherein group (i) consists of CD20, CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (FGFR 4), 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 receptors, CD229 (Ly-9), CD84 and EGFR.
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from group (i) and/or group (ii), wherein group (i) consists of CD20, CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 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, CD (IL-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, git (VSTM 3), CD230 (prion), CD235, CD261 (IL 4), CD49c (integrin a 3), ig light chain l, igM, CD119 (IFN-g R 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, TMEM8 32122 (IL-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, gitra 3 (IL 3), CD230 (prion, CD235, CD 3), CD3, CD49, c (IL 3), and IL-35 (IL-3), and (IL-3) protein (IL-3).
Provided herein is a kit for determining T cell activation, the kit comprising a substance for detecting one or more markers selected from the 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-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b and CX3CR1.
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from the 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-4rα), CCRL2, CD217, CD192 (CCR 2), CD96 (TACTILE) and CD195 (CCR 5).
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from the 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-4rα), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7rα).
Provided herein are kits for determining T cell activation comprising a substance for detecting one or more markers selected from the 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1 and CD127 (IL-7rα).
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from the group (i) and/or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX 2), CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74, and the one or more markers selected from the group (ii) consists of CD49f, CCRL2, CD124 (IL-4 ra), CD217, CD192 (CCR 2), CD355 (CRTAM), GPR56, CD195 (CCR 5) and CD96 (TACTILE).
Provided herein is a kit for determining T cell activation, the kit comprising a substance for detecting one or more markers selected from the 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 (OX 40), and/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD192 (CCR 2), CD11b and CX3CR1.
Provided herein is a kit for determining T cell activation comprising a substance for detecting one or more markers selected from the group (i) and/or group (ii), wherein group (i) consists of CD262 (DR 5, trail-R2), CD105 (endoglin), CD36L1 (SCARB 1, SR-BI), CD73 (extracellular-5' -nucleotidase), CD83, CD119 (IFN-g ra chain), CD154, CD170 (Siglec-5), notch 1 and CD360 (IL-21R), and/or said one or more markers from group (ii) is selected from CD96 (TACTILE).
Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding to one or more markers selected from the 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 strand), CD105 (endostatin), CD262 (DR 5, TRAIL-R2), CD170 (Siglec-5), CD73 (extracellular-5' -nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g-strand ）、CD148、CD150（SLAM）、CD151（PETA-3）、CD154、CD165、CD166、CD200 R、CD217、CD218a（IL-18Ra）、CD227（MUC-1）、CD245（p220/240）、CD30、CD317（BST2、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD100, CD123, CD184 (CXCR 4), CD55, TIT (VSTM 3), CD196 (CCR 6), CD230 (prion), CD261, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3 k, notch 2, CD164, CD319 (CRACC), CD355 (CRTAM), CD4, CD49a, 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, TMEM8 (IL-2), TMEM8 (IL-2 Rb), CD100, CD123, CD184 (CXCR 3), CD66, CD3, CD35 (LPR 3) and CD3, and group (ii) consisted 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.
Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding to one or more markers selected from the group (i) and/or group (ii), wherein group (i) consists of CD20, CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (FGFR 4), CD66a/c/e and TSLP R (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 receptors, CD229 (Ly-9), CD84 and CD 9.
Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding to one or more markers selected from group (i) and/or group (ii), wherein group (i) consists of CD20, CD105 (endostatin), CD107a (LAMP-1), CD109, CD120b, CD132 (co-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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 RA 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-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, git (VSTM 3), CD230 (prion), CD261, CD4, CD49 b7, light chain k, ig light chain l, igM, CD119 (IFN-g RA 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, CD (IL-2 Rb), CD3, CD35, CD261, CD49, and CD3 (DR 3), and the group (lrf) 2, 34 d (d) and (d) 2.
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 the 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-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b and CX3CR1.
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 the group consisting of 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-4rα), CCRL2, CD217, CD192 (CCR 2), CD96 (TACTILE) and CD195 (CCR 5).
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 the group consisting of 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-4rα), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7rα).
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 the group consisting of 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1 and CD127 (IL-7rα).
Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding to one or more markers selected from the group consisting of CD120b, CD83, CD357 (GITR), CD200 (OX 2), CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74 and the one or more markers selected from the group consisting of CD49f, CCRL2, CD124 (IL-4 ra), CD217, CD192 (CCR 2), CD355 (CRTAM), GPR56, CD195 (CCR 5) and CD96 (TACTILE).
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 the group consisting of 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 (OX 40), and/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD192 (CCR 2), CD11b and CX3CR1.
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 the group (i) and/or group (ii), wherein group (i) consists of CD36L (SCARB 1, SR-BI), CD262 (DR 5, trail-R2), CD105 (endothelin), CD73 (extracellular-5' -nucleotidase), CD83, CD119 (IFN-g ra chain), CD154, CD170 (Siglec-5), notch 1 and CD360 (IL-21R), and/or said one or more markers from group (ii) is selected from CD96 (TACTILE).
In some of any of the embodiments, the substance for detecting the one or more labels is one or more binding agents that bind the one or more labels. 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 are detectably labeled. In some of any of the embodiments, the one or more binding agents are fluorescently labeled.
Provided herein is a method of isolating activated T cells, the method comprising identifying a population of activated T cells according to the method in some of any of the embodiments and isolating the population.
Provided herein is a method of enriching for activated T cells, the method comprising identifying a population of activated T cells according to the method in some of any of the embodiments and selecting the population, thereby obtaining a population of cells enriched for activated T cells.
Provided herein is a method of 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 the method in some of any of the embodiments.
Provided herein are T cell populations produced according to the methods in some of any of the embodiments.
Drawings
Figure 1A shows the difference in percentage of positive cells for each marker of cd8+car+t cells calculated between T0 and T48 hour stimulation time points. The top 15 up-regulation markers and the bottom 5 down-regulation markers are shown here and are coded based on literature according to whether they are canonical or non-canonical activation markers.
Figure 1B shows the difference in positive cell percentages for each marker of cd4+ car+ T cells calculated between T0 and T48 hour stimulation time points. In this case, the CD4 CAR-T product is shown. The top 15 up-regulation markers and the bottom 5 down-regulation markers are shown here and are coded based on literature according to whether they are canonical or non-canonical activation markers.
Figure 2A shows the difference in positive cell percentages for each marker of cd8+ PBMC T cells calculated between T0 and T48 hour stimulation time points. The top 15 up-regulation markers and the bottom 5 down-regulation markers are shown here and are coded based on literature according to whether they are canonical or non-canonical activation markers.
Figure 2B shows the difference in positive cell percentages for each marker of cd4+ PBMC T cells calculated between T0 and T48 hour stimulation time points. The top 15 up-regulation markers and the bottom 5 down-regulation markers are shown here and are coded based on literature according to whether they are canonical or non-canonical activation markers.
Fig. 3A shows the same data from the previous four graphs, visualized in a different way, with the top 15 up-regulated markers and bottom 5 down-regulated markers from each of the previous four graphs clustered together and displayed on the x-axis. The CD4 components of both PBMC and car+ T cells are shown together. If the marker is blank, this does not mean that the marker is not present in the CD4 fraction, but it is not present in the top 15 and bottom 5 marker lists.
Fig. 3B shows the same data from the previous four graphs, visualized in a different manner, with the top 15 up-regulation markers and the bottom 5 down-regulation markers from each of the previous four graphs clustered together and displayed on the x-axis. The CD8 components of both PBMC and car+ T cells are shown together. If the tag is blank, this does not mean that the tag is not present in the CD8 component, but it is not present in the top 15 and bottom 5 tag lists.
FIG. 4 shows the abundance of 5 representative non-canonical activation markers within the CAR+T product, depending on whether the cell also expresses at least one canonical activation marker. Cells are divided into two bins, cells expressing at least one canonical activation marker and cells not expressing any canonical activation marker. The percentages of cells expressing the five non-canonical activation markers are shown separately in the two bins. Cells expressing at least one canonical marker are more likely to also express each non-canonical marker.
Fig. 5A-5S show pseudo-color plots of upregulated non-canonical markers generated in fig. 1-3, filtered only on car+ T cells, and plotted against three canonical activation markers.
Fig. 6A-6J show pseudo-color plots of the other canonical markers generated in fig. 1-3, filtered only on car+ T cells, and plotted against the three canonical activation markers used in fig. 4.
Fig. 7A-7K show pseudo-color plots of the non-canonical markers generated in fig. 1-3 down-regulated, filtered only on car+ T cells, and plotted against the three canonical activation markers used in fig. 4.
Figure 8 shows a venn plot of the number of markers with > 10% change filtered on all T cells (both CAR-T and PBMC) in each time point pair. Some representative markers from each group are shown.
Fig. 9A-9N show histograms of expression of 14 different cell surface markers in car+ or CAR-cells stimulated for 48 hours (t=48 h), 24 hours (T-24 h) or not stimulated (t=0 h), grouped by cd4+ T cells and cd8+ T cells.
Fig. 10A-10B show the area under the curve (AUC) of the different markers for car+ and CAR-cells, grouped per manufacturing process, where the AUC for each sample was calculated by summing the percent positivity for each marker across all time points.
Fig. 11A-11B show the data and percent positivity for each marker in the car+ chamber plotted on the y-axis, as well as the time plotted on the x-axis (in hours after stimulation). Each sample is represented as a separate line for each marker within each process.
Detailed Description
Provided herein are methods for assessing T cell activation in a cell composition, the methods comprising detecting the surface expression level 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 the duration or time of incubation settings for T cell use based on activation of the T cells. The embodiments provided are based on the identification of T cell markers that are up-or down-regulated in activated T cells. T cell markers include in many respects non-canonical markers that have not previously been reported to be associated with T cell activation or are not generally used to assess T cell activation. Embodiments provided include markers useful for assessing or determining activation of a particular subset of T cells (e.g., cd4+ or cd8+ T cells). The provided embodiments also include markers useful for assessing or determining activation of T cells engineered with a T cell signaling recombinant receptor, such as a Chimeric Antigen Receptor (CAR) or a recombinant T Cell Receptor (TCR). Also provided herein are kits for assessing T cell activation in a cell composition, the kits containing a substance for detecting the one or more markers.
Existing methods for assessing T cell activation, for example during in vitro or ex vivo T cell culture, typically 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. In general, one marker is not fully consistent with whether all existing T cells are activated, resulting in benefits in assessing T cell activation using multiple markers.
The provided methods allow for assessing T cell activation using the markers provided herein. In some aspects, the marker is a non-canonical marker. The results provided herein demonstrate that a marker (e.g., a non-canonical marker) is up-regulated in stimulated cells when compared to non-stimulated cells. The results also demonstrate that certain markers are expressed in cell subsets, 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 down-regulated upon T cell activation. The results provided herein demonstrate that after T cells have been stimulated (e.g., 72 hours, 48 hours, or 24 hours before their activation state is detected by examining the surface expression of surface markers on T cells), certain markers increase or decrease the expression of these markers based on whether the cells are activated, as verified using canonical activation markers. The results provided herein indicate that if cells are differentiated by whether they express canonical markers, cells expressing canonical markers are more likely to be positive for other markers, such as CD200 (OX 2), CD357 (GITR), CD120b, CD155 (PVR), and CD107b (LAMP-2). Furthermore, if markers identified as differentially expressed on a stimulated cell population are mapped relative to canonical markers of cell activation, a double positive cell population appears for non-canonical markers that are increased upon activation, whereas no double positive cell population appears for non-canonical markers that are decreased upon stimulation. In some embodiments, the change in marker expression is time sensitive and shows a difference in expression 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 CD 8T cell). In some embodiments, the change in marker expression is specific to cell processing, wherein the change in cell marker expression occurs more significantly in PBMC or car+ T cells.
In some aspects, the provided methods involve assessing T cell activation based on the expression or surface level of a marker such as CD20, CD105 (endostatin), 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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (DR 4, TRAIL-R1), CD304 (neuropin-1), CD49d, CD73 (extracellular felt-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 the recombinant receptor, in some embodiments are T cell activation in CD8, in some embodiments in which the CD4+ cell activation in some embodiments in the CD4+ cell activation methods involve providing methods in some embodiments in the T cell activation in the CD8+ cells are assessed.
In some aspects, the provided methods can be used to determine whether a cell is ready to be transduced. In some aspects, provided methods further comprise selecting or isolating T cells to engineer the T cells after determining activation. In some aspects, the provided methods further comprise engineering the determination and/or monitoring of activated T cells to produce a cell therapy product. In some embodiments, the provided methods can be used to monitor T cell activation, stimulation, or kinetics during incubation (e.g., under expansion conditions). In some aspects, the provided methods can be used to identify a relationship between activation status and outcome, such as how cells evolve over time. In some aspects, the provided methods allow for determining when cells can be administered to a patient.
In some embodiments, the provided methods can be used to predict the quality of T cells undergoing a manufacturing process, e.g., during or after the manufacturing process. In some aspects, the activation state determined by the provided methods may be used as a readout during manufacturing, e.g., a readout of manufacturing success or a successful manufacturing step (e.g., cell stimulation). In some embodiments, the provided methods can be used to monitor whether T cells are sufficiently activated, such as for expanding T cells to a desired threshold number, e.g., to the number required for clinical dosing of T cells for T cell therapy.
In some aspects, activation of T cells can result in differentiation of T cells. A higher proportion of early memory T cells (e.g., naive T cells) in T cell therapy may improve patient outcome (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 state of T cells, either directly or by monitoring the activation state of T cells. In some embodiments, the activation state of T cells is monitored to predict the memory state of T cells.
In some aspects, the cellular phenotype information obtained by the provided methods may be used during process development to optimize the duration of the manufacturing process or steps thereof or other conditions in order to improve the quality of the treated T cells. In some cases, this information may be used to develop a process control strategy, wherein, for example, when a predicted cell phenotype (e.g., activation state) falls outside a determined range, the conditions of one or more (e.g., current or subsequent) manufacturing steps may be changed, e.g., the duration of the current or subsequent manufacturing steps may be changed, to improve the final quality of the manufactured T cells. For example, when the activation state falls outside of a defined range during incubation, in some cases, subsequent incubation may be performed under perfusion conditions and/or in the presence of small molecules, e.g., to modulate the T cell phenotype to a desired profile.
In some aspects, activation state information obtained by the provided methods can be used to assess or reduce batch-to-batch variability of T cells undergoing a manufacturing process. In some aspects, activation status information may be used to assess or reduce inter-lot variability of pharmaceutical products produced using a manufacturing process. For example, by ensuring that T cells that are manufactured to run across different cell therapies are in a comparable activated state, the differentiation and memory states of the T cells can be maintained consistent. This may reduce variability (e.g., inter-patient variability) in the resulting T cell therapies (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 activation state of T cells before or after engineering the T cells. In some aspects, transgene expression may be higher in activated T cells than in non-activated T cells, such as after viral transduction of T cells (see, e.g., ghassemi et al, nature Biomedical Engineering (2022) 6:118-128). In some aspects, electroporation efficiency for engineering may 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, T cells are monitored prior to engineering according to the provided methods, e.g., such that engineering can be initiated once the provided methods predict that T cells are sufficiently activated to improve transgene expression. In some embodiments, T cells are monitored after engineering according to the provided methods, for example, to determine whether T cells are sufficiently activated or remain sufficiently activated after engineering to improve transgene expression.
All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If the definition set forth herein is contrary to or otherwise inconsistent with the definition set forth in the patents, applications, published applications and other publications, incorporated by reference herein, the definition set forth herein takes precedence over the definition set forth herein.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. Methods for assessing T cell activation
In some embodiments, the provided methods involve assessing the surface expression of a T cell activation marker of a T cell in a cell of the composition. In some embodiments, the provided methods are used to assess T cell activation within cells of a 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 T cells described in section II or section III.
In some embodiments, T cells are assessed by detecting surface expression of the marker. In some embodiments, T cells are evaluated by detecting expression of one or more markers. In some embodiments, the one or more markers are expressed on the surface of T cells. In some embodiments, the methods involve determining the presence or absence of one or more markers on T cells on 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 is indicative of T cell activation. In some embodiments, a decrease in the one or more markers is indicative of T cell activation. In some embodiments, the one or more markers are used to determine the extent of intracellular T cell activation of the composition. In some embodiments, the one or more markers are used to determine the number or percentage of T cells activated within the cells of the composition.
In some embodiments, activation of the T cell is determined based on expression of a combination of one or more markers in the cell of the composition. In some embodiments, T cell activation is determined based on increased expression of some of the one or more markers and decreased expression of others of the one or more markers on the cells of the composition. In some embodiments, activation of the T cell is determined based on 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 expression of a combination of 2 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 3 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 4 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 5 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 6 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 7 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 8 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 9 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 10 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 15 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 20 markers. In some embodiments, activation of T cells is determined based on expression of a combination of between 2 and 20 markers. In some embodiments, activation of T cells is determined based on expression of a combination of between 2 and 10 markers. In some embodiments, activation of T cells is determined based on expression of a combination of between 2 and 5 markers.
In some embodiments, the provided methods involve determining cd4+ T cell activation in cells of the composition. In some embodiments, the provided methods involve determining cd8+ T cell activation in cells of the composition. In some embodiments, the provided methods involve determining activation of T cells containing a recombinant receptor in 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 cells of the composition. In some embodiments, the provided methods involve determining activation of cd8+ T cells containing a recombinant receptor in cells of the composition.
In some embodiments, the cell is present in the subject. In some embodiments, cells are isolated from a subject and their activation status is assessed ex vivo. In some embodiments, 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 stimulating agents for activating cells include any of the agents described in section III.
In some embodiments, the stimulation conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (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 cells)). In some embodiments, the cells are stimulated and the phenotype is determined by whether soluble factors (e.g., cytokines or chemokines) are produced or secreted. In some embodiments, the stimulus is non-specific, i.e., not antigen-specific. In some embodiments, the cells are incubated 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 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 comprising an end value), or more than 24 hours in the presence of a stimulating condition or agent.
In some embodiments, the cells are stimulated with an agent that is an antigen or epitope thereof that is specific for a recombinant receptor or an antibody or fragment thereof that binds to and/or recognizes a recombinant receptor, or a combination thereof. In some embodiments, the recombinant receptor is a CAR and the agent is an antigen or epitope thereof specific for the CAR, or an antibody or fragment thereof that binds and/or recognizes the CAR, or a combination thereof. In certain embodiments, the cells are stimulated by incubating the cells in the presence of a target cell 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 to the CAR. In certain embodiments, the antibody or active fragment, variant, or portion thereof that binds to the CAR is an anti-idiotype (anti-ID) antibody.
In some embodiments, the stimulation conditions or stimulators include one or more agents (e.g., ligands) capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates a TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include, for example, antibodies that are bound to a solid support (e.g., beads), such as those antibodies that are specific for TCR components and/or co-stimulatory receptors (e.g., anti-CD 3, anti-CD 28), and/or one or more cytokines. In some embodiments, the one or more agents are PMA and ionomycin.
A. markers
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 cell and can be used to measure activation of the T cell. In some embodiments, the marker is identified by comparing the surface expression of the marker on stimulated T cells to unstimulated T cells. In some embodiments, the markers are identified by comparing the expression of potential markers to the expression of canonical markers on stimulated and unstimulated T cells to identify markers corresponding to activation. In some embodiments, flow cytometry is used to compare potential markers to canonical markers to identify markers. In some embodiments, the screening is performed using infinite flow cytometry (infinity flow cytometry) to identify markers by utilizing canonical markers of T cell activation as a scaffold for staining across samples, then screening potential markers across wells, each well having a unique potential marker. In some embodiments, the non-limiting cytometry screen may be used to correlate potential activation markers with canonical activation to assess which potential activation markers may be used as markers to assess T cell activation.
In some embodiments, the one or more markers used to assess T cell activation are selected from the group consisting of CD20, CD105 (endostatin), CD107a (LAMP-1), CD109, CD120b, CD132 (co-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), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 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 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, CD73 (extracellular-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 marker is one that is (i) up-regulated upon T cell stimulation or activation or (ii) down-regulated 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 cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cell is an unengineered isolated T cell. In some embodiments, T cells are engineered with recombinant receptors, such as CARs.
In some embodiments, the sample is a cd4+ car+ T cell, a cd8+ car+ T cell, a cd4+ PBMC T cell, and a cd8+ PBMC T cell. In some embodiments, the marker of group (i) is selected from the group consisting of CD20, CD105 (endostatin), CD107a (LAMP-1), CD109, CD120b, CD132 (co-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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, CD73 (extracellular-5' -nucleotidase), DR3 (TRAIL), CD186 (CXCR 6), GARP (LRRC 32), CD116, CD334 (FGFR 4), 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 receptors, CD229 (Ly-9), CD84, and EGFR.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) are selected from CD20, CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (FGFR 4), CD66a/c/e, and TSLPR (TSLP-R), and 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 (229-9), 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 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 cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cell is an unengineered isolated T cell. In some embodiments, T cells are engineered with recombinant receptors, such as CARs.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from the group consisting of CD20, CD105 (endostatin), CD107a (LAMP-1), CD109, CD120b, CD132 (co-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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (tm 3), CD230 (prion), CD235, CD261 (DR 4, IL-R1), CD304 light chain k, ig light chain l, igM, CD119 (IL-g R3), CD49d (IL-2 Rb), CD3, and CD 3; in some embodiments, the one or more markers are assessed from the group (ii) 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）、CD11b、CX3CR1、NKp80、CD127（IL-7Ra） and CD49f. In some embodiments, the one or more markers are assessed from 24 to 72 hours after stimulation, in some embodiments, the one or more markers are assessed from 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, t cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cell is an unengineered isolated T cell. In some embodiments, T cells are engineered with recombinant receptors, such as CARs.
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-4Rα）、CCRL2、CD217、CD192（CCR2）、CD96（TACTILE） and CD195 (CCR 5), and the 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. 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 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 cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cell is an unengineered isolated T cell. In some embodiments, T cells are engineered with recombinant receptors, such as CARs.
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-4Rα）、CCRL2、CD217、CD192（CCR2）、CD96（TACTILE） and CD195 (CCR 5).
In some embodiments, 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.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is 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 group (ii) is selected from CD49f, CD124 (IL-4rα), CCRL2, CD217, CD192 (CCR 2), CD96 (TACTILE), and CD195 (CCR 5). 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, T cells are engineered with recombinant receptors, such as CARs. In some embodiments, the T cell is a cd3+ car+ T cell. In some embodiments, the T cell is a cd4+ car+ T cell. In some embodiments, the T cell is a cd8+ car+ T cell. 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.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is 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) is selected from CD49f, CCRL2, CD124 (IL-4rα), CD217, CD192 (CCR 2), CD195 (CCR 5) and CD96 (TACTILE). 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, T cells are engineered with recombinant receptors, such as CARs. In some embodiments, the T cell is a cd3+ car+ T cell. In some embodiments, the T cell is a cd4+ car+ T cell. In some embodiments, the T cell is a cd8+ car+ T cell. 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.
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-4Rα), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Rα). 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 cell is a cd4+ T cell. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cell is an unengineered isolated T cell. In some embodiments, T cells are engineered with recombinant receptors, such as CARs. In some embodiments, the T cell is a cd4+ car+ T cell.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from the group consisting of CD120b, CD83, CD357 (GITR), CD200 (OX 2), CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74 and CD170 (Siglec-5), and/or the one or more markers of group (ii) is selected from the group consisting of CD49f, CCRL2, CD124 (IL-4Rα), CD217 and CD192 (CCR 2). 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 cell is a cd4+ T cell engineered with a recombinant receptor such as a CAR. In some embodiments, the T cell is a cd4+ car+ T cell.
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 the group consisting of CD96 (TACTILE), CD195 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1, and CD127 (IL-7Rα). 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 cell is a cd8+ T cell. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cell is an unengineered isolated T cell. In some embodiments, T cells are engineered with recombinant receptors, such as CARs. In some embodiments, the T cell is a cd8+ car+ T cell.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from CD120b, CD83, CD357 (GITR), CD200 (OX 2), CD134 (OX 40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR) and GPR56, and/or the one or more markers of group (ii) is selected from CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5) and CD96 (TACTILE). 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 cell is a cd8+ T cell engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cell is a cd8+ car+ T cell.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from the group consisting of CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5), and the one or more markers of group (ii) is selected from the group consisting of CD49f, CCRL2, CD124 (IL-4 Rα), 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, T cells are selective for activation of car+ T cells following stimulation with a CAR-dependent agent (e.g., an anti-idiotype antibody) or by antigen-expressing cells. 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 cell is a cd3+ car+ T cell. In some embodiments, the T cell is a cd4+ car+ T cell. In some embodiments, the T cell is a cd8+ car+ T cell. 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.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from the group consisting of CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR) and CD74, and/or the one or more markers of group (ii) is selected from the group consisting of CD49f, CCRL2 and CD124 (IL-4 Rα). In some embodiments, the one or more markers are selective for activation of cd4+ car+ T cells. In some embodiments, T cells are selective for activation of cd4+ car+ T cells following stimulation with a CAR-dependent agent (e.g., an anti-idiotype antibody) or by antigen-expressing cells. 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 cell is a cd4+ car+ T cell. In some embodiments, the one or more markers are used to assess T cell activation on cd4+ car+ T cells.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from the group consisting of CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM) and GPR56, and the one or more markers of group (ii) is selected from the group consisting of CCRL2, CD217, CD96 (TACTILE). In some embodiments, the one or more markers are selective for activation of cd8+ car+ T cells. In some embodiments, T cells are selective for activation of cd8+ car+ T cells following stimulation with a CAR dependent agent (e.g., an anti-idiotype antibody). 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 cell is a cd8+ car+ T cell. In some embodiments, the one or more markers are used to assess T cell activation on cd4+ car+ T cells.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from CD71、Notch 1、CD107a（LAMP-1）、CD166、CD245（p220/240）、CD154、Notch 2、CD165、CD120b、CD83、CD357（GITR）、CD200（OX2） and CD134 (OX 40), and/or the one or more markers of group (ii) is selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD192 (CCR 2), CD11b, and CX3CR1. 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 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 cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD71, notch 1, CD107a (LAMP-1), CD166, CD245 (p 220/240), CD154, notch 2, CD165, and CD 83), and/or the one or more markers of group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), 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 with pan T cell activation, such as anti-CD 3/anti-CD 28. 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 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 cells are T cells obtained from peripheral blood of a subject, such as T cells obtained from a PBMC sample.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD71, notch 1, CD107a (LAMP-1), CD166, CD245 (p 220/240), CD154, notch 2, and CD165, and/or the one or more markers of group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), and CD127 (IL-7 Ra). 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-CD 3/anti-CD 28. 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 cell is a cd4+ T cell. In some embodiments, the T cells are T cells obtained from peripheral blood of the subject, such as cd4+ T cells obtained from a PBMC sample.
In some embodiments, the one or more markers is selected from group (i) and/or group (ii), wherein the one or more markers of group (i) is selected from the group consisting of CD71, notch 1, CD107a (LAMP-1), CD166, notch 2, CD165, CD83, and/or the one or more markers of group (ii) is selected from the group consisting of CD11b, CX3CR1, and CD127 (IL-7 Ra). In some embodiments, the T cells are selective for assessing 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-CD 3/anti-CD 28. 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 cell is a cd8+ T cell. In some embodiments, the T cells are T cells obtained from peripheral blood of the subject, such as cd8+ T cells obtained from a PBMC sample.
In some embodiments, the one or more markers are differentially expressed on cells that express the recombinant receptor. In some embodiments, the one or more markers are differentially expressed on cells that express 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 on the cell expressing the recombinant receptor is increased as compared to the surface expression on a cell not expressing the recombinant receptor. In some embodiments, the surface expression of the one or more markers on cells expressing the recombinant receptor is reduced compared to the surface expression on cells not expressing the recombinant receptor.
In some embodiments, the one or more markers is selected from group (i) or group (ii), wherein the one or more markers of group (i) is selected from CD262 (DR 5, trail-R2), CD105 (endostatin), CD36L1 (SCARB 1, SR-BI), CD73 (extracellular-5' -nucleotidase), CD83, CD119 (IFN-g ra chain), CD154, CD170 (sig-5), notch 1, and CD360 (IL-21R), and/or the one or more markers of group (ii) is selected from CD96 (TACTILE). In some embodiments, the one or more markers are expressed in a T cell composition comprising T cells expressing the recombinant receptor. In some embodiments, the surface expression of the one or more markers of group (i) on cells expressing the recombinant receptor is increased compared to the surface expression on cells not expressing the recombinant receptor. In some embodiments, the surface expression of the one or more markers of group (ii) on cells expressing the recombinant receptor is reduced compared to the surface expression on cells not expressing the recombinant receptor.
In some embodiments, the one or more markers are surface proteins. In some embodiments, the one or more markers may be involved in a variety of cellular functions. In some embodiments, the one or more markers may be particularly related to metabolism, cell proliferation, cell signaling, immune response, apoptosis, and the like. In some embodiments, the one or more markers is selected from group (i) or group (ii), wherein the one or more markers of group (i) is selected from CD262 (DR 5, trail-R2), CD105 (endostatin), CD36L1 (SCARB 1, SR-BI), CD73 (extracellular-5' -nucleotidase), CD83, CD119 (IFN-g ra chain), CD154, CD170 (sig-5), notch 1, and CD360 (IL-21R), and/or the one or more markers of group (ii) is 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 descriptions of the markers and their roles.
TABLE 1 markers and description thereof
In some embodiments, the one or more labels are non-canonical labels. In some embodiments, the one or more markers are markers that are not normally 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 agent
In some embodiments, the T cell activation status is determined based on the amount or percentage of cells that bind to the binding agent. In some embodiments, one or more binding agents specifically bind to the one or more labels. In some embodiments, the one or more binding agents are antibodies. In some embodiments, one or more antibodies are uniquely labeled. In some embodiments, each of the one or more antibodies is uniquely fluorescently labeled. In some embodiments, measuring the unique signature of an antibody is used to quantify the surface expression of the one or more antibodies. In some embodiments, a unique fluorescent label is used to quantify the surface expression of the one or more antibodies. In some embodiments, the one or more antibodies are commercially available antibodies.
1. Antibodies to
In some embodiments, any substance used to detect a marker selected from the group consisting of CD20, CD105 (endostatin), CD107a (LAMP-1), CD109, CD120b, CD132 (co-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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (DR 4, TRAIL-R1), CD304 (neuroprotein-1), CD49d, CD73 (extracellular felt-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 is used to detect the marker.
In some embodiments, the one or more binding agents are one or more antibodies. As is well known in the art, an "antibody" is an immunoglobulin (Ig) molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, fab, fab ', F (ab') 2, fv), single chains (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen-binding fragment of the desired specificity, chimeric antibodies, nanobodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) of the desired specificity. Also included herein are miniantibodies comprising scfvs linked to CH3 domains (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.
In some embodiments, each of the one or more binding agents (e.g., antibodies) comprises a substance for binding (e.g., specifically or preferentially binding) a corresponding label. In some embodiments, binding of the binding agent (e.g., antibody) to the label can be used to detect one or more labels on the surface of the T cells, e.g., by flow cytometry, such as determining T cell activation. In some embodiments, each of the one or more binding agents (e.g., antibodies) comprises a substance for detecting one or more markers to determine T cell activation.
Binding agents (e.g., antibodies) that "specifically bind" or "preferentially bind" (used interchangeably herein) to a label are terms well known in the art. A molecule is considered to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently and more rapidly with a particular marker target for a longer duration and/or with greater affinity than if it were to react or associate with an alternative marker. An antibody specifically binds or preferentially binds a target if the antibody binds the target with greater affinity, avidity, and/or for a longer duration than if the antibody binds other substances. It will also be appreciated by reading this definition that specific binding or preferential binding is not necessarily required (although may be included). Methods for determining such specific or preferential binding are also well known in the art, such as immunoassays.
In particular embodiments, the antibody is selected from the group consisting of monoclonal antibodies, humanized antibodies, single chain antibodies, antibody fragments, 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.
In some embodiments, the antibody is detectably labeled. In some embodiments, the detectable label is a fluorescent label, a radiolabel, or an enzymatic label. In some embodiments, the one or more antibodies are fluorescently labeled with one or more fluorescent moieties.
Any of a variety of antibody binding reagents are known and may be used to detect any one or more of the above-described labels. Various commercial sources of antibody reagents include, but are not limited to Thermo Fisher、Biolegend、BD Biosciences、Abcam、Bio X Cell、Invitrogen、Sigma-Aldrich、Miltenyi Biotec、Beckman、LifeSpan BioSciences Inc、Santa Cruz Bioscience and/or Novus Biologicals.
In some embodiments, the one or more binding agents may include one or more of anti-human CD107B (LAMP-2) (e.g., clone H4B4, clone 8E2F2, clone 6A10H10, clone 02, or clone AF 488), anti-human CD120B (e.g., clone 3G7A02, clone MR2-1, clone 22221, clone utr 1, clone 80M2, clone 7G8B6, or 2H11 CR), anti-human CD357 (GITR) (e.g., clone 108-17, clone 2H4, clone 621, clone OTI9G8, clone, Clone ANC7D6, clone ANC5E3, AIT 158D, clone 4H2D6 or clone aa 26-162), anti-human CD83 (e.g., clone HB15E, clone 3G10-1F4, clone 8A4C11 or clone 6H4G 10), anti-human CD200 (OX 2) (e.g., clone A18042B, clone OX-104 or clone 6E8B 11), anti-human CD134 (OX 40) (e.g., clone Ber-ACT35, clone W4-3, clone UMAB276, clone 3G5G7 or clone OTI2F12A 2), anti-human CD200 (OX 40) (e.g., clone Ber-ACT35, clone, Anti-human CD155 (PVR) (e.g., clone TX24, clone aa314-342, clone aa220-345, clone ANC2B2, or clone ANC6A 3), anti-human CD74 (e.g., clone LN2, clone BU45, clone PIN.1, B318, clone CDLA-1, clone 2D1B11, clone M-B741, or clone 2D1B 3), 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, Clone OTI3E12, clone A6, clone mN1A or clone 4G 1), anti-human Notch 2 (e.g., clone MHN2-25, clone NOD-15, clone 8A1, clone OT13E12, clone A6, clone OTI2E7 or clone 487CT6.9.2), anti-human CD166 (e.g., clone 3A6, clone 3F8B12, clone 10F1G12, clone 4H9A5 or clone 8E12C 7), anti-human CD107a (LAMP-1) (e.g., clone H4A3, clone 6E2, clone 5H6, clone Ly1C6 or clone OTI8B 1), Anti-human CD71 (e.g., clone CY1G4, clone 1E6, clone H68.4, clone 3G291, clone DF1513, clone 10F11, clone 1A1B2, MEM-189, or clone SOM4D 10), anti-human CD245 (p 220/240) (e.g., clone DY12 or clone OTI3F 7), anti-human CD154 (e.g., clone 24-31, clone 2E2, clone 8H10F5, clone 1H4, clone 301, or clone 5A3A 9), anti-human CD165 (e.g., clone SN2 or clone AD 2), anti-human CD165 (e.g., clone D2), Anti-human CD355 (CRTAM) (e.g., clone Cr24.1, 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 6B 4), anti-human CD124 (IL-4Rα) (e.g., clone G077F6, clone 25463, clone 1D3, clone R401, clone R001, or clone Hil R-M57), anti-human CD124 (IL-4Rα), Anti-human CCRL2 (e.g., clone K097F7, clone 12K19, or clone 1B 2), anti-human CD217 (e.g., clone W15177A, clone 49M4D2, clone 004, clone 6H1B 1), anti-human CD192 (CCR 2) (e.g., clone K036C2, clone 7A7, clone 3B6B1, clone 4D12, clone 2A9-a, or clone 48607), anti-human CD96 (NK TACTILE) (e.g., clone 92.39, clone 6F9, clone 1C8, clone 5E6C12, or clone 8A11F 8), anti-human CD192 (CCR 2), Anti-human CD127 (IL-7Rα) (e.g., clone A019D5, clone AbD11590, clone HIL-7R-M21, clone IL7R/2751, clone ANC8F2 or clone 3F5D 9), anti-human CD11B (e.g., clone ICRF44, clone REA713, clone M1/70.15, clone CBRM1/5, clone M1/70, clone X-5 or clone 3A10H 5) and anti-human CX3CR1 (e.g., clone 2A9-1, clone 2B11 or clone REA 385).
In provided embodiments, the binding agent (e.g., antibody) is conjugated to a fluorescent label, such as a fluorophore. For example, the cells may be incubated with one or more fluorescently labeled antibodies. In some embodiments, any fluorescent label or fluorophore suitable for flow cytometry analysis may 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 colorants (e.g., 4', 6-diamidino-2-phenylindole (DAPI), SYT016, propidium Iodide (PI), cell membrane colorants (e.g., FMI-43), cell functional dyes (e.g., fluo-4, indo-1), and synthetic dyes (e.g., brilliant Violet (BV)). Exemplary fluorophores include, but are not limited to, hydroxycoumarin, waterfall Blue (Cascade Blue), dyight 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、Chromeo642、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.
In some embodiments, the one or more antibodies are labeled with one or more of Dylight 405, alexa Fluor 405, pacific Blue, alexa Fluor 488, fluorescein (FITC), dylight 550, phycoerythrin (PE), allophycocyanin (APC), alexa Fluor 647, dylight 650, polymannuin-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).
In some embodiments, multiple fluorophores are used for multicolor staining or labeling, wherein multiple antibodies to different markers are incubated with the cells. In some embodiments, fluorescent labels, for example, conjugated to such antibodies, are selected to minimize energy transfer between them, such as to avoid or minimize overlapping emission and absorption spectra. In some embodiments, each fluorescent label has a different emission spectrum. In some embodiments, multiple fluorescent labels may be excited with a single wavelength or multiple wavelengths, but detection occurs in areas where the peak emission spectra do not overlap. In some embodiments, one or more of the fluorescent markers may be excited by a single wavelength or the same wavelength of light, but thereby emit light of a different wavelength therefrom.
In some embodiments, each of the one or more antibodies is labeled with a unique fluorescent moiety. In some embodiments, the one or more fluorescent labels each individually comprise a fluorophore selected from the group consisting of PE-Cy7, APC, AF700, BV421, aqua, and BV 605.
C. Measuring marker surface expression
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 staining of cells involves incubation with antibodies or binding agents that specifically bind such labels, in some embodiments followed by a washing step, and separating cells that have bound to the antibodies or binding partners from those that have not bound to the antibodies or binding partners. In some aspects of such processes, a volume of cells is mixed with a quantity of the 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 ℃ and 25 ℃, such as at or about 4 ℃. In some embodiments, the staining or marking is performed for greater than 5 minutes, typically greater than 15 minutes. In some embodiments, the staining or marking 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 marking is performed for a time of, for example, or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value in between any of the foregoing values. In some embodiments, 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.
In some embodiments, the one or more labels are measured by quantifying the binding agent that binds to the one or more labels. In some embodiments, the binding agent is quantified by measuring a fluorescent label on the binding agent. In some embodiments, the binding agent is a fluorescent-labeled antibody that selectively binds to the one or more labels. In some embodiments, T cells within the cell composition are incubated with the antibody. In some embodiments, 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, T cells are incubated with antibodies that recognize markers other than the one or more markers. In some embodiments, the additional markers are exemplary markers that aid in identifying cell populations within the cell composition.
In some embodiments, antibodies that bind to the one or more markers are measured using flow cytometry. In some embodiments, antibodies that bind to the one or more markers are measured using infinite flow cytometry. In some embodiments, the antibodies are measured by Immunohistochemistry (IHC). In some embodiments, the one or more markers in the tissue are measured. In some embodiments, cells of the cell composition are fixed and stained with an antibody prior to measurement with IHC.
In some embodiments, the measurement of the surface marker is analyzed in FloJo. In some embodiments, the cell-based gate marks the population as positive or negative after quantifying the expression level with flow cytometry. In some embodiments, a cell of the cell composition is determined to be positive if the cell is in a 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 the cell is in a group with a higher level of expression than other cells in the composition.
D. assessment of activation status of T cells
In some embodiments, activation of T cells within the 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 a non-stimulated control. In some embodiments, the unstimulated control consists of T cells. In some embodiments, the unstimulated control is a cell composition that is treated in the same manner as the cell composition whose T cells are being evaluated, except that the unstimulated control is not exposed to any of the stimulating agents described in section III. In some embodiments, the unstimulated control may consist of any cell composition as described in section II, but the cell composition is not exposed to any stimulating agent (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 and the evaluated cell composition are from the same patient or donor.
In some embodiments, activation of T cells in cells of the composition is assessed by comparing the surface expression level of the one or more markers on cells of the composition to the surface expression of the one or more markers on a non-stimulated control, wherein the surface levels of the one or more markers on the cell composition and the non-stimulated control are measured simultaneously. In some embodiments, activation of the T cells is assessed by comparing the surface expression level of the one or more markers on the T cells to the surface expression of the one or more markers on a non-stimulated control, wherein the surface levels of the one or more markers on the cell composition and the non-stimulated control are measured at different times. In some embodiments, activation of T cells in cells of the composition is assessed by determining whether the level of or the percentage of each of the one or more markers positive for T cells within the cell composition is higher than the level of or the percentage of each of the one or more markers positive for unstimulated control cells. In some embodiments, activation of T cells in cells of the composition is assessed by determining whether the level of or the percentage of each of the one or more markers positive for T cells within the cell composition is lower than the level of or the percentage of each of the one or more markers positive for unstimulated control cells.
In some embodiments, activation of T cells within a cell composition is assessed by comparing the level or percentage of surface expression of the one or more activation markers on 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 T cells within the cell composition to the level or percentage of surface expression of the one or more activation markers in the reference, wherein T cells having a higher level or higher percentage of the one or more activation markers as compared to the reference are indicative of 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 T cells within the cell composition to the level or percentage of surface expression of the one or more activation markers in the reference, wherein T cells having a lower level or lower percentage of the one or more activation markers as compared to the reference are indicative of activation.
In some embodiments, the reference is an unstimulated cell composition from a combination of several donors or patients. In some embodiments, the reference is the average expression level or average percent positive cells across multiple cell compositions from the patient or donor, wherein the cell compositions are unstimulated. In some embodiments, the reference is a median expression level or median positive cell percentage 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 cell composition obtained from a different donor or patient. In some embodiments, the reference is a gating regimen, wherein the gating regimen 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.
In some embodiments, the T cell surface expression of the one or more markers is determined to be "low", "lower", or "reduced" 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 the reference (e.g., unstimulated control cell composition, average positive cell percentage in the plurality of cell compositions, median positive cell percentage in the plurality of cell compositions, maximum positive cell percentage in the plurality of cell compositions, minimum positive cell percentage in the plurality of cell compositions, etc.). In some embodiments, the T cell surface expression of the one or more markers is determined to be "low", "lower", or "reduced" 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 the reference (e.g., the mean or median fluorescence intensity of the unstimulated control cell composition, the mean or median fluorescence intensity across the plurality of cell compositions, the median mean or median fluorescence intensity across the plurality of cell compositions, the maximum mean or median fluorescence intensity across the plurality of cell compositions, the minimum mean or median fluorescence intensity across the plurality of cell compositions, etc.).
In some embodiments, the 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 the reference (e.g., unstimulated control cell composition, average positive cell percentage in the plurality of cell compositions, median positive cell percentage in the plurality of cell compositions, maximum positive cell percentage in the plurality of cell compositions, minimum positive cell percentage in the 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 the reference (e.g., mean or median fluorescence intensity of a non-stimulated control cell composition, mean or median fluorescence intensity across multiple cell compositions, median mean or median fluorescence intensity across multiple cell compositions, maximum mean or median fluorescence intensity across multiple cell compositions, minimum mean or median fluorescence intensity across multiple cell compositions, etc.).
In some embodiments, T cell activation is assessed by T cells that are positive for the one or more markers (marker + or marker Positive and negative ) or express high levels of the one or more markers (marker High height ), or that are negative for the one or more markers or express relatively low levels of the one or more markers (marker-or marker Negative of ). Thus, it is to be understood that the terms positive, pos or+ are used interchangeably herein with respect to one of the one or more markers. Also, it should be understood that the term negative (neg) or-is used interchangeably herein with respect to one of the one or more markers. Furthermore, it is understood that reference to a cell as a marker Negative of herein may refer 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 low 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 expressed at relatively low levels on certain T cell populations, but are present or expressed at relatively higher levels on certain other lymphocyte populations (e.g., NK cells). In some cases, such markers are those that are present on certain T cell populations or expressed at relatively higher levels, but are absent on certain other lymphocyte populations (e.g., NK cells or subsets thereof) or expressed at relatively lower levels.
E. Utilization of the activation state
In some embodiments, the methods comprise incubating the cell composition with a stimulating agent (such as those described in section III). In some embodiments, the methods comprise incubating the cell composition with a stimulating agent prior to detecting T cell activation using one or more markers. In some embodiments, the methods comprise incubating the cell composition with a stimulating agent after detecting T cell activation using one or more markers. In some embodiments, the method comprises determining the time for incubation of the cell composition with the stimulating agent 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 agent incubated with the cell composition based on determining T cell activation with one or more markers.
In some embodiments, the methods comprise engineering T cells after determining T cell activation using one or more markers, as described in section III. In some embodiments, the method comprises administering the vector or virus based on a determination of the activation state of the T cells. In some embodiments, the method consists of incubating the T cells with the carrier for a duration determined based on detection of one or more markers on the surface of the T cells. In some embodiments, the method consists of incubating the T cells with the carrier for a duration determined based on an assessment of the activation state of the T cells.
In some embodiments, the methods comprise incubating and/or expanding the cell composition after assessing T cell activation. In some embodiments, the method comprises determining the length of time to incubate the cell composition based on T cell activation assessed using one or more markers.
In some embodiments, the population of T cells is enriched from the sample by the provided methods, such as by isolation or selection, based on T cell activation assessed by the methods disclosed herein. In some embodiments, T cells that are positive for the one or more markers (marker + or marker Positive and negative ) or express high levels of the one or more markers (marker High height ), or that are negative for the one or more markers or express relatively low levels of the one or more markers (marker-or marker Negative of ).
In some embodiments, any known method for separation based on the one or more markers may be used. In some embodiments, the separation is affinity-based or immunoaffinity-based separation. For example, in some aspects, isolating includes isolating cells and cell populations based on the expression or expression level of the one or more markers (typically cell surface markers), e.g., by incubation with antibodies or binding partners that specifically bind to such markers, followed by a washing step typically and separating cells that have bound to the antibodies or binding partners from those cells that have not bound to the antibodies or binding partners. In some embodiments, the incubation is static (without mixing). In some embodiments, the incubation is dynamic (with mixing).
Cell composition
A. pharmaceutical product
In some embodiments, the cells of the cell composition are engineered to express recombinant receptors, such as those listed in section IV. In some embodiments, the recombinant receptor is a CAR. In some embodiments, methods of assessing T cell activation are used to determine the efficacy of cells of a cellular composition. In some embodiments, the method of assessing T cell activation is used to assess whether cells of a cell composition are 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 drug product is delivered to the patient after assessing T cell activation. In some embodiments, the cells of the composition are administered to the patient after assessing T cell activation.
B. engineered T cells
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 cells of the cell composition to assess whether the cells are ready to be engineered. In some embodiments, engineering is performed if 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.
In some embodiments, T cell activation of the cell composition is assessed prior to transduction or engineering of the T cells. In some embodiments, the cell composition is isolated from a patient to treat a disease. In some embodiments, the cell composition is a sample obtained using any of the methods in section II. In some embodiments, prior to engineering the cells, the cell composition is isolated from the patient and T cell activation is assessed 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 using any of the stimulating reagents and methods in section II prior to assessing T cell activation to prior to engineering the cell composition.
C. peripheral blood mononuclear cells
In some embodiments, the cell composition in the method of assessing T cell activation consists of Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, methods for assessing T cell activation may be used in conjunction with isolation of PBMCs. In some embodiments, the method for assessing T cell activation is used to assess T cell activation within PBMCs. In some embodiments, PBMCs are incubated with stimulating agents (such as those listed in section III B). In some embodiments, after incubating the PBMCs with the stimulating agent, the surface expression level of the one or more markers is detected to quantify T cell activation within the PBMCs.
PBMCs used in the methods described herein may be isolated by any standard means. In some embodiments, PBMCs may be isolated from a patient. In some embodiments, PBMCs may be isolated from healthy donors. In some embodiments, PBMCs may be isolated from patients before they need treatment. In some embodiments, PBMCs may be isolated from patients after they are in need of treatment. In some embodiments, PBMCs may be isolated from a donor and T cell activity assessed to determine how to use them.
Methods for generating engineered T cells
In some embodiments, the methods of assessing activation of T cells within a cell composition provided herein can be used in combination with a therapeutic composition (e.g., an export composition) that produces engineered cells (e.g., engineered cd4+ T cells and/or engineered cd8+ T cells) that express a recombinant protein, e.g., a recombinant receptor (e.g., a T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR)). In some embodiments, the methods provided herein are used in conjunction with the manufacture, generation, or generation of cell therapies, and may be used in conjunction with additional processing steps, such as isolation, separation, selection, activation, or stimulation, transduction, washing, suspending, diluting, concentrating, and/or formulating cells. In some embodiments, a method of generating or producing an engineered cell, e.g., an engineered cd4+ T cell and/or an engineered cd8+ T cell, comprises one or more of isolating a cell from a subject, preparing a cell, treating a cell, incubating a cell under stimulating conditions, and/or engineering (e.g., transducing) a cell. In some embodiments, the method comprises the processing steps performed in the order of first isolating, e.g., selecting or separating, an input cell, e.g., a primary cell, from a biological sample, incubating the input cell under stimulating conditions, engineering the input cell with a vector particle, e.g., a viral vector particle, to introduce a recombinant polynucleotide into the cell, e.g., by transduction or transfection, incubating the engineered cell (e.g., a transduced cell), e.g., to expand the cell, and collecting, harvesting all or a portion of the cell, and/or filling a container with the cell to formulate the cell as an output composition. In some embodiments, cd4+ and cd8+ T cells are manufactured independently of each other, e.g., in separate input compositions, but the manufacturing process includes the same processing steps. In some embodiments, the cd4+ and cd8+ T cells are manufactured together, for example, in the same input composition. In some embodiments, cells of the generated output composition (e.g., therapeutic cell composition) are reintroduced into the same subject either before or after cryopreservation. In some embodiments, the engineered cell export composition (e.g., therapeutic cell composition) is suitable for use in therapy (e.g., autologous cell therapy, allogeneic cell therapy). An exemplary manufacturing process is 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
In particular embodiments, the provided methods are used in conjunction with one or more input compositions that separate, select, and/or enrich cells from a biological sample to generate enriched cells (e.g., T cells). In some embodiments, provided methods include isolating cells or a composition thereof from a biological sample, such as those obtained or derived from a subject, such as a subject suffering from a particular disease or disorder or in need of or to be administered cell therapy. In some aspects, the subject is a human, such as a subject that is a patient in need of a particular therapeutic intervention (e.g., adoptive cell therapy, wherein cells are isolated, treated, and/or engineered for use in the adoptive cell therapy). Thus, in some embodiments, the cell is a primary cell, such as a primary human cell. The samples include tissues, fluids and other samples taken directly from a subject. The biological sample may be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluid (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample is a blood or blood-derived sample, or is derived from apheresis or a leukocyte apheresis product. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemias, lymphomas, lymph nodes, intestinal-related lymphoid tissue, mucosa-related lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and/or cells derived therefrom. In the context of cell therapies (e.g., adoptive cell therapies), samples include samples from autologous and allogeneic sources.
In some examples, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. In some aspects, the sample contains lymphocytes (including T cells, monocytes, granulocytes, B cells), other nucleated leukocytes, erythrocytes, and/or platelets, and in some aspects contains cells other than erythrocytes and platelets.
In some embodiments, blood cells collected from a subject are washed, e.g., to remove plasma fractions, 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 manufacturer's instructions. In some aspects, the washing step is accomplished by Tangential Flow Filtration (TFF) according to manufacturer's instructions. In some embodiments, cells are resuspended in various biocompatible buffers (e.g., PBS without Ca ++/Mg++) after washing. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.
In some embodiments, the method of preparation comprises the step of freezing (e.g., cryopreserving) the cells before or after isolation, selection and/or enrichment, and/or incubation for transduction and engineering, and/or after incubating and/or harvesting 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 chilled solution to remove plasma and platelets, for example, after a washing step. In some aspects, any of a variety of known freezing solutions and parameters may be used. In some embodiments, the cells are frozen, e.g., cryogenically frozen or cryopreserved, in a medium and/or solution having a final concentration of DMSO of or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0%, or DMSO between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8%. In particular embodiments, the cells are frozen, e.g., cryogenically frozen or cryopreserved, in a medium and/or solution having a final concentration of HSA of or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25%, or between 0.1% and-5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2%. One example involves the use of PBS containing 20% DMSO and 8% Human Serum Albumin (HSA), or other suitable cell freezing medium. It was then diluted 1:1 with medium such that the final concentrations of DMSO and HSA were 10% and 4%, respectively. The cells are then typically frozen to at or about-80 ℃ at a rate of at or about 1 °/minute and stored in the gas phase of a liquid nitrogen storage tank.
In some embodiments, the isolation of the cell or population comprises one or more preparative and/or non-affinity based cell isolation steps. In some examples, 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 a particular reagent. In some examples, cells are isolated based on one or more properties (e.g., density, adhesion characteristics, size, sensitivity to a particular component, and/or resistance). In some embodiments, the methods include density-based cell separation methods, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes and centrifuging through a Percoll or Ficoll gradient.
In some embodiments, at least a portion of the selecting step comprises incubating the cells with a selection agent. Incubation with one or more selection reagents, for example, as part of a selection method, may 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 specific molecules, such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids, in or on the cell. In some embodiments, any known method of separation based on such labels using one or more selection reagents may be used. In some embodiments, the one or more selection reagents result in an separation that is an affinity or immunoaffinity based separation. For example, in some aspects, selection includes incubation with one or more reagents for isolating cells and cell populations based on cell expression or expression levels of one or more markers (typically cell surface markers), e.g., by incubation with antibodies or binding partners that specifically bind to such markers, followed by a washing step and isolating cells that have bound to the antibodies or binding partners from those that have not bound to the antibodies or binding partners.
In some aspects of such processes, a volume of cells is mixed with a quantity of an affinity-based desired selection reagent. Immunoaffinity-based selection can be performed using any system or method that allows for an advantageous energy interaction between the isolated cells and molecules that specifically bind to the markers on the cells (e.g., antibodies or other binding partners on a solid surface (e.g., particles)). In some embodiments, the method is performed using particles, such as beads (e.g., magnetic beads), coated with a selective agent (e.g., an antibody) specific for a marker of the cell. Particles (e.g., beads) may be incubated or mixed with cells in a container (e.g., tube or bag) while shaking or mixing, wherein the ratio of cell density to particles (e.g., beads) is constant to help promote energetically favorable interactions. In other cases, the method comprises selecting cells, wherein all or a portion of the selection is performed in an internal cavity of a centrifugal chamber, e.g., under centrifugal rotation. In some embodiments, incubating the cells with a selective agent (e.g., a selective agent based on immunoaffinity) is performed in the centrifugal chamber. In certain embodiments, the separation or isolation is performed using a system, apparatus or device described in international patent application publication No. WO 2009/072003 or US 20110003380 A1. In one example, the system is a system as described in International publication No. WO 2016/073602.
In some embodiments, by implementing such selection steps or portions thereof (e.g., incubation with antibody-coated particles (e.g., magnetic beads)) in the cavity of the centrifuge chamber, the user is able to control certain parameters, such as the volume of the various solutions, the addition of solutions during processing, and timing thereof, which may provide a number of advantages over 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 may enhance the pairwise interaction between the cells being processed and the particles used for selection. In some embodiments, for example, when associated with systems, circuits, and controls as described herein, an incubation step is performed in the chamber, allowing the user to achieve agitation of the solution at one or more desired times during incubation, which may also improve interaction.
In some embodiments, at least a portion of the selecting step is performed in a centrifugal chamber, which includes incubating the cells with a selection agent. In some aspects of such processes, a volume of cells is mixed with an amount of affinity-based desired selection reagent that is much less than is typically employed when similar selections are made in a tube or container according to manufacturer's instructions for selecting the same number of cells and/or the same volume of cells. In some embodiments, the amount of the one or more selection reagents 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 one or more selection reagents used to select cells in a tube or container based incubation for the same number of cells and/or the same volume of cells according to manufacturer's instructions.
In some embodiments, for selection of cells, e.g., based on immunoaffinity selection, the cells are incubated in a 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 it is desired 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 coupled to monoclonal antibodies specific for CD4 and CD 8). In some embodiments, as described, the selection reagent is added to the cells in the chamber cavity in an amount that is generally used or will require significantly less (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%) of the amount of selection reagent that would be used to achieve about the same or similar selection efficiency for the same number of cells or the same volume of cells when selected in an oscillating or rotating tube. In some embodiments, incubation is performed with the addition of a selection buffer to the cells and selection reagent to achieve a target volume of incubation of, for example, 10 mL to 200 mL, such as 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 reagent. In some embodiments, the selection buffer and the selection reagent are pre-mixed prior to addition to the cells. In some embodiments, the selection buffer and selection reagent are added separately to the cells. In some embodiments, the selective incubation is performed under periodically mild mixing conditions, which may help promote energetically favorable interactions, allowing for the use of less total selection reagent while achieving high selection efficiency.
In some embodiments, the total duration of incubation with the selection agent is from 5 minutes to 6 hours or from about 5 minutes to about 6 hours, such as 30 minutes to 3 hours, for example at least or about at least 30 minutes, 60 minutes, 120 minutes, or 180 minutes.
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 the speed used to precipitate 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 an RCF at the sample or wall of a chamber or other container, which is 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 such low speed rotation and a subsequent rest period, e.g., rotation and/or rest for 1,2, 3, 4,5, 6, 7, 8, 9, or 10 seconds, e.g., about 1 or 2 seconds, followed by rest for about 5, 6, 7, or 8 seconds.
In some embodiments, this process is performed within a completely closed system integral to the chamber. In some embodiments, this process (and in some aspects one or more additional steps, such as a pre-wash step to wash the cell-containing sample, such as a apheresis sample) is performed in an automated manner such that cells, reagents, and other components are aspirated and pushed out of the chamber at the appropriate time and centrifuged to complete the washing and binding steps in a single closed system using an automated procedure.
In some embodiments, after incubating and/or mixing the cells with one or more selection reagents, the incubated cells are subjected to separation to select the cells based on the presence or absence of the one or more specific reagents. In some embodiments, the separation is performed in the same closed system, wherein the cells are incubated with the selection agent. In some embodiments, after incubation with the selection reagent, the incubated cells (including cells in which the selection reagent has been bound) are transferred into a system for separation of cells based on immunoaffinity. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column.
Such isolation steps may be based on positive selection (where cells that have bound an agent (e.g., an antibody or binding partner) are retained for further use) and/or negative selection (where cells that have not bound an agent (e.g., an antibody or binding partner) are retained). In some examples, both fractions are retained for further use. In some aspects, negative selection may be particularly useful in the absence of antibodies useful for specifically identifying cell types in heterogeneous populations, such that isolation is preferably based on markers expressed by cells other than the desired population.
In some embodiments, the process step further comprises negative and/or positive selection of the incubated cells, e.g., using a system or apparatus that can perform affinity-based selection. In some embodiments, the separation is performed by enriching a specific cell population via positive selection, or depleting a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed on the positively or negatively selected cells (marker+) or at a relatively high level (marker High height ), respectively. Multiple rounds of the same selection step (e.g., positive or negative selection steps) may be performed. In certain embodiments, the positive or negative selected fraction is subjected to a selection process, such as by repeating the positive or negative selection step. In some embodiments, the selection is repeated twice, three times, four times, five times, six times, seven times, eight times, nine times, 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.
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 (such as those expressing a marker) refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Likewise, negative selection, removal, or depletion of particular types of cells (such as those expressing a marker) refers to reducing the number or percentage of such cells, but need not result in complete removal of all such cells.
In some examples, multiple rounds of separation steps are performed, wherein fractions from positive or negative selection of one step are subjected to another separation step, such as subsequent positive or negative selection. In some examples, a single isolation step may deplete cells expressing multiple markers simultaneously, such as by incubating the cells with multiple antibodies or binding partners (each antibody or binding partner being 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 the various cell types. In certain embodiments, one or more separation steps are repeated and/or performed more than once. In some embodiments, the positive or negative selection fractions resulting from the separation step are subjected to the same separation step, such as by repeating the positive or negative selection step. In some embodiments, a single isolation step is repeated and/or performed more than once, e.g., 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 selecting 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.
For example, in some aspects, specific subsets of T cells are isolated by positive or negative selection techniques, such as cells positive for one or more surface markers or expressing high levels of the surface markers, e.g., 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 antibody or binding partner may be conjugated (e.g., directly or indirectly) to a solid support or matrix (e.g., magnetic beads 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 [ sic ] M-450 CD3/CD 28T cell expander and/or ExpACT [ sic ] beads).
In some embodiments, T cells are isolated from a PBMC sample by negative selection for a marker expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes such as CD 14). In some aspects, a cd4+ or cd8+ selection step is used to isolate cd4+ helper T cells and cd8+ cytotoxic T cells. Such cd4+ and cd8+ populations may 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 cells, memory T cells, and/or effector T cell subpopulations.
In some embodiments, the 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 transplantation after administration, which is particularly robust in some aspects 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.
In embodiments, memory T cells are present in both cd62l+ and CD 62L-subsets of cd8+ peripheral blood lymphocytes. PBMCs may be enriched or depleted against CD62L-cd8+ and/or cd62l+cd8+ fractions, for example using anti-CD 8 and anti-CD 62L antibodies.
In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR, CD28, CD3 and/or CD 127, and in some aspects, on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a cd8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD 62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting from a negative cell fraction selected based on CD4 expression, which is subjected to negative selection based on expression of CD14 and CD45RA and positive selection based on CD 62L.
In some aspects this selection is made simultaneously, while in other aspects it is made sequentially in any order. In some aspects, the same CD4 expression-based selection step used to prepare the population or subpopulation of cd8+ T cells is also used to generate the population or subpopulation of cd4+ T cells such that both positive and negative fractions from the CD 4-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, the selection of the cd4+ T cell population and the selection of the cd8+ T cell population are performed simultaneously. In some embodiments, the selection of the cd4+ T cell population and the cd8+ T cell population is performed sequentially in either order. In some embodiments, methods for selecting cells may include those methods as described in published U.S. application No. US 20170037369. In some embodiments, the selected cd4+ T cell population and the selected cd8+ T cell population may be combined after selection. In some aspects, the selected cd4+ T cell population and the selected cd8+ T cell population may be combined in a bioreactor bag as described herein. In some embodiments, the population of selected CD4+ T cells and the population of selected CD8+ T cells are treated separately, as according to the provided methods, whereby the population of selected CD4+ T cells is enriched for CD4+ T cells and incubated with a stimulating agent (e.g., anti-CD 3/anti-CD 28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR) and incubated under conditions that expand T cells, and the population of selected CD8+ T cells is enriched for CD8+ T cells and incubated with a stimulating agent (e.g., anti-CD 3/anti-CD 28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR) (e.g., the same recombinant protein as used to engineer CD4+ T cells from the same donor), and incubated under conditions that expand T cells.
In certain embodiments, a biological sample (e.g., a sample of PBMCs or other leukocytes) is subjected to selection of cd4+ T cells, wherein both negative and positive fractions are retained. In certain embodiments, the cd8+ T cells are selected from the negative fraction. In some embodiments, the biological sample is subjected to selection of cd8+ T cells, wherein both negative and positive fractions are retained. In certain embodiments, the cd4+ T cells are selected from the negative fraction.
In a specific example, a PBMC sample or other leukocyte sample is subjected to selection of cd4+ T cells, wherein both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19 and positive selection based on the marker characteristics of central memory T cells (such as CD62L or CCR 7), wherein the positive and negative selections are performed in any order.
By identifying a population of cells with 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, the naive cd4+ T lymphocytes are cd45ro-, cd45ra+, cd62l+, or cd4+ T cells. In some embodiments, the central memory cd4+ T cells are cd62l+ and cd45ro+. In some embodiments, effector CD4+ T cells are CD 62L-and CD45RO-.
In one example, to enrich for cd4+ T cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix (e.g., magnetic or paramagnetic beads) to allow separation of cells for positive and/or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, volume 58: METASTASIS RESEARCH Protocols, volume 2: cell Behavior In Vitro and In Vivo, pages 17-25 S.A. Brooks and U.S. Schumacher editions Humana Press Inc., totolva, N.J.).
In some aspects, the incubated cell sample or composition to be isolated is incubated with a selection reagent containing a 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 cell population that is desired to be isolated (e.g., desired to be selected negatively or positively).
In some embodiments, the magnetic particles or beads comprise magnetically responsive material bound to a specific binding member (e.g., an antibody or other binding partner). Many well-known magnetically responsive materials for use in magnetic separation methods are known, such as those described in Molday, U.S. Pat. No. 4,452,773 and in european patent specification EP 452342B, which are hereby incorporated by reference. Colloidal sized particles such as those described in Owen U.S. patent nos. 4,795,698 and Liberti et al, U.S. patent No. 5,200,084, may also be used.
Incubation is typically performed under conditions whereby the antibody or binding partner, or a molecule that specifically binds to such antibody or binding partner attached to a magnetic particle or bead (e.g., a secondary antibody or other agent), specifically binds to a cell surface molecule if present on a cell within the sample.
In certain embodiments, the magnetically responsive particles are coated in a primary or other binding partner, secondary antibody, lectin, 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, cells are labeled with a primary antibody or binding partner instead of beads, and then magnetic particles coated with a cell type specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with a biotinylated primary or secondary antibody.
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 unlabeled cells. For positive selection, cells attracted by the magnet were retained, and for negative selection, cells not attracted (unlabeled cells) were retained. In some aspects, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative fractions are retained and further processed or subjected to additional separation steps.
In some embodiments, affinity-based selection is via Magnetic Activated Cell Sorting (MACS) (Miltenyi Biotech, obu, ca). Magnetically Activated Cell Sorting (MACS) (e.g., a clinic MACS system) enables high purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode in which non-target and target species are eluted sequentially after application of an external magnetic field. That is, cells attached to the magnetized particles remain in place, while unattached species are eluted. Then, after the first elution step is completed, the species that are trapped in the magnetic field and prevented from eluting are released in some way so that they can be eluted and recovered. In certain embodiments, the non-target cells are labeled and depleted from a heterogeneous cell population.
In some embodiments, the magnetically responsive particles remain attached to the cells, which are then incubated, cultured and/or engineered, and in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, 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 competitive non-labeled antibodies, magnetizable particles or antibodies conjugated with cleavable linkers, and the like. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the isolating and/or selecting produces one or more import compositions of enriched 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, the separate input compositions are isolated, selected, enriched, and/or obtained from separate biological samples collected, obtained, and/or obtained from the same subject.
In certain embodiments, the one or more input compositions are or include a composition enriched for 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 a particular embodiment, the T cell enriched input composition consists essentially of cd3+ T cells.
In certain embodiments, the one or more input compositions are or include a composition enriched for cd4+ 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% cd4+ T cells. In certain embodiments, the input composition of 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. In some embodiments, the composition enriched for T cells consists essentially of cd4+ T cells.
In certain embodiments, the one or more compositions are or comprise cd8+ T cells, which are or comprise 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 are 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% of cd4+ T cells, and/or does not contain or is substantially free of cd4+ T cells. In some embodiments, the composition enriched for T cells consists essentially of cd8+ T cells.
In some embodiments, the one or more input compositions of enriched T cells are frozen, e.g., cryopreserved and/or cryogenically frozen, after isolation, selection and/or enrichment. In some embodiments, the one or more input compositions are frozen, e.g., cryopreserved and/or cryogenically frozen, prior to any step of incubating, activating, stimulating, engineering, transducing, transfecting, incubating, amplifying, harvesting, and/or formulating the composition of the cells. In particular embodiments, the one or more cryogenically frozen input compositions are stored, for example, at or about-80 ℃ for 12 hours to 7 days, 24 hours to 120 hours, or 2 days to 5 days. In particular embodiments, the one or more cryogenically frozen input compositions are stored at or about-80 ℃ for an amount of time less than 10 days, 9 days, 8 days, 7 days, 6 days or 5 days, 4 days, 3 days, 2 days or 1 day. In some embodiments, one or more cryogenically frozen input compositions are stored or stored at or about-80 ℃ for about 1, 2, 3, 4, 5, or 6 days.
B. Activation and stimulation of cells
In some embodiments, the provided methods are used in combination with incubating the cells under stimulating conditions. In some embodiments, the stimulation conditions include activating or stimulating and/or conditions capable of activating or stimulating a signal (e.g., a signal generated from a TCR and/or co-receptor) in a cell (e.g., a cd4+ T cell or a cd8+ T cell). In some embodiments, the stimulation conditions include one or more steps of culturing, incubating, activating, propagating the cells with a stimulating agent (e.g., an agent that activates or stimulates and/or is capable of activating or stimulating a signal in the cells) and/or in the presence of a stimulating agent. In some embodiments, the stimulating agent stimulates and/or activates the TCR and/or co-receptor. In certain embodiments, the stimulating agent is an agent as described in section II-B-1.
In certain embodiments, one or more compositions enriched for 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 certain embodiments, after one or more compositions of enriched T cells have been isolated, selected, enriched, or obtained from a biological sample, the one or more compositions are incubated under stimulating conditions. In certain embodiments, the one or more compositions are input compositions. In certain embodiments, the one or more input compositions have been previously cryogenically frozen and stored and thawed prior to incubation.
In certain embodiments, the one or more compositions enriched for T cells are or comprise two separate compositions, e.g., separate input compositions, of enriched T cells. In particular embodiments, 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 incubated under stimulation conditions, respectively. In certain embodiments, the two separate compositions comprise a composition enriched for cd4+ T cells. In a particular embodiment, the two separate compositions comprise a composition enriched for cd8+ T cells. In some embodiments, the two separate compositions enriched for cd4+ T cells and enriched for cd8+ T cells are incubated under stimulation conditions, respectively.
In some embodiments, a single composition enriched for T cells is incubated under stimulating conditions. In certain embodiments, the single composition is a composition enriched for cd4+ T cells. In some embodiments, the single composition is a composition enriched for cd4+ and cd8+ T cells that have been pooled from separate compositions prior to incubation.
In some embodiments, the composition enriched for cd4+ T cells incubated under stimulation 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 about 100% cd4+ T cells. In certain embodiments, the composition of enriched cd4+ T cells incubated under stimulation 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.
In some embodiments, the composition enriched for cd8+ T cells incubated under stimulation 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 at or about 100% cd8+ T cells. In certain embodiments, the composition of enriched cd8+ T cells incubated under stimulation 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% of cd4+ T cells, and/or is free or substantially free of cd4+ T cells.
In some embodiments, separate compositions enriched for cd4+ and cd8+ T cells are combined into a single composition and incubated under stimulating conditions. In certain embodiments, the separate stimulated compositions of enriched cd4+ and enriched cd8+ T cells are combined into a single composition after incubation has been performed and/or completed. In some embodiments, the separate stimulated compositions of stimulated cd4+ T and stimulated cd8+ T cells are treated separately after incubation has been performed and/or completed, as in accordance with the provided methods, whereby a population of stimulated cd4+ T cells (e.g., incubated with a stimulating anti-CD 3/anti-CD 28 magnetic bead stimulating agent) is transduced with a viral vector encoding a recombinant protein (e.g., CAR) and incubated under conditions that expand T cells, and a population of stimulated cd8+ T cells (e.g., incubated with a stimulating anti-CD 3/anti-CD 28 magnetic bead stimulating agent) is transduced with a viral vector encoding a recombinant protein (e.g., CAR) (e.g., the same recombinant protein as used to engineer cd4+ T cells from the same donor) and incubated under conditions that expand T cells.
In some embodiments, incubating under stimulation conditions may include culturing, incubating, stimulating, activating, propagating, including by incubating in the presence of stimulation conditions, e.g., conditions designed to induce proliferation, expansion, activation, and/or survival of cells in the population, mimicking antigen exposure, and/or priming cells for genetic engineering (e.g., for introduction of recombinant antigen receptors). In particular embodiments, the stimulation conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (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 designed to activate cells)).
In some aspects, the stimulation and/or incubation under stimulation conditions is performed according to a variety of techniques, such as those described in U.S. Pat. No. 6,040,1 77;Klebanoff (2012) J Immunther.35 (9) to Riddell et al 651-660; terakura et al (2012) blood.1:72-82; and/or Wang et al (2012) J Immunther.35 (9) 689-701.
In some embodiments, cells (e.g., T cells), cell compositions, and/or cell populations, such as CD4 + and CD8 + T cells, or a combination, population, or subpopulation thereof, are expanded by adding feeder cells, such as non-dividing Peripheral Blood Mononuclear Cells (PBMCs), to a culture-initiating composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells per T lymphocyte in the initial population to be expanded), and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may comprise gamma irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, feeder cells are added to the medium prior to the addition of the T cell population.
In some embodiments, the stimulation conditions include a temperature suitable for growth of human T lymphocytes, for example at least about 25 degrees celsius, typically at least about 30 degrees celsius, and typically at or about 37 degrees celsius. In some embodiments, the temperature transition is achieved during the incubation, such as from 37 degrees celsius to 35 degrees celsius. Optionally, the incubation may further comprise adding non-dividing EBV transformed Lymphoblastoid Cells (LCLs) as feeder cells. The LCL may 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 (e.g., a ratio of LCL feeder cells to naive T lymphocytes of at least about 10:1).
In embodiments, populations of antigen-specific CD4 + and CD8 + may 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 may also be used.
In particular embodiments, the stimulating conditions include incubating, culturing, and/or incubating the cells with a stimulating agent. In certain embodiments, the stimulating agent is an agent as described in section II-B-1. In certain embodiments, the stimulating agent comprises or includes a bead. Exemplary stimulating agents are or include anti-CD 3/anti-CD 28 magnetic beads. In certain embodiments, when the cells are contacted with and/or incubated with a stimulating agent, the incubation, culturing, and/or initiation of culturing the cells under stimulating conditions occurs. In certain embodiments, the cells are incubated before, during, and/or after genetically engineering the cells (e.g., introducing the recombinant polynucleotide into the cells, such as by transfection or transduction).
In some embodiments, the enriched T cell composition is incubated at a ratio of stimulating agent and/or beads (e.g., anti-CD 3/anti-CD 28 magnetic beads) to cells of 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 stimulating agent and/or bead 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 stimulating agent to cells is about 1:1 or 1:1. In some embodiments, the ratio is determined after assessing T cell activation using the methods provided herein.
In certain embodiments, incubating the cells at a ratio (e.g., a ratio of 1: 1) of less than 3:1 or less than 3 stimulating agents (e.g., anti-CD 3/anti-CD 28 magnetic beads) per cell 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 stimulating agent (e.g., anti-CD 3/anti-CD 28 magnetic beads) at a bead to cell ratio of less than 3 (or 3:1 or less than 3 beads per cell). In certain embodiments, incubating the cells at a ratio of less than 3:1 or less than 3 beads per cell (e.g., a ratio of 1:1) reduces the amount of cell death (e.g., as a result of activation-induced cell death) that occurs during incubation.
In particular embodiments, the enriched T cell composition is incubated with the stimulating agent (e.g., anti-CD 3/anti-CD 28 magnetic beads) at a stimulating agent and/or bead to cell ratio of less than 3:1 (e.g., 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, apoptosis, or apoptosis, for a period of 1 day, 2 days, 3 days, 4 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 incubation. In particular embodiments, the T cell enriched composition is incubated with the stimulating agent at a stimulating agent and/or bead to cell ratio of less than 3:1 (e.g., 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 incubation.
In certain embodiments, the T cell enriched composition is incubated with a stimulating agent (e.g., anti-CD 3/anti-CD 28 magnetic beads) at a ratio of beads to cells of less than 3:1 (e.g., a ratio of 1:1), and cells of the composition have a viability 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 have undergone an exemplary and/or alternative procedure in which the T cell enriched composition is incubated with a stimulating agent at a ratio of 3:1 or greater.
In some embodiments, the enriched T cell composition incubated with the stimulating agent 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 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 10 7 cells/mL, 5 x 10 7 cells/mL or 1 x 10 8 cells/mL. In some embodiments, the enriched T cell composition incubated with the stimulating agent comprises about 0.5 x 10 6 cells/mL, 1 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 2 x 10 6 cells/mL, 2.5 x 10 6 cells/mL, 3 x 10 6 cells/mL, 3.5 x 10 6 cells/mL, 4 x 10 6 cells/mL, 4.5 x 10 6 cells/mL, 5 x 10 6 cells/mL, 5.5 x 10 6 cells/mL, 6 x 10 6 cells/mL, 6.5 x 10 6 cells/mL, 7 x 10 6 cells/mL, 7.5 x 10 6 cells/mL, 8 x 10 6 cells/mL, 8.5 x 10 6 cells/mL, 9 x 10 6 cells/mL, 9.5 x 10 6 cells/mL, or 10 x 10 6 cells/mL, such as about 2.4 x 10 6 cells/mL.
In some embodiments, the T cell enriched composition is incubated with the stimulating agent at a temperature of from about 25 to about 38 ℃, such as from about 30 to about 37 ℃, for example, at or about 37±2 ℃. In some embodiments, the T cell enriched composition is incubated with the stimulating agent at a CO 2 level of from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example, or about 5% ± 0.5%. In some embodiments, the T cell enriched composition is incubated with the stimulating agent at a temperature of or about 37 ℃ and/or at a CO 2 level of or about 5%.
In particular embodiments, the stimulation conditions include incubating, culturing, and/or incubating the enriched T cell composition with and/or in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind and/or are capable of binding to receptors expressed by and/or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the 4- α -helix bundle family of cytokines include, but are not limited to, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include IL-2. In some embodiments, the stimulation conditions include incubating the composition of enriched T cells (e.g., enriched cd4+ T cells or enriched cd8+ T cells) in the presence of a stimulating agent (anti-CD 3/anti-CD 28 magnetic beads) and in the presence of one or more recombinant cytokines as described.
In certain embodiments, the CD4+ T cell enriched composition 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 sufficiently produce IL-2 in an amount that allows for growth, division, and expansion throughout the process of a composition for producing output cells (e.g., engineered cells suitable for use in cell therapy). In some embodiments, incubating the cd4+ T cell enriched composition in the presence of recombinant IL-2 under stimulation 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 enriched cd4+ T cell composition 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 enriched cd4+ T cell composition 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 not incubating the enriched cd4+ T cell composition in the presence of recombinant IL-2.
In certain embodiments, the amount or concentration of the one or more cytokines is measured and/or quantified in International Units (IU). The international units can be used to quantify vitamins, hormones, cytokines, vaccines, blood products and similar bioactive substances. In some embodiments, IU is or includes a measure of biological agent efficacy by comparison to an international reference standard (e.g., WHO first international standard for IL-2 (WHO 1st International Standard for Human IL-2), 86/504) having a particular weight and intensity. International units are the only accepted and standardized method of reporting bioactive units that are published and derived from international collaborative research work. In certain embodiments, the IU of the composition, sample, or source of cytokine may be obtained by a product comparison test with a similar WHO standard product. For example, in some embodiments, the IU/mg of a composition, sample or source of human recombinant IL-2, IL-7 or IL-15 is compared to the WHO standard IL-2 product (NIBSC code: 86/500), the WHO standard IL-17 product (NIBSC code: 90/530), and the WHO standard IL-15 product (NIBSC code: 95/554), respectively.
In some embodiments, biological activity in IU/mg is equivalent to (ED 50)-1 x 106 in ng/ml. In particular embodiments ED 50 of recombinant human IL-2 or IL-15 is equivalent to the concentration required for half maximal stimulation of cell proliferation (XTT cleavage) using CTLL-2 cells. In certain embodiments 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 of IL-2 are discussed in Wadhwa et al, journal of Immunological Methods (2013), 379 (1-2): 1-7; and Gearing and Thorpe, journal of Immunological Methods (1988), 114 (1-2): 3-9; details related to the determination and calculation of IU of IL-15 are discussed in Soman et al Journal of Immunological Methods (2009) 1-2): 83-94, which is hereby incorporated by reference in its entirety.
In certain embodiments, the compositions enriched for CD8+ T cells are 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, IL-2, IL-7 and/or IL-15 is recombinant. In certain embodiments, IL-2, IL-7 and/or IL-15 is human. In particular embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and/or IL-15. In some aspects, the incubation of the enriched T cell composition further comprises the presence of a stimulating agent, such as anti-CD 3/anti-CD 28 magnetic beads.
In some embodiments, the cells are incubated with 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. In some embodiments, IU/mL of cytokine incubated with the cells is based on assessing T cell activation using any of the methods provided herein.
In some embodiments, the T cell enriched 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 T cell enriched composition is incubated with recombinant IL-2 at or 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 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 T cell enriched composition is a cd8+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd8+ T cells, wherein the cd4+ T cells are not enriched and/or wherein the cd4+ T cells are negatively selected or depleted from the composition. In some embodiments, the T cell enriched composition is a cd4+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd4+ T cells, wherein the cd8+ T cells are not enriched and/or wherein the cd8+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-7 and/or recombinant IL-15 as in the amounts described. In some embodiments, the enriched CD8+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-15 as in the amounts described.
In some embodiments, the T cell enriched composition is incubated with 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 T cell enriched composition is incubated with recombinant IL-7 at a concentration of at or about 50 IU/ml、100 IU/ml、150 IU/ml、200 IU/ml、250 IU/ml、300 IU/ml、350 IU/ml、400 IU/ml、450 IU/ml、500 IU/ml、550 IU/ml、600 IU/ml、650 IU/ml、700 IU/ml、750 IU/ml、800 IU/ml、750 IU/ml、750 IU/ml、750 IU/ml or 1,000 IU/ml. In a particular embodiment, 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 may also be incubated with recombinant IL-2 and/or recombinant IL-15 as in the amounts described. In certain embodiments, the T cell enriched composition is enriched for cd4+ T cells, wherein the cd8+ T cells are not enriched and/or wherein the 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.
In some embodiments, the T cell enriched composition is incubated with 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 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 T cell enriched composition is incubated with recombinant IL-15 at 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 at 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 T cell enriched composition is a cd8+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd8+ T cells, wherein the cd4+ T cells are not enriched and/or wherein the cd4+ T cells are negatively selected or depleted from the composition. In some embodiments, the T cell enriched composition is a cd4+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd4+ T cells, wherein the cd8+ T cells are not enriched and/or wherein the cd8+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-15 may also be incubated with recombinant IL-7 and/or recombinant IL-2 as in the amounts described. In some embodiments, the enriched CD8+ T cell composition incubated with recombinant IL-15 may also be incubated with recombinant IL-2 as in the amounts described.
In certain embodiments, the cells (e.g., enriched cd4+ T cells and/or enriched cd8+ T cells) are incubated with the stimulating agent in the presence of one or more antioxidants. In some embodiments, antioxidants include, but are not limited to, one or more antioxidants including tocopherol, tocotrienol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alpha-tocoquinone, trolox (6-hydroxy-2, 5,7, 8-tetramethylchroman-2-dicarboxylic acid), butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, beta-carotene, selenium, ubiquinone, syphilin (luetin), S-adenosylmethionine, glutathione, taurine, N-acetylcysteine (NAC), citric acid, L-carnitine, BHT, thioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione (glutathione), cystamine and cystathionine (cysstathionine), and/or glycine-histidine. In some aspects, incubating the enriched T cell composition (e.g., enriched cd4+ T cells and/or enriched cd8+ T cells) with an antioxidant further comprises the presence of a stimulating agent (e.g., anti-CD 3/anti-CD 28 magnetic beads) and one or more recombinant cytokines (as described).
In some embodiments, the one or more antioxidants are or include sulfur-containing oxidizing agents. In certain embodiments, the sulfur-containing antioxidant may include a thiol-containing antioxidant and/or an antioxidant that exhibits one or more sulfur moieties, for example, within the ring structure. In some embodiments, the sulfur-containing antioxidant may include, for example, N-acetylcysteine (NAC) and 2, 3-Dimercaptopropanol (DMP), L-2-oxo-4-thiazolidine formate (OTC), and lipoic acid. In a particular embodiment, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified to a derivatized glutathione in one or more steps within the cell. In particular embodiments, glutathione precursors may include, but are not limited to, N-acetylcysteine (NAC), L-2-oxothiazolidine-4-carboxylic acid (procaysteine), lipoic acid, S-allylcysteine, or methioninesulfonium chloride.
In some embodiments, incubating the cells (e.g., enriched for cd4+ T cells and/or enriched for cd8+ T cells) under stimulation conditions comprises incubating the cells in the presence of one or more antioxidants. In certain embodiments, the cells are stimulated with the stimulating agent in the presence of one or more antioxidants. In some embodiments, the cells are incubated in the presence of one or more antioxidants between 1 ng and 100 ng/ml, between 10 ng and 1 μg/ml, between 100 ng 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. In some embodiments, the cells are incubated in the presence of one or more antioxidants 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. In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In certain embodiments, the one or more antioxidants are or include glutathione precursors.
In some embodiments, the one or more antioxidants is or includes N-acetylcysteine (NAC). In some embodiments, incubating the cells (e.g., enriched for cd4+ T cells and/or enriched for cd8+ T cells) under stimulation conditions comprises incubating the cells in the presence of NAC. In a particular embodiment, the cells are stimulated with the stimulating agent in the presence of NAC. In some embodiments, cells are incubated in the presence of NAC 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. In some embodiments, the cells are incubated in the presence of NAC 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. In some embodiments, the cells are incubated with or with about 0.8 mg/ml. In certain embodiments, incubating the composition enriched for T cells (e.g., enriched for cd4+ T cells and/or enriched for cd8+ T cells) in the presence of one or more antioxidants, e.g., NAC, reduces activation in the cells as compared to cells incubated in an alternative and/or exemplary process in the absence of the antioxidants.
In some embodiments, the composition or cell (e.g., enriched for cd4+ T cells and/or enriched for cd8+ T cells) is incubated in the presence of a stimulating condition or stimulating agent 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 elicit cells for genetic engineering (e.g., for the introduction of recombinant antigen receptors). Exemplary stimulating agents (e.g., anti-CD 3/anti-CD 28 magnetic beads) are described below. Incubation with the stimulating agent may also be performed in the presence of one or more stimulating 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, the CD4+ T cell enriched composition is incubated with a stimulating agent (i.e., recombinant IL-2, recombinant IL-7, recombinant IL-15) and NAC in amounts as described under stimulating conditions. In some embodiments, the cd8+ T cell enriched composition is incubated with a stimulating agent (i.e., recombinant IL-2, recombinant IL-15) and NAC in amounts as described under stimulating conditions.
In some embodiments, the conditions for stimulation and/or activation may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (e.g., nutrient, amino acid, antibiotic, ion, and/or stimulating factor (e.g., cytokine, chemokine, antigen, binding partner, fusion protein, recombinant soluble receptor, and any other agent designed to activate a cell)).
In some aspects, incubation is performed according to a variety of techniques, such as those described in U.S. Pat. No. 6,040,1 77;Klebanoff (2012) J Immunther.35 (9) to Riddell et al 651-660, terakura et al (2012) blood.1:72-82, and/or Wang et al (2012) J Immunther.35 (9) 689-701.
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 the 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 comprises mixing with one or more agents to induce stimulation and/or activation. In some embodiments, cells (e.g., selected cells) are mixed with a stimulating condition or agent in a centrifugal chamber. In some aspects of such processes, a volume of cells is mixed with an amount of one or more stimulation conditions or agents that are much smaller than those typically used when performing similar stimulation in a cell culture plate or other system.
In some embodiments, the stimulus is added to the cells in the chamber cavity in an amount that is generally used or will require a significantly smaller amount (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%) of the amount of stimulus than would be used to achieve about the same or similar efficiency of selection for the same cell number or same cell volume, e.g., when selected in a periodically oscillating or rotating tube or bag, but not mixed in the centrifuge chamber. In some embodiments, incubation is performed with the addition of an incubation buffer to the cells and the stimulating agent to achieve a target volume of incubation, 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) reagent. In some embodiments, the incubation buffer and the stimulating agent are pre-mixed prior to adding the cells. In some embodiments, the incubation buffer and the stimulating agent are added to the cells separately. In some embodiments, the stimulation incubation is performed under periodically mild mixing conditions, which may help promote energetically favorable interactions and thereby allow for less overall stimulator to be used while achieving stimulation and activation of the cells.
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 the speed used to precipitate 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 an RCF at the sample or wall of a chamber or other container, which is 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 such low speed rotation and a subsequent rest period, e.g., rotation and/or rest for 1,2, 3, 4,5, 6, 7, 8, 9, or 10 seconds, e.g., about 1 or 2 seconds, followed by rest for about 5, 6, 7, or 8 seconds.
In some embodiments, for example, the total duration of incubation with the stimulating agent is between or between about 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, between 18 hours and 30 hours, or between 12 hours and 24 hours, such as at least or at least about or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, further incubation is performed for a time between or about between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours and includes the endpoints.
In some embodiments, the cells are cultured, incubated, and/or incubated under stimulating conditions, e.g., as described in section II-C, prior to and/or during the step for introducing the polynucleotide (e.g., a polynucleotide encoding a recombinant receptor) into the cells, e.g., by transduction and/or transfection. In certain embodiments, cells are cultured, incubated, and/or incubated under stimulation for an amount of time 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 1 day and 8 days, between 1 day and 3 days, between 4 days and 6 days, or between 4 days and 5 days prior to genetic engineering. In some embodiments, the cells are incubated for 2 days or about 2 days prior to engineering.
In certain embodiments, the cells are incubated with and/or in the presence of a stimulating agent prior to and/or during genetically engineering the cells. In certain embodiments, the cells are incubated with and/or in the presence of a stimulating agent for an amount of time 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 less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, or for an amount of time 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 genetically engineering the cells. In particular embodiments, the cells are incubated with and/or in the presence of a stimulating agent 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 agent for 4 days or about 4 days. In particular embodiments, the cells are incubated with and/or in the presence of a stimulating agent for 5 days or about 5 days. In certain embodiments, the cells are incubated with and/or in the presence of a stimulating agent for less than 7 days.
In some embodiments, incubating the cells under stimulation conditions comprises incubating the cells with a stimulating agent as described in section II-B-1. In some embodiments, the stimulating agent comprises or includes beads, such as paramagnetic beads, and the cells are incubated with the stimulating agent at a ratio of less than 3:1 (beads: cells), such as a ratio of 1:1. In certain embodiments, the cells are incubated with the stimulating agent in the presence of one or more cytokines and/or one or more antioxidants. In some embodiments, the CD4+ T cell enriched composition is incubated with the stimulating agent in the presence of recombinant IL-2, IL-7, IL-15 and NAC at a ratio of 1:1 (beads: cells). In certain embodiments, the CD8+ T cell enriched composition is incubated with the stimulating agent in a ratio of 1:1 (beads: cells) in the presence of recombinant IL-2, IL-15, and NAC. In some embodiments, the stimulating agent is removed and/or isolated from the cells within 6 days, 5 days, or 4 days, or within about 6 days, 5 days, or 4 days from the start or initiation of incubation, e.g., from the time the stimulating agent is added to or contacted with the cells.
1. Stimulating agent
In some embodiments, incubating the enriched cell composition under stimulation conditions is or includes incubating and/or contacting the enriched cell composition with a stimulating agent capable of activating and/or expanding T cells. In some embodiments, the stimulating agent is capable of stimulating and/or activating one or more signals in the cell. In some embodiments, the one or more signals are mediated by a receptor. In particular embodiments, the one or more signals are, or are associated with, a change in the level or amount of signal transduction and/or second messengers (e.g., cAMP and/or intracellular calcium), a change in the amount, cellular localization, conformation, phosphorylation, ubiquitination, and/or truncation of one or more cellular proteins, and/or a change in cellular activity (e.g., transcription, translation, protein degradation, cellular morphology, activation state, and/or cell division). In particular embodiments, the stimulating agent activates and/or is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more co-stimulatory molecules.
In certain embodiments, the stimulating agent comprises particles (e.g., beads) 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 a bead. In some embodiments, the beads are biocompatible, i.e., are composed of a material suitable for biological use. In some embodiments, the beads are non-toxic to cultured cells (e.g., cultured T cells). In some embodiments, the beads may be any particle capable of attaching an agent in a manner that allows interaction between the agent and the cells.
In some embodiments, the stimulating agent comprises one or more agents capable of activating and/or expanding T cells, such as pan-T cell activating agents. In some embodiments, the pan-T cell activating agent comprises anti-CD 3/anti-CD 28 beads. In some embodiments, the pan-T cell activating reagent comprises an anti-CD 3/anti-CD 28 streptavidin oligomerization reagent.
In some embodiments, the stimulating agent comprises one or more agents capable of activating and/or expanding cells (e.g., T cells) that are bound to or otherwise attached to the bead, such as to or attached to the surface of the bead. In certain embodiments, the beads are acellular particles. In particular embodiments, the beads may include colloidal particles, microspheres, nanoparticles, magnetic beads, and the like. In some embodiments, the beads are agarose beads. In certain embodiments, the beads are agarose gel beads.
In certain embodiments, the stimulating agent comprises monodisperse beads. In certain embodiments, the monodisperse beads comprise size dispersions having a standard deviation of diameter from each other of less than 5%.
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, an agent as contemplated herein may include, but is not limited to, RNA, DNA, a protein (e.g., an enzyme), an antigen, a polyclonal antibody, a monoclonal antibody, an antibody fragment, a carbohydrate, a lipid lectin, or any other biological molecule having 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 may be directly or indirectly attached to the bead by various methods known and available in the art. The attachment may be covalent, non-covalent, electrostatic or hydrophobic, and may be achieved by various attachment means including, for example, chemical means, mechanical means or enzymatic means. In some embodiments, a biomolecule (e.g., biotinylated anti-CD 3 antibody) may be indirectly attached to the bead via another biomolecule (e.g., an anti-biotin antibody) that is directly attached to the bead.
In some embodiments, the stimulating agent comprises a bead and one or more agents that interact directly with macromolecules on the cell surface. In certain embodiments, the beads (e.g., paramagnetic beads) interact with the cells 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 cells. 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 biomolecule) as described herein, followed by the addition of a second agent (e.g., a secondary antibody (e.g., a biotinylated anti-CD 3 antibody) or other second biomolecule (e.g., streptavidin)), whereby the secondary antibody or other second biomolecule specifically binds to such primary antibody or other biomolecule on the particle.
In some embodiments, the stimulating agent contains one or more agents (e.g., antibodies) that are attached to beads (e.g., paramagnetic beads) and that specifically bind ：CD2、CD3、CD4、CD5、CD8、CD25、CD27、CD28、CD29、CD31、CD44、CD45RA、CD45RO、CD54（ICAM-1）、CD127、MHCI、MHCII、CTLA-4、ICOS、PD-1、OX40、CD27L（CD70）、4-1BB（CD137）、4-1BBL、CD30L、LIGHT、IL-2R、IL-12R、IL-1R、IL-15R;IFN-γR、TNF-αR、IL-4R、IL-10R、CD18/CDl la（LFA-1）、CD62L（L- selectins to one or more of the following macromolecules on cells (e.g., T cells), 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 of these macromolecules or fragments thereof. In some embodiments, the agent (e.g., antibody) attached to the bead specifically binds to one or more of the following macromolecules on the cell (e.g., T cell), CD28, CD62L, CCR, CD27, CD127, CD3, CD4, CD8, CD45RA, and/or CD45RO. In some embodiments, the one or more agents attached to the bead are antibodies. Such antibodies can include polyclonal antibodies, monoclonal antibodies (including full length antibodies with immunoglobulin Fc regions), antibody compositions with multi-epitope specificity, multi-specific 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 stimulating agent is an antibody fragment (including antigen binding fragments), such as a Fab, fab '-SH, fv, scFv, or (Fab') 2 fragment. It is understood that constant regions of any isotype can be used for antibodies contemplated herein, including IgG, igM, igA, igD and IgE constant regions, and that such constant regions can be obtained from any human or animal species (e.g., murine species).
In some embodiments, the agent is an antibody that binds to and/or recognizes one or more components of a T cell receptor. In certain embodiments, the agent is an anti-CD 3 antibody. In certain embodiments, the agent is an antibody that binds to and/or recognizes a co-receptor. In some embodiments, the stimulating agent comprises an anti-CD 28 antibody. In certain embodiments, the stimulatory agent comprises an anti-CD 3 antibody and an anti-CD 28 antibody. In some embodiments, the antibody is a Fab. In some embodiments, the stimulators contain an anti-CD 3 Fab and an anti-CD 28 Fab.
In some embodiments, the stimulators are anti-CD 3/anti-CD 28 streptavidin oligomerization reagents, as described in PCT publication No. WO/2015/158868 or WO 2019/197949. In some embodiments, the streptavidin is recombinant strepitacin, which is mutant streptavidin. In some embodiments, strepitacin is a mutant streptavidin comprising a mutation Val-Thr-Ala-Arg or Ile-Gly-Ala-Arg at positions 44-47 of wild-type streptavidin, thereby replacing the wild-type amino acid Glu-Ser-Ala-Val. In some embodiments, the stimulating agent is soluble. In some embodiments, the agent is a soluble agent of an anti-CD 3/CD28 Fab fragment linked to a recombinant strepitactin backbone. In some embodiments, the stimulating agent is ExpamerTM.
In some embodiments, the stimulators are anti-CD3/anti-CD 28 beads (e.g., DYNABEADS cube M-450 CD3/CD 28T cell expander and/or ExpACT cube).
In some embodiments, the beads have a diameter of greater than about 0.001 μm, greater than about 0.01 μm, greater than about 0.1 μm, greater than about 1.0 μm, greater than about 10 μm, greater than about 50 μm, greater than about 100 μm, or greater than about 1000 μm and no more than about 1500 μm. In some embodiments, the beads have a diameter of about 1.0 μm to about 500 μm, about 1.0 μm to about 150 μm, about 1.0 μm to about 30 μm, about 1.0 μm to about 10 μm, about 1.0 μm to about 5.0 μm, about 2.0 μm to about 5.0 μm, or about 3.0 μm to about 5.0 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In some embodiments, the beads have a diameter of at least or at least about or about 0.001 µm、0.01 µm、0.1 µm、0.5 µm、1.0 µm、1.5 µm、2.0 µm、2.5 µm、3.0 µm、3.5 µm、4.0 µm、4.5 µm、5.0 µm、5.5 µm、6.0 µm、6.5 µm、7.0 µm、7.5 µm、8.0 µm、8.5 µm、9.0 µm、9.5 µm、10 µm、12 µm、14 µm、16 µm、18 µm、 or 20 μm. In certain embodiments, the beads have a diameter of at or about 4.5 μm. In certain embodiments, the beads have a diameter of at or about 2.8 μm.
In some embodiments, the beads have a density 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 3, or greater than 5g/cm 3. In some embodiments, the beads have a density of between about 0.001 g/cm 3 and about 100 g/cm 3, between about 0.01 g/cm 3 and about 50 g/cm 3, Between about 0.1 g/cm 3 and about 10 g/cm 3, between about 0.1 g/cm 3 and about.5 g/cm 3, Between about 0.5 g/cm 3 and about 1 g/cm 3, between about 0.5 g/cm 3 and about 1.5 g/cm 3, Between about 1 g/cm 3 and about 1.5 g/cm 3, between about 1 g/cm 3 and about 2 g/cm 3, Or between about 1g/cm 3 and about 5 g/cm 3. In some embodiments, the beads have a density of about 0.5 g/cm 3, 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 beads is about 1.6 g/cm 3. In a particular embodiment, the density of the beads or particles is about 1.5 g/cm 3. In certain embodiments, the density of the particles is about 1.3 g/cm 3.
In certain embodiments, the plurality of beads have a uniform density. In certain embodiments, the uniform density comprises a standard deviation of density of less than 10%, less than 5%, or less than 1% of the average bead density.
In some embodiments, the beads have a surface area of from about 0.001 m 2/gram of particles (m 2/g) to about 1,000 m 2/g, from about.010 m 2/g to about 100m 2/g, About 0.1m 2/g to about 10 m 2/g, about 0.1m 2/g to about 1m 2/g, About 1m 2/g to about 10m 2/g, about 10m 2/g to about 100m 2/g, About 0.5m 2/g to about 20 m 2/g, Between about 0.5 m 2/g to about 5m 2/g or about 1m 2/g to about 4m 2/g. In some embodiments, the surface area of the particles or beads is from about 1m 2/g to about 4m 2/g.
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 certain 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.
In particular embodiments, the beads comprise a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core comprises a metal. In some embodiments, the metal may 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), ferrite (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and a metal alloy (e.g., coTaZn). In some embodiments, the magnetic core comprises one or more of ferrite, 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 (Fe 3O 4), maghemite (γfe2o3), or pyrite (Fe 3S 4). In some embodiments, the core comprises iron oxide (e.g., fe 3O4).
In certain embodiments, the beads contain a magnetic core, paramagnetic core, and/or superparamagnetic core covered by a surface-functionalized coating (coat or coating). In some embodiments, the coating may contain a material that may include, but is not limited to, a polymer, polysaccharide, silica, fatty acid, protein, carbon, agarose, sepharose, or a combination thereof. In some embodiments, the polymer may be polyethylene glycol, poly (lactic-co-glycolic acid), polyglutaridehyde, polyurethane, polystyrene, or polyvinyl alcohol. In certain embodiments, the outer coating (coat or coating) comprises polystyrene. In certain embodiments, the outer coating is surface functionalized.
In some embodiments, the stimulating agent comprises a bead comprising a metal oxide core (e.g., an iron oxide core) and a coating, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coating comprises at least one polysaccharide (e.g., aminodextran), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In certain embodiments, the one or more agents include an anti-CD 3 antibody and an anti-CD 28 antibody or antigen-binding fragment thereof. In some embodiments, the stimulating agent comprises an anti-CD 3 antibody, an anti-CD 28 antibody, and an anti-biotin antibody. In some embodiments, the stimulating agent comprises an anti-biotin antibody. In some embodiments, the beads have a diameter of about 3 μm to about 10 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In certain embodiments, the beads have a diameter of about 3.5 μm.
In some embodiments, the stimulating agent 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 beads are monodisperse paramagnetic (e.g., superparamagnetic) beads comprising paramagnetic (e.g., superparamagnetic) iron cores (e.g., cores comprising magnetite (Fe 3O4) and/or maghemite (γfe 2O3)) and polystyrene coatings (coat or coating). In some embodiments, the beads are non-porous. In some embodiments, the bead comprises 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 include an anti-CD 3 antibody and an anti-CD 28 antibody. In some embodiments, the stimulating agent is or comprises anti-CD 3/anti-CD 28 magnetic beads. In some embodiments, the one or more agents include an anti-CD 3 antibody and/or an anti-CD 28 antibody, as well as an antibody or antigen fragment thereof capable of binding to a labeled antibody (e.g., biotinylated antibody) (e.g., labeled anti-CD 3 or anti-CD 28 antibody). In certain embodiments, the beads have a density of about 1.5 g/cm 3 and a surface area of about 1m 2/g to about 4m 2/g. In a particular embodiment, the beads are monodisperse superparamagnetic beads having a diameter of about 4.5 μm and a density of about 1.5 g/cm 3. In some embodiments, the beads are monodisperse superparamagnetic beads having an average diameter of about 2.8 μm and a density of about 1.3 g/cm 3.
In some embodiments, the T cell enriched composition is incubated with the stimulating agent at a bead to cell ratio of at or about 3 : 1、2.5 : 1、2 : 1、1.5 : 1、1.25 : 1、1.2 : 1、1.1 : 1、1 : 1、0.9 : 1、0.8 : 1、0.75 : 1、0.67 : 1、0.5 : 1、0.3 : 1 or 0.2:1. In particular embodiments, the ratio of 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 stimulating agent to cells is about 1:1 or 1:1.
C. engineered cells
In some embodiments, the provided methods involve engineering cells with recombinant antigen receptors. 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 a receptor, including by virus (e.g., retrovirus or lentivirus), transduction, transposon, and electroporation.
Cells expressing the receptor and administered by the provided methods include engineered cells. Genetic engineering generally involves introducing nucleic acids encoding recombinant or engineered components into a composition containing cells, such as by retroviral transduction, transfection or transformation.
In some embodiments, the methods provided herein are used in combination with one or more compositions for engineering enriched T cells. In certain embodiments, engineering is or includes introducing 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. The introduction of a nucleic acid molecule encoding a recombinant protein (e.g., recombinant receptor) into a cell can be performed using any of a number of known vectors. Such vectors include viral and nonviral systems, including lentiviral and gamma retroviral systems, as well as transposon-based systems, such as PiggyBac or sleep Beauy-based gene transfer systems. Exemplary methods include those for transferring nucleic acids encoding a receptor, including by virus (e.g., retrovirus or lentivirus), transduction, transposon, and electroporation. In some embodiments, engineering produces one or more engineered compositions enriched for T cells.
In certain embodiments, one or more compositions enriched for T cells are engineered, e.g., transduced or transfected, prior to incubating the cells under conditions that promote proliferation and/or expansion, e.g., as provided by the methods of section III-D. In certain embodiments, one or more compositions enriched for T cells are engineered after stimulation, activation, and/or incubation under stimulation conditions, as described in the methods provided in section III-B. In particular embodiments, the one or more compositions are stimulated compositions. In certain embodiments, the one or more stimulated compositions have been previously cryogenically frozen and stored, and thawed prior to engineering.
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, 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 enriched for cd4+ T cells. In a particular embodiment, the two separate compositions comprise a composition enriched for cd8+ T cells. In some embodiments, the two separate compositions enriched for cd4+ T cells and enriched for cd8+ T cells are separately genetically engineered, such as after incubation under stimulation conditions as described above. In some embodiments, a single composition enriched for T cells is genetically engineered. In certain embodiments, the single composition is a composition enriched for cd4+ T cells. In some embodiments, the single composition is a composition enriched for cd4+ and cd8+ T cells that have been pooled from separate compositions prior to engineering.
In some embodiments, the composition of engineered, e.g., transduced or transfected enriched cd4+ T cells (e.g., 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 at or about 100% cd4+ T cells. In certain embodiments, the engineered composition enriched in cd4+ 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.
In some embodiments, the composition of engineered, e.g., transduced or transfected enriched cd8+ 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 at or about 100% cd8+ T cells. In certain embodiments, the engineered composition enriched in cd8+ 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.
In some embodiments, separate compositions enriched for cd4+ and cd8+ T cells are combined into a single composition and genetically engineered, e.g., transduced or transfected. In certain embodiments, the separate engineered compositions enriched for cd4+ and enriched for cd8+ T cells are combined into a single composition after genetic engineering has been performed and/or completed. In particular embodiments, separate compositions enriched for cd4+ and enriched for cd8+ T cells (e.g., separate compositions stimulated for cd4+ and cd8+ T cells) are separately engineered and treated separately for T cell incubation and/or expansion after genetic engineering has been performed and/or completed.
In some embodiments, the 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, contacting may be achieved by centrifugation, such as rotary inoculation (spinoculation) (e.g., centrifugal inoculation). In some embodiments, the composition containing the cells, virus particles, and agent may be spun, 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). In some embodiments, the rotation is performed with 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., or about 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 or about 693 g, as measured, for example, at an inner or outer wall of a chamber or cavity. The term "relative centrifugal force" or RCF is generally understood to be an effective force exerted on an object or substance (e.g. a cell, sample or pellet and/or a point in a rotated chamber or other container) relative to the gravitational force of the earth at a specific point in space as compared to the axis of rotation. The values may be determined using well known formulas that take into account gravity, rotational speed and radius of rotation (distance from the axis of rotation and the object, substance or particle measuring the RCF). In some embodiments, at least a portion of the contacting, incubating, and/or engineering of the cells (e.g., cells from the enriched cd4+ T cells or the stimulated composition of enriched cd8+ T cells) with the virus is performed under rotation 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 rotation is between 600 g and 700 g, e.g., at or about 693 g.
In certain embodiments, at least a portion of the engineering, transduction, and/or transfection is performed under rotation, e.g., rotary seeding and/or centrifugation. In some embodiments, the rotation is performed, about performed, or at least about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, form 1, 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 at least 7 days. In some embodiments, the rotation is performed or performed for about 60 minutes. In certain embodiments, the rotation is performed for about 30 minutes. In some embodiments, the rotation is between 600 g and 700 g, for example at or about 693 g for about 30 minutes.
In certain embodiments, the number of living 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 living cells to be engineered, transduced and/or transfected is about or less than about 300 x 10 6 cells.
In certain embodiments, at least a portion of the engineering, transduction, and/or transfection is performed at a volume (e.g., a rotary seeding volume) of 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 about 30 mL. In certain embodiments, the cell pellet volume after rotary seeding 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 is about 10 mL.
In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response (e.g., proliferation, survival, and/or activation), e.g., as measured by expression of a cytokine or activation marker, and then transducing the activated cells and expanding in culture to an amount 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.
In some embodiments, the method for genetic engineering is performed by contacting one or more cells of the composition with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor). In some embodiments, contacting can be achieved by centrifugation, such as rotary seeding (e.g., centrifugal seeding). 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, as well as various kits for use in such systems. Exemplary chambers, systems, and processing instruments and cabinets are described, for example, 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 respective contents of which are incorporated herein by reference in their entirety. Exemplary kits for use with such systems include, but are not limited to, disposable kits sold under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2 by BioSafe SA.
In some embodiments, the system is included with and/or placed in association with other instruments, including instruments for operating, automating, controlling and/or monitoring the transduction step and one or more various other processing steps performed in the system (e.g., one or more processing steps performed by a centrifugal chamber system as described herein or in international publication No. WO 2016/073602 may be used or combined). In some embodiments, such instruments are housed in cabinets. In some embodiments, the instrument comprises a cabinet comprising a housing containing control circuitry, a centrifuge, a cover, a motor, a pump, a sensor, a display, and a user interface. Exemplary devices are described in U.S. patent No. 6,123,655, U.S. patent No. 6,733,433, and U.S. patent No. 2008/0171951.
In some embodiments, the system comprises a series of containers, such as bags, tubing, stopcocks, clamps, connectors, and centrifugal chambers. In some embodiments, the container (e.g., a bag) comprises one or more containers (e.g., bags) containing the cells and viral vector particles to be transduced in the same container or in separate containers (e.g., the same bag or separate bags). In some embodiments, the system further comprises one or more containers (e.g., bags) containing a medium, such as a diluent and/or wash solution, that is drawn into the chamber and/or other components during the process to dilute, resuspend, and/or wash the components and/or compositions. The vessel may be connected at one or more locations in the system, for example at locations corresponding to the input line, diluent line, wash line, waste line and/or output line.
In some embodiments, the chamber is associated with a centrifuge that is capable of effecting rotation of the chamber, e.g., about its axis of rotation. Transduction of the bound cells and/or in one or more other processing steps, rotation may occur before, during and/or after incubation. Thus, in some embodiments, one or more of the individual processing steps are performed under rotation (e.g., under a particular force). The chamber is typically rotatable vertically or substantially vertically such that the chamber is placed vertically during centrifugation, and the side walls and shaft are vertical or substantially vertical, and the one or more end walls are horizontal or substantially horizontal.
In some embodiments, the cell-containing composition and the viral vector particle-containing composition, and optionally air, may be combined or mixed prior to providing the composition to the cavity. In some embodiments, the cell-containing composition and the viral vector particle-containing composition, and optionally air, are provided separately in a cavity and combined and mixed therein. In some embodiments, the cell-containing composition, the viral vector particle-containing composition, and optionally air may be provided to the internal cavity in any order. In any of such embodiments, the composition comprising the cell and viral vector particles is an input composition once combined or mixed together, whether the input composition is combined and/or mixed inside or outside the centrifugal chamber, and/or whether the cell and viral vector particles are provided to the centrifugal chamber together or separately (e.g., simultaneously or sequentially).
In some embodiments, in the transduction method, uptake of a volume of gas (e.g., air) is performed prior to incubating the cells and viral vector particles (e.g., spinning). In some embodiments, uptake of a volume of gas (e.g., air) is performed during incubation (e.g., rotation) of the cells and viral vector particles in the transduction method.
In some embodiments, the liquid volume of the cells or viral vector particles comprising the transduction composition, and optionally the volume of air, may be a predetermined volume. The volume may be a volume programmed into the system and/or controlled by circuitry associated with the system.
In some embodiments, the intake of the transduction composition and optionally a gas (e.g., air) is controlled manually, semi-automatically, and/or automatically until a desired or predetermined volume has been ingested into the interior cavity of the chamber. In some embodiments, a sensor associated with the system may detect liquid and/or gas flowing into and out of the centrifugal chamber, for example, via its color, flow rate, and/or density, and may communicate with associated circuitry to stop or continue ingestion as needed until such desired or predetermined volume of ingestion has been achieved. In some aspects, a sensor that is programmed or only capable of detecting liquid in the system, rather than gas (e.g., air), may be enabled to allow gas (e.g., air) to pass into the system without stopping ingestion. In some such embodiments, when gas (e.g., air) ingestion is desired, an opaque tube may be placed in the line near the sensor. In some embodiments, the intake of a gas (e.g., air) may be controlled manually.
In aspects of the provided methods, the internal cavity of the centrifugal chamber is subjected to high-speed rotation. In some embodiments, the rotation is effected before, simultaneously with, after or intermittently with the intake of the liquid input composition and optionally air. In some embodiments, the rotation is achieved after ingestion of the liquid input composition and optionally air. In some embodiments, the rotation is by centrifugation of the centrifugal chamber at the inner surface of the sidewall of the interior cavity and/or at the surface layer of the cells at or about 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 relative centrifugal force. In some embodiments, the rotation is by centrifugation with a force of greater than or about 1100 g, such as, for example, 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 or about 1600 g force.
In some embodiments, the transduction method comprises spinning or centrifuging the transduction composition and optionally air in a centrifuge 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 spun or centrifuged in the centrifuge chamber for greater than 5 minutes, but no more than 60 minutes, no more than 45 minutes, no more than 30 minutes, or no more than 15 minutes. In particular embodiments, transduction comprises spinning or centrifugation for or for about 60 minutes.
In some embodiments, the transduction method comprises spinning or centrifuging the transduction composition and optionally air in a centrifuge chamber for a time between or between about 10 minutes and 60 minutes, between 15 minutes and 45 minutes, between 30 minutes and 60 minutes, or between 45 minutes and 60 minutes, each comprising an end value, and the spinning or centrifuging is performed with a force 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 at the inner surface of the inner cavity sidewall and/or at the cell surface layer. In particular embodiments, the transduction method comprises rotating or centrifuging the transduction composition (e.g., cells and viral vector particles) at 1600 g or about 1600 g for 60 minutes or about 60 minutes.
In some embodiments, the gas (e.g., air) in the cavity of the chamber is vented from the chamber. In some embodiments, the gas (e.g., air) is vented to a container that is operatively connected to the centrifugal chamber as part of a closed system. In some embodiments, the container is a free or empty container. In some embodiments, air (e.g., gas) in the cavity of the chamber is exhausted through a filter that is operatively connected to the interior cavity of the chamber via a sterile tubing set line. In some embodiments, the air is expelled using a manual, semi-automatic, or automatic process. In some embodiments, air is expelled from the cavity prior to, simultaneously with, intermittently with, or subsequent to delivering (expression) the output composition containing the incubated cells and viral vector particles (e.g., cells that have begun to transduce or cells that have been transduced with viral vectors) from the cavity of the chamber.
In some embodiments, transduction and/or other incubation is performed as or as part of a continuous or semi-continuous process. In some embodiments, the continuous process involves continuous ingestion of the cells and viral vector particles, such as the transduction composition (either as a single pre-existing composition, or by continuous drawing into the same vessel (e.g., cavity), mixing portions thereof), and/or continuous delivery or evacuation of liquid from the vessel, and optionally evacuation of gas (e.g., air), during at least a portion of incubation (e.g., while centrifugation). In some embodiments, the continuous ingestion and continuous delivery are performed at least partially simultaneously. In some embodiments, continuous uptake occurs during part of the incubation, e.g., during part of the centrifugation, and continuous transport occurs during separate parts of the incubation. The two may be alternated. Thus, continuous ingestion and delivery while incubation is performed may allow for the processing (e.g., transduction) of a larger total volume of sample.
In some embodiments, the incubation is part of a continuous process, the method comprising during at least a portion of the incubation, effecting continuous ingestion of the transduction composition into the cavity during rotation of the chamber and during a portion of the incubation, effecting continuous delivery of liquid from the cavity and optionally venting of gas (e.g., air) through the at least one opening during rotation of the chamber.
In some embodiments, semi-continuous incubation is performed by alternating between effecting ingestion of the composition into the cavity, incubating, transporting liquid from the cavity and optionally evacuating gas (e.g., air) from the cavity, such as to an output container, and then ingesting 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 incubation, effecting uptake of the transduction composition into the cavity through the at least one opening, and, after incubation, effecting delivery of fluid from the cavity, effecting uptake of another transduction composition comprising cells and viral vector particles into the internal cavity, and incubating the other transduction composition in the internal cavity under conditions whereby cells in the other transduction composition are transduced by the vector. The process can continue in an iterative fashion for many additional rounds. In this regard, semi-continuous or continuous methods may allow for the production of even larger volumes and/or numbers of cells.
In some embodiments, a portion of the transduction incubation is performed in a centrifuge chamber, which is performed under conditions that include rotation or centrifugation.
In some embodiments, the method comprises incubating, wherein another portion of the incubation of the cells and viral vector particles occurs without rotation or centrifugation, typically after the at least one portion of the incubation comprising 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 will be 72 hours or about 72 hours.
In some such embodiments, further incubation is effected under conditions such that the viral vector integrates into the host genome of one or more cells. It is assessed or determined whether incubation has resulted in integration of the viral vector particles into the host genome, and therefore the conditions for further incubation are determined empirically to be within the level of the skilled person. In some embodiments, viral vector integration into the host genome can be assessed by measuring the expression level of a recombinant protein (e.g., a heterologous protein) encoded by a nucleic acid contained in the viral vector particle genome after incubation. The expression level of the recombinant molecule may be assessed using a variety of well known methods, for example in the case of cell surface proteins, for example by affinity-based methods (e.g. immunoaffinity-based methods), for example by flow cytometry. In some examples, expression is measured by detecting a transduction marker and/or a reporter construct. In some embodiments, nucleic acids encoding truncated surface proteins are included in vectors and used as markers for their expression and/or enhancement.
In some embodiments, the composition comprising the cells, the vector (e.g., viral particles), and the agent may be rotated, typically with 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). In some embodiments, the rotation is performed with a force (e.g., relative centrifugal force) from 100 g to 3200 g or from about 100 g to about 3200 g (e.g., or about 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) as measured, for example, at an inner or outer wall of the chamber or cavity. The term "relative centrifugal force" or RCF is generally understood to be an effective force exerted on an object or substance (e.g. a cell, sample or pellet and/or a point in a rotated chamber or other container) relative to the gravitational force of the earth at a specific point in space as compared to the axis of rotation. The values may be determined using well known formulas that take into account gravity, rotational speed and radius of rotation (distance from the axis of rotation and the object, substance or particle measuring the RCF).
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 into a bioreactor bag assembly for culturing the genetically engineered cells, e.g., for culturing or expanding the cells, as described above.
In certain embodiments, the composition enriched for T cells is engineered, e.g., transduced or transfected, in the presence of a transduction adjuvant. In some embodiments, the composition of enriched T cells is engineered in the presence of one or more polycations. In some embodiments, transduction is performed in the presence of one or more transduction adjuvants, e.g., incubating the T cell enriched composition with viral vector particles. In certain embodiments, the T cell enriched composition is transfected in the presence of one or more transduction adjuvants, e.g., incubated with a non-viral vector. In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of gene delivery, such as by increasing the amount, fraction, and/or percentage of engineered (e.g., transduced or transfected) cells in the composition. 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 the polycation contain or express the 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 the recombinant transduction adjuvant than alternative and/or exemplary methods of engineering cells in the absence of the transduction adjuvant.
In some embodiments, the composition of enriched 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 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.
In some embodiments, the cells are engineered in the presence of cytokines, e.g., recombinant human cytokines, 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.
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 certain embodiments, the composition enriched for T cells is engineered in the presence of recombinant IL-2 at a concentration of at or 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 engineered in or in the presence of about 85 IU/ml. In some embodiments, the T cell population is a cd4+ T cell population. In certain embodiments, the T cell enriched composition is enriched for cd4+ T cells, wherein the cd8+ T cells are not enriched and/or wherein the cd8+ T cells are negatively selected or depleted from the composition. In a particular embodiment, the T cell enriched composition is a cd8+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd8+ T cells, wherein the cd4+ T cells are not enriched and/or wherein the cd4+ T cells are negatively selected or depleted from the composition.
In some embodiments, the composition enriched for 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 T cell enriched composition is engineered in the presence of IL-7 at or about 50 IU/ml、100 IU/ml、150 IU/ml、200 IU/ml、250 IU/ml、300 IU/ml、350 IU/ml、400 IU/ml、450 IU/ml、500 IU/ml、550 IU/ml、600 IU/ml、650 IU/ml、700 IU/ml、750 IU/ml、800 IU/ml、750 IU/ml、750 IU/ml、750 IU/ml or 1,000 IU/ml. In a particular embodiment, the T cell enriched composition is engineered in or in the presence of about 600 IU/ml IL-7. In some embodiments, a composition engineered in the presence of recombinant IL-7 is enriched for a population of T cells (e.g., cd4+ T cells). In certain embodiments, the T cell enriched composition is enriched for cd4+ T cells, wherein the cd8+ T cells are not enriched and/or wherein the cd8+ T cells are negatively selected or depleted from the composition.
In some embodiments, the composition enriched for T cells is engineered in the presence of recombinant IL-15, e.g., human recombinant IL-15, the recombinant IL-15 concentration is 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 T cell enriched composition is engineered in the presence of IL-15 at 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 of IL-15. In some embodiments, the enriched T cell composition is engineered in or in about 10 IU/ml IL-15. In some embodiments, the enriched T cell composition is incubated in or at about 10 IU/ml recombinant IL-15. In some embodiments, a 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 T cell enriched composition is a cd8+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd8+ T cells, wherein the cd4+ T cells are not enriched and/or wherein the cd4+ T cells are negatively selected or depleted from the composition. In some embodiments, the T cell enriched composition is a cd4+ T cell enriched composition. In certain embodiments, the T cell enriched composition is enriched for cd4+ T cells, wherein the cd8+ T cells are not enriched and/or wherein the cd8+ T cells are negatively selected or depleted from the composition.
In certain embodiments, compositions enriched for CD8+ T cells are engineered in the presence of IL-2 and/or IL-15. In certain embodiments, the composition enriched for CD4+ T cells is engineered in the presence of IL-2, IL-7, and/or IL-15. In some embodiments, IL-2, IL-7 and/or IL-15 is recombinant. In certain embodiments, IL-2, IL-7 and/or IL-15 is human. In particular embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and/or IL-15.
In certain embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, antioxidants include, but are not limited to, one or more antioxidants including tocopherol, tocotrienol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alpha-tocoquinone, trolox (6-hydroxy-2, 5,7, 8-tetramethylchroman-2-dicarboxylic acid), butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, beta-carotene, selenium, ubiquinone, syphilin, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine (NAC), citric acid, L-carnitine, BHT, thioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine, and cystathionine, and/or glycine-histidine.
In some embodiments, the one or more antioxidants are or include sulfur-containing oxidizing agents. In certain embodiments, the sulfur-containing antioxidant may include a thiol-containing antioxidant and/or an antioxidant that exhibits one or more sulfur moieties, for example, within the ring structure. In some embodiments, the sulfur-containing antioxidant may include, for example, N-acetylcysteine (NAC) and 2, 3-Dimercaptopropanol (DMP), L-2-oxo-4-thiazolidine formate (OTC), and lipoic acid. In a particular embodiment, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified to a derivatized glutathione in one or more steps within the cell. In particular embodiments, glutathione precursors may include, but are not limited to, N-acetylcysteine (NAC), L-2-oxothiazolidine-4-carboxylic acid (procaysteine), lipoic acid, S-allylcysteine, or methioninesulfonium chloride.
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 one or more antioxidants between 1 ng and 100 ng/ml, between 10 ng and 1 μg/ml, between 100 ng 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 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. In some embodiments, the cells are engineered in the presence of the one or more antioxidants 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. In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In certain embodiments, the one or more antioxidants are or include glutathione precursors.
In some embodiments, the cells are engineered in the presence of NAC. In some embodiments, the cells are engineered in the presence of NAC between 1 ng and 100 ng/ml, between 10 ng and1 μg/ml, between 100 ng and 10 μg/ml, between 1 μg/ml and 100 μg/ml, between 10 μg/ml and1 mg/ml, between 100 μg/ml and1 mg/ml, between 1,500 μg/ml and2 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. In some embodiments, the cells are engineered in the presence of NAC 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. In some embodiments, the cells are engineered with or with about 0.8 mg/ml.
In some embodiments, the composition of enriched T cells (e.g., 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, the composition enriched for T cells (e.g., stimulated T cells, e.g., stimulated cd4+ T cells or stimulated cd8+ T cells) is transduced, e.g., incubated with a viral vector particle, in the presence of one or more polycations. In certain embodiments, a composition of enriched T cells (e.g., stimulated T cells, e.g., stimulated cd4+ T cells or stimulated cd8+ T cells) is transfected with a non-viral vector (e.g., incubated 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, such as by increasing the amount, fraction, and/or percentage of engineered (e.g., transduced or transfected) cells in the composition. 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 the polycation contain or express the 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 the recombinant polynucleotide than alternative and/or exemplary methods of engineering cells in the absence of the polycation.
In certain embodiments, for example, the cell-enriched composition, e.g., the cd4+ T cell-enriched or cd8+ T cell-enriched (e.g., stimulated T cell thereof) composition, is engineered in the presence of a low concentration or amount of polycation relative to an exemplary and/or alternative method of engineering cells in the presence of a polyanion. In certain embodiments, the enriched cells, such as a composition of stimulated T cells (e.g., stimulated cd4+ T cells or stimulated cd8+ T cells), are engineered in the presence of an amount or concentration of 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 polycation used in the exemplary and/or alternative process of engineering cells. In some embodiments, the enriched cells, such as a composition of stimulated T cells (e.g., stimulated cd4+ T cells or stimulated cd8+ T cells), are 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, the composition of enriched cells (e.g., stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells) is engineered in the presence of the polycation at 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.
In certain embodiments, engineering the composition of enriched cells, such as 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, apoptosis, or apoptosis). In some embodiments, the composition of enriched T cells (e.g., 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 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, 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, apoptosis, or apoptosis, after the engineering step is completed. In some embodiments, the composition is engineered in the presence of a low concentration or amount of polycation compared to alternative and/or exemplary methods of engineering cells in the presence of a higher amount or concentration of polycation (e.g., greater than 50 μg/ml, 100 μg/ml, 500 μg/ml, or 1,000 μg/ml), and the cells of the composition have a viability 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 undergoing the exemplary and/or alternative processes.
In some embodiments, the polycation is positively charged. In certain embodiments, the polycation reduces the repulsive force between the cell and the vector (e.g., viral or non-viral vector) and mediates contact and/or binding of the vector to the cell surface. In some embodiments, the polycation is a polybrene, DEAE-dextran, protamine sulfate, poly-L-lysine, or cationic liposome.
In a particular embodiment, the polycation is protamine sulfate. In some embodiments, the composition of enriched T cells (e.g., stimulated T cells, e.g., cd4+ T cells or cd8+ T cells) is engineered in the presence of less than or about 500 μg/ml, less than or about 400 μg/ml, less than or about 300 μg/ml, less than or about 200 μg/ml, less than or about 150 μg/ml, less than or about 100 μg/ml, less than or about 90 μg/ml, less than or about 80 μg/ml, less than or about 75 μg/ml, less than or about 70 μg/ml, less than or about 60 μg/ml, less than or about 50 μg/ml, less than or about 40 μg/ml, less than or about 30 μg/ml, less than or about 25 μg/ml, less than or about 20 μg/ml, or less than or about 15 μg/ml, or less than or about 10 μg/ml of protamine sulfate. In particular embodiments, the enriched cells, such as a composition of stimulated T cells (e.g., stimulated cd4+ T cells or stimulated cd8+ T cells), are engineered in the presence of protamine sulfate at 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、50 µg/ml、55 µg/ml、60 µg/ml、75 µg/ml、80 µg/ml、85 µg/ml、90 µg/ml、95 µg/ml、100 µg/ml、105 µg/ml、110 µg/ml、115 µg/ml、120 µg/ml、125 µg/ml、130 µg/ml、135 µg/ml、140 µg/ml、145 µg/ml or 150 μg/ml.
In some embodiments, the engineered composition enriched for 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 about 100% cd4+ T cells. In certain embodiments, the composition of engineered 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.
In some embodiments, the composition of engineered 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 at or about 100% cd8+ T cells. In certain embodiments, the composition of engineered 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.
In some embodiments, engineering the cells includes 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 carrier, performing, or performing for about or 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 carrier for or for about 24 hours, 36 hours, 48 hours, 60 hours, 72 hours or 84 hours, 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, or about 72 hours, or about 2 days.
In some embodiments, the engineering is performed at a temperature of from about 25 to about 38 ℃, such as from about 30 to about 37 ℃, from about 36 to about 38 ℃, or at or about 37±2 ℃. In some embodiments, the enriched T cell composition is engineered at a CO 2 level of from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example, at or about 5% ± 0.5%. In some embodiments, the T cell enriched composition is engineered at a temperature of or about 37 ℃ and/or at a CO 2 level of or about 5%.
In some embodiments, the cells are incubated after one or more steps for genetically engineering, e.g., transducing or transfecting, the cells (e.g., cd4+ and/or cd8+ T cells) to contain the polynucleotide encoding the recombinant receptor. In some embodiments, incubating may include culturing, incubating, stimulating, activating, amplifying, and/or propagating. In some such embodiments, further culturing is effected under conditions such that the viral vector integrates into the host genome of one or more cells. Incubation and/or engineering may be performed in a culture vessel such as a unit, chamber, well, column, tube, set of tubes, valve, vial, petri dish, bag or other container for culturing or incubating cells. In some embodiments, the composition or cell is incubated in the presence of a stimulating condition or a stimulating agent. Such conditions include those designed to induce proliferation, expansion, activation and/or survival of cells in the population, mimic antigen exposure and/or elicit cells for genetic engineering (e.g., for the introduction of recombinant antigen receptors).
In some embodiments, the further incubation is performed at a temperature above room temperature, for example, above or above about 25 ℃, such as typically above or above about 32 ℃, 35 ℃, or 37 ℃. In some embodiments, further incubation is effected at a temperature of or about 37oC ± 2oC, for example at a temperature of or about 37oC.
In some embodiments, further incubation is performed under conditions for stimulating and/or activating the cells, which may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (e.g., nutrient, amino acid, antibiotic, ion, and/or stimulating factor (e.g., cytokine, chemokine, antigen, binding partner, fusion protein, recombinant soluble receptor, and any other agent designed to activate the cells)).
In some embodiments, the stimulation conditions or agents include one or more agents (e.g., stimulatory and/or auxiliary agents), such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates a TCR/CD3 intracellular signaling cascade in the T cell, e.g., is an agent suitable for delivering a primary signal, e.g., to initiate activation of an ITAM-induced signal (e.g., those specific for a TCR component), and/or to promote a co-stimulatory signal (e.g., a co-stimulatory signal specific for a T cell co-stimulatory receptor), e.g., anti-CD 3, anti-CD 28, or anti-41-BB (e.g., optionally bound to a solid support such as a bead), and/or one or more cytokines. The stimulators include anti-CD3/anti-CD 28 beads (e.g., DYNABEADS cube M-450 CD3/CD 28T cell expander and/or ExpACT cube beads). Optionally, the amplification method may further comprise the step of adding an anti-CD 3 and/or anti-CD 28 antibody to the culture medium. In some embodiments, the stimulatory agent includes IL-2 and/or IL-15, for example, IL-2 concentration of at least about 10 units/mL.
In some embodiments, the stimulation conditions or stimulators include one or more agents (e.g., ligands) capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates a TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include, for example, antibodies that are bound to a solid support (e.g., beads), such as those antibodies that are specific for TCR components and/or co-stimulatory receptors (e.g., anti-CD 3, anti-CD 28), and/or one or more cytokines. Optionally, the amplification method may further comprise the step of adding anti-CD 3 and/or anti-CD 28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulatory agent includes IL-2 and/or IL-15, for example, IL-2 concentration of at least about 10 units/mL, at least about 50 units/mL, at least about 100 units/mL, or at least about 200 units/mL.
The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (e.g., nutrient, amino acid, antibiotic, ion, and/or stimulating factor (e.g., cytokine, chemokine, antigen, binding partner, fusion protein, recombinant soluble receptor, and any other agent intended to activate a cell)).
In some aspects, incubation is performed according to a variety of techniques, such as those described in U.S. Pat. No. 6,040,1 77;Klebanoff (2012) J Immunther.35 (9) to Riddell et al 651-660, terakura et al (2012) blood.1:72-82, and/or Wang et al (2012) J Immunther.35 (9) 689-701.
In some embodiments, the further incubation is performed in the same vessel or apparatus in which the contacting is performed. In some embodiments, further incubation is performed without rotation or centrifugation, which is typically performed after the at least a portion of incubation performed under rotation (e.g., in combination with centrifugation or rotary inoculation). In some embodiments, the further incubation is performed outside the stationary phase, e.g., outside the chromatographic matrix, e.g., in solution.
In some embodiments, the further incubation is performed in a different container or device from the container or device in which the contacting is performed, for example by transferring (e.g., automatically transferring) the cell composition into a different container or device after the contacting with the viral particles and the agent.
In some embodiments, further culturing or incubation is performed, for example, to facilitate ex vivo amplification 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. In some embodiments, further culturing or incubating is performed 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.
In some embodiments, for example, the total duration of incubation with the stimulating agent is between or between about 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours, such as at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, further incubation is performed for a time between or about between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours and includes the endpoints.
In some embodiments, the methods provided herein do not include further culturing or incubation, e.g., do not include an ex vivo amplification step, or include a significantly shorter ex vivo amplification step.
In some embodiments, the stimulating agent is removed and/or isolated from the cells prior to engineering. In certain embodiments, the stimulating agent is removed and/or isolated from the cells after engineering. In certain embodiments, the stimulating 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 stimulating agent is a stimulating agent as described in section I-B-1. In certain embodiments, the stimulating agent is removed and/or isolated from the cells as described in section I-B-2.
1. Carrier and method
In some embodiments, cells (e.g., T cells) are genetically engineered to express recombinant receptors. In some embodiments, engineering is by introducing one or more polynucleotides encoding recombinant receptors or portions or components thereof. Polynucleotides encoding recombinant receptors are also provided, as are vectors or constructs comprising such nucleic acids and/or polynucleotides.
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 gamma retroviral vector.
In some embodiments, the polynucleotide encoding the recombinant receptor comprises at least one promoter operably linked to control expression of the recombinant receptor. In some examples, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant receptor. In some embodiments, the polynucleotide may contain regulatory sequences (such as transcription and translation initiation and termination codons) specific for the type of host (e.g., bacterial, fungal, plant, or animal) into which the polynucleotide is to be introduced, as appropriate and in consideration of whether the polynucleotide is DNA-based or RNA-based. In some embodiments, the polynucleotide may 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 may contain a non-native promoter operably linked to a nucleotide sequence encoding a recombinant receptor and/or one or more additional polypeptides. In some embodiments, the promoter is selected from the group consisting of RNA pol I, pol II, or pol III promoters. 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., a U6 or H1 promoter). In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as the Cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and promoters found in the long terminal repeat of murine stem cell viruses. Other known promoters are also contemplated.
In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, the simian virus 40 early promoter (SV 40), cytomegalovirus immediate early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter (EF 1 alpha), mouse phosphoglycerate kinase 1 Promoter (PGK), and chicken beta-actin promoter (CAGG) coupled to the CMV early enhancer. In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises a MND promoter, which is a synthetic promoter containing the U3 region of the modified MoMuLV LTR with a myeloproliferative sarcoma 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 may include, but are not limited to, the human elongation factor 1 alpha (EF 1 alpha) promoter or modified forms thereof or MND promoters.
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 an analog thereof, or is capable of binding or recognition by a Lac repressor or a tetracycline repressor analog thereof. In some embodiments, the polynucleotide does not include regulatory elements, e.g., a promoter.
In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., chimeric Antigen Receptor (CAR)) contains a signal sequence encoding a signal peptide. Non-limiting illustrative examples of signal peptides include, for example, the GMCSFR a chain signal peptide shown in SEQ ID NO. 10 and encoded by the nucleotide sequence shown in SEQ ID NO. 9, the CD 8a signal peptide shown in SEQ ID NO. 11, or the CD33 signal peptide shown in SEQ ID NO. 12.
In some embodiments, the polynucleotide contains a nucleic acid sequence 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 comprise a transduction marker, a surrogate marker, and/or a resistance marker or a selection marker. Additional nucleic acid sequences introduced, for example, encoding one or more additional polypeptides, include nucleic acid sequences that may improve the efficacy of therapy, such as by promoting viability and/or function of the transferred cells, nucleic acid sequences that provide genetic markers for selection and/or evaluation of cells (e.g., assessing survival or localization in vivo), nucleic acid sequences that improve safety, such as by sensitizing cells to in vivo negative selection, 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. Pat. No. 6,040,177.
In some embodiments, the marker is a transduction marker or a surrogate marker. The transduction markers or surrogate markers can be used to detect cells into which a polynucleotide (e.g., a polynucleotide encoding a recombinant receptor) has been introduced. In some embodiments, the transduction marker may indicate or confirm modification to the cell. In some embodiments, the surrogate marker is a protein prepared to co-express with a recombinant receptor (e.g., CAR) on the cell surface. In particular embodiments, such surrogate markers are surface proteins that have been modified to have little or no activity. In certain embodiments, the surrogate markers are encoded by the same polynucleotide encoding the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a 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 ribosome jump, such as the 2A sequence. In some cases, an extrinsic marker gene may be associated with an engineered cell for allowing detection or selection of the cell, and in some cases, may also be used to promote cell elimination and/or cell suicide.
Exemplary surrogate markers may include truncated forms of a cell surface polypeptide, such as truncated forms that are nonfunctional and do not transduce or are incapable of transducing a signal or are generally transduced by a full length form of the cell surface polypeptide, and/or are not internalized or are incapable of internalization. Exemplary truncated cell surface polypeptides include truncated forms of a growth factor or other receptor, such as truncated human epidermal growth factor receptor 2 (tHER), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences shown in SEQ ID NO: 2 or 3), or Prostate Specific Membrane Antigen (PSMA) or modified forms thereof, such as truncated PSMA (tPSMA). In some aspects, the tgfr may contain an epitope recognized by the antibody cetuximab (Erbitux) or other therapeutic anti-EGFR antibody or binding molecule, which may be used to identify or select cells that have been engineered with the tgfr construct and the encoded foreign protein, and/or to eliminate or isolate cells expressing the encoded foreign protein. See U.S. Pat. No. 8,802,374 and Liu et al, nature Biotech.2016, month 4; 34 (4): 430-434). In some aspects, the marker (e.g., surrogate marker) comprises all or part (e.g., truncated form) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated non-human CD 19). Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence shown in SEQ ID NO: 2 or 3 or an amino acid sequence exhibiting 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.
In some embodiments, the label is or comprises a detectable protein, such as a fluorescent protein, e.g., green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP) (e.g., superfolder GFP (sfGFP)), red Fluorescent Protein (RFP) (e.g., tdTomato, mCherry, mStrawberry, asRed, dsRed, or DsRed 2), 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 protein. In some embodiments, the label is or comprises an enzyme (e.g., luciferase), a lacZ gene from E.coli, alkaline phosphatase, secreted Embryonic Alkaline Phosphatase (SEAP), chloramphenicol Acetyl Transferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), beta-galactosidase, chloramphenicol Acetyl Transferase (CAT), beta-Glucuronidase (GUS), or variants thereof. In some aspects, the expression of the enzyme may be detected by adding a substrate that may be detected based on the expression and functional activity of the enzyme.
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 selection marker is an antibiotic resistance gene. In some embodiments, the resistance marker or selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the resistance marker or selectable marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene or a gecomycin resistance gene or modified form thereof.
Any of the recombinant receptors and/or one or more additional polypeptides described herein may be encoded by one or more polynucleotides comprising one or more nucleic acid sequences encoding the recombinant receptors in any combination, orientation, or arrangement. For example, one, two, three, or more polynucleotides may encode one, two, three, or more different polypeptides (e.g., recombinant receptors or portions or components thereof) and/or one or more additional polypeptides (e.g., markers and/or effector molecules). In some embodiments, a polynucleotide comprises a nucleic acid sequence encoding a recombinant receptor (e.g., CAR) or a portion or component thereof, and a nucleic acid sequence encoding one or more additional polypeptides. In some embodiments, one vector or construct contains a nucleic acid sequence encoding a recombinant receptor (e.g., CAR) or a portion or component thereof, and a separate vector or construct contains a nucleic acid sequence encoding one or more additional polypeptides. In some embodiments, the nucleic acid sequence encoding the recombinant receptor and the nucleic acid sequence encoding one or more additional polypeptides are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is present upstream of the nucleic acid encoding the one or more additional polypeptides. In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding the one or more additional polypeptides.
In certain instances, a polynucleotide comprises a nucleic acid sequence encoding two or more different polypeptide chains, e.g., a recombinant receptor and one or more additional polypeptides, e.g., a marker and/or an effector molecule. In some embodiments, nucleic acid sequences encoding two or more different polypeptide chains (e.g., a recombinant receptor and one or more additional polypeptides) are present in two separate polynucleotides. For example, two separate polynucleotides are provided, and each may be transferred or introduced separately into a cell for expression in the cell. In some embodiments, the nucleic acid sequence encoding the marker and the nucleic acid sequence encoding the recombinant receptor are present or inserted at different locations within the genome of the cell. In some embodiments, the nucleic acid sequence encoding the marker and the nucleic acid sequence encoding the recombinant receptor are operably linked to two different promoters.
In some embodiments, such as those in which the polynucleotide comprises first and second nucleic acid sequences, the coding sequences encoding each of the different polypeptide chains may be operably linked to the same or different promoters. In some embodiments, the nucleic acid molecule may contain a promoter that drives expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules may be polycistronic (bicistronic or tricistronic), see, e.g., U.S. patent No. 6,060,273. In some embodiments, the nucleic acid sequence encoding the recombinant receptor and the nucleic acid sequence encoding one or more additional polypeptides are operably linked to the same promoter and are optionally separated by an Internal Ribosome Entry Site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jump (e.g., the 2A element). For example, an exemplary tag and optional ribosome jump sequence can be any of those disclosed in PCT publication No. WO 2014031687.
In some embodiments, the transcriptional unit may be engineered to contain an IRES bicistronic unit that allows the gene product (e.g., encoding a recombinant receptor and additional polypeptide) to be co-expressed by information from a single promoter. Alternatively, in some cases, a single promoter may direct expression of RNAs that contain two or three genes (e.g., encoding a marker and encoding a recombinant receptor) separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin) in a single Open Reading Frame (ORF). Thus, the ORF encodes a single polypeptide that is processed into separate proteins during translation (in the case of 2A) or post-translationally. In some cases, the peptide (e.g., T2A) may result in synthesis of a peptide bond at the C-terminus of the ribosome skip (ribosome skip) 2A element, which results in separation between the 2A sequence end and the next peptide downstream (see, e.g., de Felipe. GENETIC VACCINES AND Ther. 2:13 (2004) and de Felipe et al Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that may be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 8), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 7), leptospira mingii beta tetrad virus (T2A, e.g., SEQ ID NO: 1 or 4), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 5 or 6), as described in U.S. patent publication No. 20070116690.
In some embodiments, the polynucleotide encoding the recombinant receptor and/or additional polypeptide is contained in a vector, or may be cloned into one or more vectors. In some embodiments, one or more vectors may be used to transform or transfect a host cell, e.g., for an engineered cell. Exemplary vectors include vectors designed for introduction, propagation and amplification or for expression or both, such as plasmids and viral vectors. In some aspects, the vector is an expression vector, e.g., a recombinant expression vector. In some embodiments, the recombinant expression vector may be prepared using standard recombinant DNA techniques.
In some embodiments, the vector may be a pUC series (FERMENTAS LIFE SCIENCES), pBluescript series (Stratagene, lajoba, california), pET series (Novagen, madison, wis.), pGEX series (PHARMACIA BIOTECH, uppsala, sweden) or pEX series (Clontech, paruoldo, california). In some cases, phage vectors such as λg10, λgt11, λ ZapII (Stratagene), λembl4, and λnm1149 may also be used. In some embodiments, plant expression vectors may be used and include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
In some embodiments, the polynucleotide encoding the recombinant receptor and/or one or more additional polypeptides is introduced into a composition comprising cultured cells, such as by retroviral transduction, transfection, or transformation.
In some embodiments, the vector is a viral vector, such as a retroviral vector. In some embodiments, polynucleotides encoding recombinant receptors and/or one or more additional polypeptides are introduced into cells by retroviruses or lentiviral vectors or by transposons (see, e.g., baum et al (2006) Molecular Therapy: the Journal of THE AMERICAN Society of Gene therapy.13:1050-1063; frecha et al (2010) Molecular Therapy 18:1748-1757; and Hackett et al (2010) Molecular Therapy 18:674-683).
In some embodiments, the vector comprises a viral vector, such as a retrovirus or lentivirus, a non-viral vector or a transposon, such as a sleeping beauty transposon system, a vector derived from simian virus 40 (SV 40), adenovirus, adeno-associated virus (AAV), a lentiviral vector or a retroviral vector, such as a gamma-retroviral vector, a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Stem Cell Virus (MSCV), spleen Focus Forming Virus (SFFV), or adeno-associated virus (AAV).
In some embodiments, electroporation is used to introduce ONE or more polynucleotides into T cells (see, e.g., chicaybam et al, (2013) PLoS ONE 8 (3): e60298; and Van Tedeloo et al (2000) GENE THERAPY (16): 1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition (see, e.g., manuri et al (2010) Hum Gene Ther 21 (4): 427-437; shalma et al (2013) Molec Ther Nucl Acids, e74; and Huang et al (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic material (e.g., polynucleotides and/or vectors) in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, john Wiley & Sons, new york. N.y.), protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microprojectile bombardment (Johnston, nature, 346: 776-777 (1990)), and strontium phosphate DNA co-precipitation (Brash et al, mol, cell biol., 7: 2031-2034 (1987)), as well as other approaches described in, for example, international patent application publication No. WO 2014055668 and U.S. Pat. No. 7,446,190.
In some embodiments, one or more polynucleotides or vectors encoding recombinant receptors and/or one or more additional polypeptides may be introduced into a cell (e.g., a T cell) during or after amplification. For example, the introduction of one or more polynucleotides or one or more vectors may be performed using any suitable retroviral vector. The resulting genetically engineered cells can then be freed from the initial stimulus (e.g., anti-CD 3/anti-CD 28 stimulus) and subsequently stimulated in the presence of a second type of stimulus (e.g., by a recombinant receptor introduced de novo). The second type of stimulus may include an antigen stimulus in the form of a peptide/MHC molecule, a cognate (cross-linked) ligand of a genetically introduced receptor (e.g., the natural antigen and/or ligand of a CAR), or any ligand (e.g., an antibody) that binds directly within the framework of a new receptor (e.g., by recognizing a constant region within the receptor). See, for example, cheadle et al, "CHIMERIC ANTIGEN receptors for T-cell based therapy" Methods Mol biol.2012; 907:645-66 or Barrett et al, CHIMERIC ANTIGEN Receptor Therapy for Cancer Annual Review of Medicine, volume 65:333-347 (2014).
In some cases, vectors may be used that do not require activating cells (e.g., T cells). In some such cases, the cells may be selected and/or transduced prior to activation. Thus, the cells may be engineered before or after culturing the cells, and in some cases during the same time or at least a portion of the culturing.
A. Viral vector particles
In some embodiments, one or more polynucleotides are introduced into the cell using recombinant infectious viral particles (e.g., vectors derived from simian virus 40 (SV 40), adenovirus, adeno-associated virus (AAV)). In some embodiments, one or more polynucleotides are introduced into T cells using recombinant lentiviral vectors or retroviral vectors (e.g., gamma-retroviral vectors) (see, e.g., koste et al (2014) GENE THERAPY, month 4, 3, doi: 10.1038/gt.2014.25; carlens et al (2000) Exp Hematol (10): 1137-46; alonso-Camino et al (2013) Mol Ther Nucl Acids 2, e93; park et al, trends Biotechnol.2011, month 11, 29 (11): 550-557).
In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector has a Long Terminal Repeat (LTR), such as a retroviral vector derived from moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Stem Cell Virus (MSCV), spleen Focus Forming Virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. The retroviruses are typically amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces retroviral gag, pol and/or env sequences. A number of exemplary retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740, 6,207,453, 5,219,740; miller and Rosman (1989) BioTechniques 7:980-990; miller, A.D. (1990) Human GENE THERAPY 1:5-14; scarpa et al (1991) Virology 180:849-852; burns et al (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur.Opin. Genet. Development.3:102-109).
Methods of lentiviral transduction are known. Exemplary Methods are described, for example, in Wang et al (2012) J. Immunother35 (9): 689-701, cooper et al (2003) blood.101:1637-1644, verhoeyen et al (2009) Methods Mol biol.506:97-114, and CAVALIERI et al (2003) blood.102 (2): 497-505.
In some embodiments, the viral vector particles contain a genome derived from a retroviral genome-based vector (e.g., derived from a lentiviral genome-based vector). In some aspects of the provided viral vectors, a heterologous nucleic acid encoding a recombinant receptor (e.g., an antigen receptor, such as a CAR) is contained and/or located between the 5 'LTR and 3' LTR sequences of the vector genome.
In some embodiments, the viral vector genome is a lentiviral genome, such as an HIV-1 genome or an SIV genome. For example, lentiviral vectors have been generated by attenuating virulence genes multiple times, e.g., genes env, vif, vpu and nef can be deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al, (1996 and 1998), zufferey et al, (1997), dull et al, 1998, U.S. Pat. No. 6,013,516, and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry a base sequence for incorporation of foreign nucleic acids for selection and for transferring the nucleic acids into host cells. Known lentiviruses can be readily obtained from a custody institution or collection such as the american type culture collection ("ATCC"; university of marasas, virginia (University blvd)), 10801, 20110-2209) or isolated from known sources using conventional techniques.
Non-limiting examples of lentiviral vectors include those derived from lentiviruses, such as human immunodeficiency virus 1 (HIV-1), HIV-2, simian Immunodeficiency Virus (SIV), human T-lymphotropic virus 1 (HTLV-1), HTLV-2, or equine infectious anemia virus (E1 AV). For example, lentiviral vectors have been generated by attenuating HIV virulence genes multiple times, e.g., deleting genes env, vif, vpr, vpu and nef, making the vector safer for therapeutic purposes. Lentiviral vectors are known in the art, see Naldini et al, (1996 and 1998), zufferey et al, (1997), dull et al, 1998, U.S. Pat. No. 6,013,516, and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry a base sequence for incorporation of foreign nucleic acids for selection and for transferring the nucleic acids into host cells. Known lentiviruses can be readily obtained from a custody institution or collection such as the american type culture collection ("ATCC"; university of marasas, virginia (University blvd)), 10801, 20110-2209) or isolated from known sources using conventional techniques.
In some embodiments, the viral genome vector may contain sequences of the 5 'and 3' LTRs of a retrovirus (e.g., lentivirus). In some aspects, the viral genome construct may contain sequences from the 5 'and 3' ltrs of the lentivirus, and in particular may contain the R and U5 sequences from the 5 'ltrs of the lentivirus, as well as the inactivated or self-inactivated 3' ltrs from the lentivirus. The LTR sequence may be an LTR sequence of any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequence is an HIV LTR sequence.
In some embodiments, the nucleic acid of a viral vector (e.g., an HIV viral vector) lacks additional transcriptional units. The vector genome may contain an inactivated or self-inactivated 3' LTR (Zufferey et al J Virol 72:9873, 1998; miyoshi et al J Virol 72:8150, 1998). For example, a deletion in the U3 region of the 3' LTR of the nucleic acid used to generate viral vector RNA may be used to generate a self-inactivating (SIN) vector. This deletion can then be transferred to the 5' LTR of proviral DNA during reverse transcription. Self-inactivating vectors typically have deletions from the enhancer and promoter sequences of the 3 'Long Terminal Repeat (LTR) that are copied into the 5' LTR during vector integration. In some embodiments, sufficient sequence may be deleted, including removal of the TATA box, to eliminate transcriptional activity of the LTR. This can prevent the generation of full-length vector RNA in transduced cells. In some aspects, the U3 element of the 3' LTR contains deletions of its enhancer sequence, TATA box, sp1, and NF-. Kappa.B sites. Due to self-inactivating 3'LTR, proviruses produced after entry and reverse transcription contain an inactivated 5' LTR. This may improve safety by reducing the risk of mobilizing the vector genome and the effect of the LTR on nearby cell promoters. The self-inactivating 3' LTR may be constructed by any method known in the art. In some embodiments, this does not affect the vector titer or the in vitro or in vivo properties of the vector.
Optionally, the U3 sequence from the lentiviral 5' LTR may be replaced with a promoter sequence (e.g., a heterologous promoter sequence) in the viral construct. This may increase the titer of the virus recovered from the packaging cell line. Enhancer sequences may also be included. Any enhancer/promoter combination that increases expression of the viral RNA genome in the packaging cell line may be used. In one example, a CMV enhancer/promoter sequence is used (U.S. patent No. 5,385,839 and U.S. patent No. 5,168,062).
In certain embodiments, the risk of insertional mutagenesis can be minimized by constructing the retroviral vector genome (e.g., lentiviral vector genome) to be integration defective. A variety of approaches can be used to generate non-integrated vector genomes. In some embodiments, one or more mutations may be engineered into the integrase component of the pol gene such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself may be modified to prevent integration by, for example, mutating or deleting one or both attachment sites, or to render the 3' LTR Proximal Polypurine Tract (PPT) nonfunctional by deletion or modification. In some embodiments, non-genetic pathways may be used, including pharmacological agents that inhibit one or more functions of the integrase. These methods are not mutually exclusive, that is, more than one of the methods may be used at a time. For example, both the integrase and the attachment site may be nonfunctional, or both the integrase and the PPT site may be nonfunctional, or both the attachment site and the PPT site may be nonfunctional, or both may be nonfunctional. Such methods and viral vector genomes are known and available (see Philpott and Thrasher, human GENE THERAPY 18:483, 2007; engelman et al J Virol 69:2729, 1995; brown et al J Virol 73:9011 (1999); WO 2009/076524; mcWilliams et al J Virol 77:11150, 2003; poll and Levin J Virol 70:5288, 1996).
In some embodiments, the vector contains sequences for propagation in a host cell (e.g., a prokaryotic host cell). In some embodiments, the nucleic acid of the viral vector contains one or more origins of replication for propagation in prokaryotic cells (e.g., bacterial cells). In some embodiments, vectors comprising a prokaryotic origin of replication may also contain genes whose expression confers a detectable or selectable marker, such as resistance.
The viral vector genome is typically constructed in the form of a plasmid, which can be transfected into a packaging cell line or a production cell line. Any of a variety of known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome. In some embodiments, at least two components are involved in preparing the viral-based gene delivery system, a first, packaging plasmid, comprising structural proteins and enzymes necessary for the production of viral vector particles, and a second, viral vector itself, i.e., genetic material to be transferred. Biosafety protection can be incorporated in the design of one or both of these components.
In some embodiments, the packaging plasmid may contain all retroviral (e.g., HIV-1) proteins except for the envelope proteins (Naldini et al, 1998). In other embodiments, the viral vector may lack additional viral genes (e.g., those associated with virulence, such as vpr, vif, vpu and nef and/or Tat (the main transactivator of HIV)). In some embodiments, lentiviral vectors (e.g., HIV-based lentiviral vectors) contain only the genes of the three parent viruses gag, pol, and rev, which reduces or eliminates the possibility of reconstitution of the wild-type virus by recombination.
In some embodiments, the viral vector genome is introduced into a packaging cell line that contains all components required to package viral genomic RNA transcribed from the viral vector genome into viral particles. Alternatively, the viral vector genome may comprise one or more genes encoding viral components in addition to the one or more sequences of interest (e.g., recombinant nucleic acids). However, in some aspects, to prevent replication of the genome in the target cell, endogenous viral genes required for replication are removed and provided separately in the packaging cell line.
In some embodiments, the packaging cell line is transfected with one or more plasmid vectors containing components necessary to produce the particles. In some embodiments, packaging cell lines are transfected with a plasmid containing a viral vector genome (including LTR, cis-acting packaging sequences, and target sequences, i.e., nucleic acids encoding antigen receptors (e.g., CARs)), and one or more helper plasmids encoding viral enzymes and/or structural components (e.g., gag, pol, and/or rev). In some embodiments, various genetic components of retroviral vector particles are isolated using a variety of vectors. In some such embodiments, providing the packaging cell with a separate vector reduces the likelihood of recombination events that might otherwise produce replication competent viruses. In some embodiments, a single plasmid vector with all retroviral components may be used.
In some embodiments, retroviral vector particles (e.g., lentiviral vector particles) are pseudotyped to increase the transduction efficiency of a host cell. For example, in some embodiments, retroviral vector particles (e.g., lentiviral vector particles) are pseudotyped with VSV-G glycoprotein, which provides a broad cell host range, thereby expanding the cell types that can be transduced. In some embodiments, packaging cell lines are transfected with a plasmid or polynucleotide encoding a non-native envelope glycoprotein to include, for example, a amphotropic, amphotropic or amphotropic envelope, such as sindbis virus envelope, GALV or VSV-G.
In some embodiments, the packaging cell line provides components, including viral regulatory proteins and structural proteins, required for trans-action in packaging viral genomic RNA into lentiviral vector particles. In some embodiments, the packaging cell line may be any cell line capable of expressing a lentiviral protein and producing a functional lentiviral vector particle. In some aspects, suitable packaging cell lines include 293 (ATCC CCL X), 293T, heLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cells.
In some embodiments, the packaging cell line stably expresses one or more viral proteins. For example, in some aspects, packaging cell lines containing gag, pol, rev and/or other structural genes but without the LTRs and packaging components may be constructed. In some embodiments, packaging cell lines may be transiently transfected with nucleic acid molecules encoding one or more viral proteins, viral vector genomes containing nucleic acid molecules encoding heterologous proteins, and/or nucleic acids encoding envelope glycoproteins.
In some embodiments, the viral vector and packaging plasmid and/or helper plasmid are introduced into the packaging cell line by transfection or infection. Packaging cell lines produce viral vector particles containing viral vector genomes. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran, and liposome transfection methods, electroporation, and microinjection.
When the recombinant plasmid and retroviral LTRs and packaging sequences are introduced into a particular cell line (e.g., by calcium phosphate precipitation), the packaging sequences may allow for packaging of the RNA transcripts of the recombinant plasmid into viral particles, which may then be secreted into the culture medium. In some embodiments, the recombinant retrovirus-containing medium is then collected, optionally concentrated, and used for gene transfer. For example, in some aspects, after co-transfection of the packaging plasmid and transfer vector into the packaging cell line, the viral vector particles are recovered from the culture medium and titrated by standard methods used by those skilled in the art.
In some embodiments, retroviral vectors, such as lentiviral vectors, may be produced in packaging cell lines (e.g., the exemplary HEK 293T cell line) by introducing a plasmid to allow for the production of lentiviral particles. In some embodiments, the packaging cells are transfected and/or contain polynucleotides encoding gag and pol, as well as polynucleotides encoding recombinant receptors (e.g., antigen receptors, such as CARs). In some embodiments, the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a rev protein. In some embodiments, the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a non-native envelope glycoprotein (e.g., VSV-G). In some such embodiments, the cell supernatant contains the recombinant lentiviral vector about two days after transfection of the cells (e.g., HEK 293T cells), which can be recovered and titrated.
The recovered and/or produced retroviral vector particles can be used to transduce target cells using the methods as described. Once in the target cell, the viral RNA is reverse transcribed, enters the nucleus and is stably integrated into the host genome. Expression of a recombinant protein (e.g., an antigen receptor, such as a CAR) can be detected one or two days after viral RNA integration.
In some embodiments, provided methods relate to methods of transducing cells by contacting (e.g., incubating) a cell composition comprising a plurality of cells with a viral particle. In some embodiments, the cells to be transfected or transduced are or comprise primary cells obtained from the subject, e.g., cells enriched and/or selected from the subject.
In some embodiments, the concentration of cells to be transduced in the composition is 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 water stop 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 10 7 cells/mL, 5 x 10 7 cells/mL, or 1 x 10 8 cells/mL.
In some embodiments, the viral particles are provided in a ratio of copies of the viral vector particles or units of Infection (IU) thereof to the total number of cells to be transduced (IU/cells). For example, in some embodiments, the viral particles are present during contact as viral vector particles that are or about or at least about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU per cell.
In some embodiments, the titer of the viral vector particles is or is between about 1 x 10 6 IU/mL and 1 x 10 8 IU/mL, such as between about 5 x 10 6 IU/mL and 5 x 10 7 IU/mL, such as at least 6 x 106 IU/mL、7 x 106IU/mL、8 x 106 IU/mL、9 x 106 IU/mL、1 x 107 IU/mL、2 x 107 IU/mL、3 x 107 IU/mL、4 x 107 IU/mL or 5 x 10 7 IU/mL.
In some embodiments, transduction may be achieved at a multiplicity of infection (MOI) of less than 100, such as typically less than 60, 50, 40, 30, 20, 10, 5 or less.
In some embodiments, the methods involve contacting or incubating the cells with a viral particle. In some embodiments, the contacting is performed for 30 minutes to 72 hours, such as 30 minutes to 48 hours, 30 minutes to 24 hours, or 1 hour to 24 hours, for example at least or about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, or more.
In some embodiments, the contacting is performed in solution. In some embodiments, the cells and virus particles are contacted in a volume of from 0.5 mL to 500 mL or from about 0.5 mL to about 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100mL, 0.5 mL to 50mL, 0.5 mL to 10 mL, 0.5 mL to 5mL, 5mL to 500 mL, 5mL to 200 mL, 5mL to 100mL, 5mL to 50mL, 5mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100mL, 10 mL to 50mL, 50mL to 500 mL, 50mL to 200 mL, 50mL to 100mL, 100mL to 500 mL, 100mL to 200 mL, or 200 mL to 500 2.
In certain embodiments, the input cells are treated, incubated, or contacted with particles comprising binding molecules that bind to or recognize recombinant receptors encoded by viral DNA.
In some embodiments, incubating the cells with the viral vector particles results in or produces an output composition comprising cells transduced with the viral vector particles.
B. non-viral vectors
In some embodiments, the recombinant nucleic acid is transferred into T cells by electroporation (see, e.g., chicaybam et al, (2013) PLoS ONE 8 (3): e 60298; and Van Tedeloo et al (2000) GENE THERAPY (16): 1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition (see, e.g., manuri et al (2010) Hum Gene Ther 21 (4): 427-437; shalma et al (2013) Molec Ther Nucl Acids, e74; and Huang et al (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, john Wiley & Sons, N.Y.), protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microprojectile bombardment (Johnston, nature, 346: 776-777 (1990)), and strontium phosphate DNA co-precipitation (Brash et al, mol. Cell biol., 7: 2031-2034 (1987)).
Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International patent application publication No. WO 2014055668 and U.S. Pat. No. 7,446,190.
In some embodiments, the recombinant nucleic acid is transferred into a T cell via a transposon. Transposons (transposable elements) are DNA moveable segments that can move from one locus to another within the genome. These elements move via a conserved "cut-and-paste" mechanism, the transposase catalyzes the excision of the transposon from its original location and facilitates its re-integration elsewhere in the genome. If the transposase is provided by another transposase gene, the element lacking the transposase can be mobilized. Thus, transposons can be used to incorporate foreign DNA into the host genome without the use of viral transduction systems. Examples of transposons suitable for use with mammalian cells (e.g., human primary leukocytes) include, but are not limited to, sleeping Beauty (sleep) and PiggyBacs.
Transposon-based transfection is a two-component system consisting of a transposase and a transposon. In some embodiments, the system comprises a transposon engineered to comprise foreign DNA (also referred to herein as cargo DNA), such as a gene encoding a recombinant receptor, flanked by inverted repeat/direct repeat (IR/DR) sequences recognized by a concomitant transposase. In some embodiments, the non-viral plasmid encodes a transposase under the control of a promoter. Transfection of the plasmid into the host cell results in transient expression of the transposase, whereby during the initial period following transfection, the transposase is expressed at a sufficient level to integrate the transposon into genomic DNA. In some embodiments, the transposase itself is not integrated into genomic DNA, and thus the expression of the transposase decreases over time. In some embodiments, the transposase expression is expressed by the host cell at a level sufficient for integration of the corresponding transposon for less than about 4 hours, less than about 8 hours, less than about 12 hours, less than about 24 hours, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 2 weeks, less than about 3 weeks, less than about 4 weeks, less than about weeks, or less than about 8 weeks. In some embodiments, cargo DNA introduced into the host genome is not subsequently removed from the host genome, at least because the host does not express an endogenous transposase capable of excising cargo DNA.
Sleeping Beauty (SB) is a synthetic member of the Tc/1-water arm superfamily of transposons, reconstructed from dormant elements found in the salmonidae fish genome. SB transposon-based transfection is a two-component system consisting of a transposase and a transposon containing an inverted repeat/direct repeat (IR/DR) sequence that results in precise integration into the TA dinucleotide. Transposons are designed with the expression cassette of interest flanked by IR/DR. SB transposases bind to specific binding sites located on sleeping beauty transposon IR. SB transposases mediate integration of transposons, which are mobile elements encoding a cargo sequence flanked on both sides by inverted terminal repeats having catalytic enzyme (SB) binding sites. Stable expression is obtained when SB inserts the gene sequence into the vertebrate chromosome at the TA target dinucleotide by a cut-and-paste mechanism. This system has been used to engineer a variety of vertebrate cell types, including human primary peripheral blood leukocytes. In some embodiments, the cell is contacted with, incubated with, and/or treated with a SB transposon that comprises a cargo gene (e.g., a gene encoding a recombinant receptor or CAR) flanked by SB IR sequences. In particular embodiments, the cells to be transfected are contacted with, incubated with, and/or treated with a plasmid comprising a SB transposon that comprises a cargo gene (e.g., a gene encoding a CAR) flanked by SB IR sequences. In certain embodiments, the plasmid further comprises a gene encoding an SB transposase flanked by no SB IR sequences.
PiggyBac (PB) is another transposon system that may be used to integrate cargo DNA into the genomic DNA of a host (e.g., a human). The PB transposase recognizes PB transposon-specific Inverted Terminal Repeats (ITRs) located at both ends of the transposon, and efficiently removes the content from the original site and integrates the content into TTAA chromosomal site. The PB transposon system enables mobilization of the gene of interest between two ITRs in the PB vector into the target genome. The PB system has been used to engineer a variety of vertebrate cell types, including human primary cells. In some embodiments, the cells to be transfected are contacted with, incubated with, and/or treated with a PB transposon that comprises a cargo gene (e.g., a gene encoding a CAR) flanked by PB IR sequences. In particular embodiments, the cells to be transfected are contacted with, incubated with, and/or treated with a plasmid comprising a PB transposon that comprises a cargo gene (e.g., a gene encoding a CAR) flanked by PB IR sequences. In certain embodiments, the plasmid further comprises a gene encoding an SB transposase flanked by no PB IR sequences.
In some embodiments, the various elements of transposon/transposase used in the subject methods, such as one or more SB or PB vectors, can be produced by standard methods of restriction enzyme cleavage, ligation, and molecular cloning. One approach for constructing the subject vector includes the following steps. First, a purified nucleic acid fragment containing the desired component nucleotide sequence as well as foreign sequences is cleaved with a restriction endonuclease from an initial source (e.g., a vector comprising a transposase gene). The fragments containing the desired nucleotide sequence are then separated from the different sized unwanted fragments using conventional separation methods (e.g., by agarose gel electrophoresis). The desired fragments are excised from the gel and ligated together in the appropriate configuration to produce a circular nucleic acid or plasmid containing the desired sequences (e.g., sequences corresponding to the various elements of the subject vector as described above). The thus constructed circular molecule is then amplified in a prokaryotic host (e.g., E.coli) if desired. The procedures involved in these steps, cleavage, plasmid construction, cell transformation and plasmid generation are well known to those skilled in the art, and the enzymes required for restriction and ligation are commercially available. (see, e.g., R.Wu, eds., methods in Enzymology, volume 68, ACADEMIC PRESS, new York (1979); T.Maniatis, E.F. Fritsch, and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982); catalog nos. 1982-83, NEW ENGLAND Biolabs, inc.; catalog nos. 1982-83, bethesda Research Laboratories, inc.; examples of how to construct vectors for use in the subject methods are provided in the experimental section below. Preparation of representative sleeping beauty transposon systems is also disclosed in WO 98/40510 and WO 99/25817).
In some embodiments, transduction with a transposon containing a cargo DNA sequence flanked by inverted repeat/direct repeat (IR/DR) sequences recognized by a transposase is performed with a plasmid comprising a transposase gene and a plasmid comprising a transposon. In certain embodiments, the cargo DNA sequence encodes a heterologous protein, such as a recombinant T cell receptor or CAR. In some embodiments, the plasmid comprises a transposase and a transposon. In some embodiments, the transposase is under the control of a ubiquitous promoter or any promoter suitable for driving expression of the transposase in a target cell. Ubiquitous promoters include, but are not limited to, EF1a, CMB, SV40, PGK1, ubc, human beta-actin, CAG, TRE, UAS, ac, caMKIIa, and U6. In some embodiments, the cargo DNA comprises a selection cassette that allows for selection of cells that stably integrate the cargo DNA into genomic DNA. Suitable selection cassettes include, but are not limited to, selection cassettes encoding kanamycin resistance genes, spectinomycin resistance genes, streptomycin resistance genes, ampicillin resistance genes, carbenicillin resistance genes, hygromycin resistance genes, bleomycin resistance genes, erythromycin resistance genes, and polymyxin B resistance genes.
In some embodiments, the component for transduction with a transposon (e.g., a plasmid comprising SB transposase and SB transposon) is introduced into the target cell. Any convenient protocol may be employed, wherein the protocol may introduce the system components into the target cells in vitro or in vivo, depending on the location of the target cells. For example, where the target cells are isolated cells, the system may be introduced directly into the cells under cell culture conditions that allow for viability of the target cells, e.g., by using standard transformation techniques. Such techniques include, but are not necessarily limited to, viral infection, transformation, conjugation, protoplast fusion, electroporation, particle gun techniques, calcium phosphate precipitation, direct microinjection, viral vector delivery, and the like. The choice of method generally depends on the type of cell to be transformed and the environment in which the transformation occurs (i.e., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al Short Protocols in Molecular Biology, 3rd edition, wiley & Sons, 1995.
In some embodiments, the SB transposon and the source of SB transposase are introduced into a target cell of a multicellular organism (e.g., a mammal or human) under conditions sufficient to excise the inverted repeat flanking nucleic acid from the vector carrying the transposon and subsequently integrate the excised nucleic acid into the genome of the target cell. Some embodiments further comprise the step of ensuring that the requisite transposase activity is present in the target cell along with the introduced transposon. Depending on the structure of the transposon vector itself, i.e. whether the vector comprises a region encoding a product having transposase activity, the method may further comprise introducing a second vector encoding the requisite transposase activity into the target cell.
In some embodiments, the amount of transposon-containing vector nucleic acid and the amount of vector nucleic acid encoding a transposase introduced into the cell are sufficient to provide the desired excision and insertion of the transposon nucleic acid into the target cell genome. Thus, the amount of vector nucleic acid introduced should provide a sufficient amount of transposase activity and a sufficient copy number of the nucleic acid desired to be inserted into the target cell. The amount of vector nucleic acid introduced into the target cell varies depending on the efficiency of the particular introduction protocol employed (e.g., the particular ex vivo administration protocol employed).
Once the vector DNA has been combined with the requisite transposase into the target cell, the region of the vector nucleic acid flanked by inverted repeats (i.e., the vector nucleic acid located between the inverted repeats recognized by the sleeping American transposase) is excised from the vector via the provided transposase and inserted into the genome of the target cell. Thus, after introducing the vector DNA into the target cell, a transposase-mediated excision of the exogenous nucleic acid carried by the vector is then performed and inserted into the genome of the target cell. In particular embodiments, the vector is integrated into the genome of 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 15%, or at least 20% of a cell transfected with the SB transposon and/or SB transposase. In some embodiments, the integration of the nucleic acid into the target cell genome is stable, i.e., the vector nucleic acid remains present in the target cell genome for more than a transient period of time, and a portion of the chromosomal genetic material is transferred to the progeny of the target cell.
In certain embodiments, transposons are used to integrate nucleic acids (i.e., polynucleotides) of various sizes into the genome of a target cell. In some embodiments, the DNA size inserted into the target cell genome using the subject methods ranges from about 0.1 kb to 200 kb, from about 0.5 kb to 100 kb, from about 1.0 kb to about 8.0 kb, from about 1.0 to about 200 kb, from about 1.0 to about 10 kb, from about 10 kb to about 50 kb, from about 50 kb to about 100 kb, or from about 100 kb to about 200 kb. In some embodiments, the DNA size inserted into the target cell genome using the subject methods ranges from about 1.0 kb to about 8.0 kb. In some embodiments, the DNA size inserted into the target cell genome using the subject methods ranges from about 1.0 kb to about 200 kb. In particular embodiments, the DNA size inserted into the target cell genome using the subject methods ranges from about 1.0 kb to about 8.0 kb.
D. Cell culture and/or expansion
In some embodiments, provided methods include one or more steps for incubating cells (e.g., incubating cells under conditions that promote proliferation and/or expansion). In some embodiments, following the step of genetic engineering (e.g., introduction of a recombinant polypeptide into a cell by transduction or transfection), the cell is incubated under conditions that promote proliferation and/or expansion. In certain embodiments, the cells are incubated under stimulating conditions and the cells are incubated after transduction or transfection with a recombinant polynucleotide (e.g., a polynucleotide encoding a recombinant receptor). In some embodiments, the incubating produces one or more incubation compositions enriched for T cells.
In certain embodiments, one or more compositions of enriched T cells (including stimulated and transduced T cells) (separate compositions of such cd4+ and cd8+ T cells) are incubated prior to formulating the cells, e.g., under conditions that promote proliferation and/or expansion. In some aspects, the methods of culturing (e.g., for promoting proliferation and/or amplification) include methods as provided herein in section I-F. In certain embodiments, after one or more compositions enriched for T cells have been engineered (e.g., transduced or transfected), the one or more compositions are incubated. In certain embodiments, the one or more compositions are engineered compositions. In certain embodiments, the one or more engineered compositions have been previously cryogenically frozen and stored, and thawed prior to incubation.
In certain embodiments, the one or more compositions of engineered T cells are or comprise two separate compositions of enriched T cells. In particular embodiments, two separate compositions of enriched T cells introduced with recombinant receptor (e.g., CAR) are separately incubated under conditions that promote proliferation and/or expansion of the cells, e.g., two separate compositions of enriched T cells selected, isolated, and/or enriched from the same biological sample. In some embodiments, the condition is a stimulation condition. In certain embodiments, the two separate compositions comprise compositions enriched for cd4+ T cells (e.g., engineered cd4+ T cells that have incorporated nucleic acid encoding a recombinant receptor and/or express a recombinant receptor). In particular embodiments, the two separate compositions include compositions enriched for cd8+ T cells (e.g., engineered cd8+ T cells introduced with nucleic acid encoding a recombinant receptor and/or expressing a recombinant receptor). In some embodiments, the two separate compositions enriched for cd4+ T cells and enriched for cd8+ T cells (e.g., engineered cd4+ T cells and engineered cd8+ T cells) are separately incubated, e.g., under conditions that promote proliferation and/or expansion. In some embodiments, a single composition enriched for T cells is incubated. In certain embodiments, the single composition is a composition enriched for cd4+ T cells. In some embodiments, the single composition is a composition enriched for cd4+ and cd8+ T cells that have been pooled from separate compositions prior to incubation.
In some embodiments, a composition of enriched cd4+ T cells (e.g., engineered cd4+ T cells), e.g., incubated under conditions that promote proliferation and/or expansion, 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 about 100% cd4+ T cells. In some embodiments, the composition comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or at least about 100% of cd4+ T cells that express the recombinant receptor and/or that have been transduced or transfected with a recombinant polynucleotide encoding the recombinant receptor. In certain embodiments, the cultured composition enriched in 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.
In some embodiments, a composition of enriched cd8+ T cells (e.g., engineered cd8+ T cells), e.g., incubated under conditions that promote proliferation and/or expansion, 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 about 100% cd8+ T cells. In particular embodiments, the composition comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or at or about 100% cd8+ T cells expressing a recombinant receptor and/or that have been transduced or transfected with a recombinant polynucleotide encoding a recombinant receptor. In certain embodiments, the composition of enriched cd8+ T cells incubated under stimulation 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% of cd4+ T cells, and/or is free or substantially free of cd4+ T cells.
In some embodiments, separate compositions enriched for cd4+ and cd8+ T cells (e.g., separate compositions of engineered cd4+ and engineered cd8+ T cells) are combined into a single composition and incubated, for example, under conditions that promote proliferation and/or expansion. In certain embodiments, the separately incubated compositions enriched for cd4+ and enriched for cd8+ T cells are combined into a single composition after incubation has been performed and/or completed. In certain embodiments, separate compositions enriched for cd4+ and cd8+ T cells (e.g., separate compositions of engineered cd4+ and engineered cd8+ T cells) are incubated separately, e.g., under conditions that promote proliferation and/or expansion.
In some embodiments, the cells (e.g., engineered cells) are incubated in a volume of medium that is, is about, or is at least 100 mL、200 mL、300 mL、400 mL、500 mL、600 mL、700 mL、800 mL、900 mL、1,000 mL、1,200 mL、1,400 mL、1,600 mL、1,800 mL、2,000 mL、2,200 mL、 or 2,400 mL. In some embodiments, cells are incubated at an initial volume that is later adjusted to a different volume. In certain embodiments, the volume is adjusted later during incubation. In certain embodiments, the volume increases from an initial volume during incubation. In certain embodiments, the volume is increased as the cells achieve density during incubation. In certain embodiments, the initial volume is at or about 500 mL.
In certain embodiments, the volume increases from the initial volume as the cells achieve a density or concentration during incubation. In particular embodiments, the volume is increased when the cells achieve a density and/or concentration of about, or at least 0.1 x 10 6 cells/ml, 0.2 x 10 6 cells/ml, 0.4 x 10 6 cells/ml, 0.6 x10 6 cells/ml, 0.8 x10 6 cells/ml, 1 x10 6 cells/ml, 1.2 x10 6 cells/ml, 1.4 x 10 6 cells/ml, 1.6x10 6 cells/ml, 1.8x10 6 cells/ml, 2.0x10 6 cells/ml, 2.5 x10 6 cells/ml, 3.0x10 6 cells/ml, 3.5x10 6 cells/ml, 4.0x10 6 cells/ml, 4.5 x10 6 cells/ml, 5.0 x10 6 cells/ml, 6 x10 6 cells/ml, 8 x10 6 cells/ml, or 10 x 10 6 cells/ml. In some embodiments, the volume increases from the initial volume when the cells achieve a density and/or concentration of at least, or about, 0.6 x 10 6 cells/ml. In some embodiments, the density and/or concentration is of living cells in culture. In particular embodiments, the volume is increased when the cells achieve a density and/or concentration of about, or at least 0.1 x 10 6 viable cells/ml, 0.2 x 10 6 viable cells/ml, 0.4 x 10 6 viable cells/ml, 0.6 x 10 6 viable cells/ml, 0.8 x 10 6 viable cells/ml, 1 x 10 6 viable cells/ml, 1.2 x 10 6 viable cells/ml, 1.4 x 10 6 viable cells/ml, 1.6x10 6 viable cells/ml, 1.8x10 6 viable cells/ml, 2.0x10 6 viable cells/ml, 2.5 x 10 6 viable cells/ml, 3.0x10 6 viable cells/ml, 3.5x10 6 viable cells/ml, 4.0x10 6 viable cells/ml, 4.5 x 10 6 living cells/ml, 5.0x10 6 living cells/ml, 6 x 10 6 living cells/ml, 8 x 10 6 living cells/ml, Or 10 x 10 6 living cells/ml. In some embodiments, when the living cells are achieved at a density and/or concentration of at least or about 0.6 x 10 6 living cells/ml, the volume is increased from the initial volume. In some embodiments, the density and/or concentration of cells or living cells may be determined or monitored during incubation, such as by using methods as described, including optical methods, including Digital Holographic Microscopy (DHM) or Differential Digital Holographic Microscopy (DDHM).
In some embodiments, the cells achieve a density and/or concentration and the volume is increased, increased by about or by at least 100 mL、200 mL、300 mL、400 mL、500 mL、600 mL、700 mL、800 mL、900 mL、1,000 mL、1,200 mL、1,400 mL、1,600 mL、1,800 mL、2,000 mL、2,200 mL or 2,400 mL. In some embodiments, the volume is increased by 500 mL. In particular embodiments, the volume is increased to a volume of, about, or at least 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1,000 mL, 1,200 mL, 1,400 mL, 1,600 mL, 1,800 mL, 2,000 mL, 2,200 mL, or 2,400 mL. In certain embodiments, the volume increases to a volume of 1,000 mL. In certain embodiments, the volume increases at a rate of at least, or about every 1,2, 3, 4,5, 6, 7, 8, 9, or 10 minutes of 5 mL, 10mL, 20 mL, 25 mL, 30mL, 40 mL, 50 mL, 60 mL, 70 mL, 75 mL, 80 mL, 90 mL, or 100 mL. In certain embodiments, the rate is at or about 50 mL per 8 minutes.
In some embodiments, the composition of enriched T cells (e.g., engineered T cells) is incubated under conditions that promote proliferation and/or expansion. In some embodiments, such conditions may be designed to induce proliferation, expansion, activation, and/or survival of cells in a population. In particular embodiments, the stimulation conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (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 designed to promote growth, division, and/or expansion of cells)).
In some embodiments, the incubation is performed under conditions that generally include a temperature suitable for the growth of primary immune cells (e.g., 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, the T cell enriched composition is incubated at a temperature of 25 to 38 ℃, such as 30 to 37 ℃, for example, at or about 37±2 ℃. In some embodiments, incubation is performed for a period of time until the culturing (e.g., incubating or expanding) produces the desired or threshold density, concentration, number, or dose of cells. In some embodiments, incubation is performed for a period of time until the culture (e.g., incubation or expansion) produces a desired or threshold density, concentration, number, or dose of living cells. In some embodiments, the incubation is greater than or greater than about or for about or 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or more. In some embodiments, the density, concentration, and/or number or dose of cells may be determined or monitored during incubation, such as by using methods as described, including optical methods, including Digital Holographic Microscopy (DHM) or Differential Digital Holographic Microscopy (DDHM).
In some embodiments, the stimulating agent is removed and/or isolated from the cells prior to incubation. In certain embodiments, the stimulating 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 some embodiments, the stimulating agent is a stimulating agent as described herein, for example, in section I-B-1. In certain embodiments, the stimulating agent is removed and/or isolated from the cells as described herein (e.g., in section I-B-2).
In certain embodiments, a composition of enriched T cells (e.g., engineered T cells) is incubated in the presence of one or more cytokines (e.g., a separate composition of engineered cd4+ T cells and engineered cd8+ T cells). In certain embodiments, the one or more cytokines are recombinant cytokines. In certain embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind and/or are capable of binding to receptors expressed by and/or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the 4- α -helix bundle family of cytokines include, but are not limited to, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include recombinant IL-2.
In certain embodiments, the composition enriched for cd4+ T cells (e.g., engineered cd4+ T cells) is incubated with recombinant IL-2. In some embodiments, incubating a composition enriched for cd4+ T cells (e.g., engineered cd4+ T cells) in the presence of recombinant IL-2 increases the probability or likelihood that 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 enriched in cd4+ T cells (e.g., engineered cd4+ T cells) in the presence of recombinant IL-2 increases the probability and/or likelihood that an output composition enriched in cd4+ T cells (e.g., engineered cd4+ T cells suitable for cell therapy) will be produced from the composition enriched in 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%, or at least 200% cd4+ as compared to an alternative and/or exemplary method of not incubating the composition enriched in the presence of recombinant IL-2.
In some embodiments, the cells (e.g., separate compositions of engineered cd4+ T cells and engineered cd8+ T cells) are incubated with cytokines, e.g., recombinant human cytokines, at a concentration of between 1 IU/ml and 2,000 IU/ml, between 10 IU/ml and 100 IU/ml, between 50 IU/ml and 500 IU/ml, between 100 IU/ml and 200 IU/ml, between 500 IU/ml and 1400 IU/ml, between 250 IU/ml and 500 IU/ml, or between 500 IU/ml and 2,500 IU/ml.
In some embodiments, the enriched T cell composition (e.g., the separate compositions of engineered CD4+ T cells and engineered CD8+ T cells) is incubated with recombinant IL-2, e.g., human recombinant IL-2, at a concentration of between 2 IU/ml and 500 IU/ml, between 10 IU/ml and 250 IU/ml, between 100 IU/ml and 500 IU/ml, or between 100 IU/ml and 400 IU/ml. In particular embodiments, the composition enriched in T cells is incubated with IL-2 at or about 50 IU/ml, 75 IU/ml, 100 IU/ml, 125 IU/ml, 150 IU/ml, 175 IU/ml, 200 IU/ml, 225 IU/ml, 250 IU/ml, 300 IU/ml, or 400 IU/ml. In some embodiments, the enriched T cell composition is incubated with recombinant IL-2 at a concentration of 200 IU/ml. In some embodiments, the T cell enriched composition is a cd4+ T cell enriched composition, such as an engineered cd4+ T cell composition. In particular embodiments, the T cell enriched composition is a cd8+ T cell enriched composition, such as an engineered cd8+ T cell composition.
In some embodiments, a composition enriched for T cells (e.g., a separate composition of engineered CD4+ T cells and CD8+ T cells) is incubated with IL-7, e.g., human recombinant IL-7, at a concentration of between 10 IU and 5,000 IU/ml, between 500 IU and 2,000 IU/ml, between 600 IU and 1,500 IU/ml, between 500 IU and 2,500 IU/ml, between 750 IU and 1,500 IU/ml, or between 1,000 and 2,000 IU/ml. In particular embodiments, the T cell enriched composition is incubated with IL-7 at or about 100 IU/ml、200 IU/ml、300 IU/ml、400 IU/ml、500 IU/ml、600 IU/ml、700 IU/ml、800 IU/ml、900 IU/ml、1,000 IU/ml、1,200 IU/ml、1,400 IU/ml or 1,600 IU/ml. In some embodiments, the cells are incubated in the presence of recombinant IL-7 at a concentration of at or about 1,200 IU/ml. In some embodiments, the composition enriched in T cells is a composition enriched in cd4+ T cells (e.g., engineered cd4+ T cells).
In some embodiments, a composition enriched for T cells (e.g., a separate composition of engineered CD4+ T cells and CD8+ T cells) is incubated with IL-15, e.g., human recombinant IL-15, at a concentration of between 0.1 IU/ml and 200 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 T cell enriched composition is incubated with IL-15 at 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、50 IU/ml、100 IU/ml or 200 IU/ml. In a particular embodiment, the T cell enriched composition is incubated with recombinant IL-15 at a concentration of 20 IU/ml. In some embodiments, the composition enriched in T cells is a composition enriched in cd4+ T cells (e.g., engineered cd4+ T cells). In particular embodiments, the T cell enriched composition is a cd8+ T cell enriched (e.g., engineered cd8+ T cell) composition.
In certain embodiments, the composition enriched for cd8+ T cells (e.g., engineered cd8+ T cells) is incubated in the presence of IL-2 and/or IL-15 (e.g., in an amount as described). In certain embodiments, the composition enriched for CD4+ T cells (e.g., engineered CD4+ T cells) is incubated in the presence of IL-2, IL-7, and/or IL-15 (e.g., in the amounts described). In some embodiments, IL-2, IL-7 and/or IL-15 is recombinant. In certain embodiments, IL-2, IL-7 and/or IL-15 is human. In particular embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and/or IL-15.
In certain embodiments, the incubation is performed in a closed system. In certain embodiments, the incubation is performed in a closed system under sterile conditions. In certain embodiments, incubation is performed in the same closed system as one or more steps of the provided system. In some embodiments, the T cell enriched composition is removed from the closed system and placed in and/or connected to a bioreactor for cultivation. Examples of suitable bioreactors for incubation include, but are not limited to GE Xuri W, GE Xuri W5, sartorius BioSTAT RM 20|50, finesse SmartRocker bioreactor systems, and Pall XRS bioreactor systems. In some embodiments, the bioreactor is used to perfuse and/or mix cells during at least a portion of the incubation step.
In some embodiments, cells cultured in a closed, connected bioreactor and/or under control of the bioreactor undergo faster expansion during culturing than cells cultured without the bioreactor (e.g., cells cultured under static conditions (e.g., without mixing, rocking, movement, and/or perfusion). In some embodiments, cells cultured in a closed, connected bioreactor and/or under control of the bioreactor reach or achieve a threshold expansion, cell count, and/or density within 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 60 hours, 48 hours, 36 hours, 24 hours, or 12 hours. In some embodiments, cells grown in a closed, linked bioreactor and/or under control of the bioreactor reach or achieve a threshold expansion, cell count and/or density of 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, as compared to cells grown in an exemplary and/or alternative process that do not grow cells in a closed, linked bioreactor and/or under control of the bioreactor.
In some embodiments, the mixing is or includes rocking and/or motion. In some cases, the bioreactor may be subject to motion or rocking, which in some aspects may increase oxygen transfer. Moving the bioreactor may include, but is not limited to, rotation along a horizontal axis, rotation along a vertical axis, rocking movement along a horizontal axis of tilt (tilted or incorporated) of the bioreactor, or any combination thereof. In some embodiments, at least a portion of the incubation is performed with rocking. The rocking speed and rocking angle can be adjusted to achieve the desired agitation. In some embodiments, the rocking angle is 20o, 19o, 18o, 17o, 16o, 15o, 14o, 13o, 12o, 11o, 10o, 9o, 8o, 7o, 6o, 5o, 4o, 3o, 2o, or 1o. In certain embodiments, the rocking angle is between 6o-16 o. In other embodiments, the rocking angle is between 7o and 16 o. In other embodiments, the rocking angle is between 8o and 12 o. In some embodiments, the rocking rate is 1、2、3、4、5、6、7、8、9、10、11、1 12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40 rpm. in some embodiments, the rocking rate is between 4 rpm and 12 rpm, for example between 4 rpm and 6 rpm, inclusive.
In some embodiments, the bioreactor is maintained at or near 37 ℃ and at or near 5% CO 2 level and has a steady air flow rate of about or at least 0.01L/min, 0.05L/min, 0.1L/min, 0.2L/min, 0.3L/min, 0.4L/min, 0.5L/min, 1.0L/min, 1.5L/min, or 2.0L/min or greater than 2.0L/min. In certain embodiments, at least a portion of the incubation is performed under perfusion conditions, such as at a rate of 290 ml/day, 580 ml/day, and/or 1160 ml/day (e.g., depending on the timing associated with initiation of incubation and/or the density of the incubated cells). In some embodiments, at least a portion of cell culture expansion is performed with a rocking motion, e.g., at an angle between 5o and 10o (e.g., 6 o), at a constant rocking speed, e.g., a speed between 5 RPM and 15 RPM (e.g., 6 RPM or 10 RPM).
In some embodiments, the at least a portion of the incubating step is performed under constant perfusion (e.g., slow steady rate perfusion). In some embodiments, the perfusion is or includes an outflow of liquid (e.g., spent media) and an inflow of fresh media. In certain embodiments, the perfusion is replaced with fresh medium with spent medium. In some embodiments, at least a portion of the incubation is performed under perfusion having a steady rate of about or at least 100 ml/day, 200 ml/day, 250 ml/day, 275 ml/day, 290 ml/day, 300 ml/day, 350 ml/day, 400 ml/day, 450 ml/day, 500 ml/day, 550 ml/day, 575 ml/day, 580 ml/day, 600 ml/day, 650 ml/day, 700 ml/day, 750 ml/day, 800 ml/day, 850 ml/day, 900 ml/day, 950 ml/day, 1000 ml/day, 1100 ml/day, 1160 ml/day, 1200 ml/day, 1400 ml/day, 1600 ml/day, 1800 ml/day, 2000 ml/day, ml/day, or 2400 ml/day.
In particular embodiments, incubation is initiated without perfusion and perfusion is initiated after a set and/or predetermined amount of time (e.g., at or about or at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours or more than 72 hours after initiation of incubation). In certain embodiments, perfusion begins when the density or concentration of cells reaches a set or predetermined density or concentration. In some embodiments, perfusion begins when the cultured cells reach a density or concentration of about, or at least 0.1 x 10 6 cells/ml, 0.2 x 10 6 cells/ml, 0.4 x 10 6 cells/ml, 0.6 x10 6 cells/ml, 0.8 x10 6 cells/ml, 1 x10 6 cells/ml, 1.2 x10 6 cells/ml, 1.4 x 10 6 cells/ml, 1.6x10 6 cells/ml, 1.8x10 6 cells/ml, 2.0x10 6 cells/ml, 2.5 x 10 6 cells/ml, 3.0x10 6 cells/ml, 3.5x10 6 cells/ml, 4.0x10 6 cells/ml, 4.5 x 10 6 cells/ml, 5.0 x 10 6 cells/ml, 6 x 10 6 cells/ml, 8 x 10 6 cells/ml or 10 x 10 6 cells/ml. In certain embodiments, perfusion begins when the density or concentration of living cells reaches a set or predetermined density or concentration. In some embodiments, perfusion begins when the cultured living cells reach a density or concentration of about, or at least 0.1 x 10 6 living cells/ml, 0.2 x 10 6 living cells/ml, 0.4 x 10 6 living cells/ml, 0.6 x 10 6 viable cells/ml, 0.8 x 10 6 viable cells/ml, 1 x 10 6 viable cells/ml, 1.2 x 10 6 viable cells/ml, 1.4 x 10 6 viable cells/ml, 1.6x10 6 viable cells/ml, 1.8x10 6 viable cells/ml, 2.0x10 6 viable cells/ml, 2.5 x 10 6 viable cells/ml, 3.0x10 6 viable cells/ml, 3.5x10 6 viable cells/ml, 4.0x10 6 viable cells/ml, 4.5 x 10 6 living cells/ml, 5.0 x 10 6 living cells/ml, 6 x 10 6 living cells/ml, 8 x 10 6 living cells/ml or 10 x 10 6 living cells/ml.
In certain embodiments, perfusion is performed at different rates during incubation. For example, in some embodiments, the rate of perfusion depends on the density and/or concentration of the cultured cells. In certain embodiments, the rate of perfusion is increased when the cells reach a set or predetermined density or concentration. The perfusion rate may be changed during incubation, for example, from a steady perfusion rate to an increased steady perfusion rate, once, twice, three times, four times, five times, more than ten times, more than 15 times, more than 20 times, more than 25 times, more than 50 times, or more than 100 times. In some embodiments, the steady perfusion rate is increased when the cells reach a set or predetermined cell density or concentration of about, or at least 0.6X10 6 cells/ml, 0.8X10 6 cells/ml, 1X 10 6 cells/ml, 1.2 x 10 6 cells/ml, 1.4 x 10 6 cells/ml, 1.6 x 10 6 cells/ml, 1.8 x 10 6 cells/ml, 2.0 x 10 6 cells/ml, 2.5 x 10 6 cells/ml, 3.0 x 10 6 cells/ml, 3.5 x 10 6 cells/ml, 4.0 x 10 6 cells/ml, 4.5 x 10 6 cells/ml, 5.0 x 10 6 cells/ml, 6 x 10 6 cells/ml, 8 x 10 6 cells/ml or 10 x 10 6 cells/ml. In some embodiments, the steady perfusion rate is increased when the cells reach a set or predetermined viable cell density or concentration of about, or at least 0.6X10 6 viable cells/ml, 0.8X10 6 viable cells/ml, 1X 10 6 viable cells/ml, 1.2 x 10 6 viable cells/ml, 1.4 x 10 6 viable cells/ml, 1.6 x 10 6 viable cells/ml, 1.8 x 10 6 viable cells/ml, 2.0 x 10 6 living cells/ml, 2.5 x 10 6 living cells/ml, 3.0 x 10 6 living cells/ml, 3.5 x 10 6 living cells/ml, 4.0 x 10 6 viable cells/ml, 4.5 x 10 6 viable cells/ml, 5.0 x 10 6 viable cells/ml, 6 x 10 6 viable cells/ml, 8X 10 6 living cells/ml or 10X 10 6 living cells/ml. In some embodiments, the density and/or concentration of cells or living cells during incubation (e.g., under perfusion) may be determined or monitored, such as by using methods as described, including optical methods, including Digital Holographic Microscopy (DHM) or Differential Digital Holographic Microscopy (DDHM).
In some embodiments, incubation is initiated without perfusion and perfusion is initiated when the density or concentration of cells reaches a set or predetermined density or concentration. In some embodiments, perfusion begins at a rate of about or at least 100 ml/day, 200 ml/day, 250 ml/day, 275 ml/day, 290 ml/day, 300 ml/day, 350 ml/day, 400 ml/day, 450 ml/day, 500 ml/day, 550 ml/day, 575 ml/day, 580 ml/day, 600 ml/day, 650 ml/day, 700 ml/day, 750 ml/day, and, 800 ml/day, 850 ml/day, 900 ml/day, 950 ml/day, 1000 ml/day, 1100 ml/day, 1160 ml/day, 1200 ml/day, 1400 ml/day, 1600 ml/day, 1800 ml/day, 2000 ml/day, 2200 ml/day, or 2400 ml/day. In some embodiments, perfusion begins when the cultured cells or cultured living cells reach a density or concentration of about, or at least 0.1 x 10 6 cells/ml, 0.2 x 10 6 cells/ml, 0.4 x 10 6 cells/ml, 0.6 x10 6 cells/ml, 0.8 x10 6 cells/ml, 1 x10 6 cells/ml, 1.2 x10 6 cells/ml, 1.4 x 10 6 cells/ml, 1.6x10 6 cells/ml, 1.8x10 6 cells/ml, 2.0x10 6 cells/ml, 2.5 x 10 6 cells/ml, 3.0x10 6 cells/ml, 3.5x10 6 cells/ml, 4.0x10 6 cells/ml, 4.5 x 10 6 cells/ml, 5.0 x 10 6 cells/ml, 6 x 10 6 cells/ml, 8 x 10 6 cells/ml or 10 x 10 6 cells/ml.
In certain embodiments, at least a portion of the incubation is performed at a rate of perfusion and when the density or concentration of cells reaches a set or predetermined density or concentration, the rate of perfusion is increased to a rate of about or at least 100 ml/day, 200 ml/day, 250 ml/day, 275 ml/day, 290 ml/day, 300 ml/day, 350 ml/day, 400 ml/day, 450 ml/day, 500 ml/day, 550 ml/day, 575 ml/day, 580 ml/day, 600 ml/day, 650 ml/day, 700 ml/day, 750 ml/day, 800 ml/day, 850 ml/day, 900 ml/day, 950 ml/day, 1000 ml/day, 1100 ml/day, 1160 ml/day, 1200 ml/day, 1400 ml/day, 1600 ml/day, 1800 ml/day, 2000 ml/day, 2200 ml/day, or 2400 ml/day. In some embodiments, perfusion begins when the cultured cells or cultured living cells reach a density or concentration of about, or at least 0.1 x 10 6 cells/ml, 0.2 x 10 6 cells/ml, 0.4 x 10 6 cells/ml, 0.6 x10 6 cells/ml, 0.8 x10 6 cells/ml, 1 x10 6 cells/ml, 1.2 x10 6 cells/ml, 1.4 x 10 6 cells/ml, 1.6x10 6 cells/ml, 1.8x10 6 cells/ml, 2.0x10 6 cells/ml, 2.5 x 10 6 cells/ml, 3.0x10 6 cells/ml, 3.5x10 6 cells/ml, 4.0x10 6 cells/ml, 4.5 x 10 6 cells/ml, 5.0 x 10 6 cells/ml, 6 x 10 6 cells/ml, 8 x 10 6 cells/ml or 10 x 10 6 cells/ml. In some embodiments, the perfusion is performed when the cells are incubated at a volume of about or at least 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL. In some embodiments, the volume is 1000 mL.
In certain embodiments, incubation is initiated without perfusion or at a rate, and when the density or concentration of cells reaches a concentration of, or is about, or is at least 0.61 x 10 6 cells/ml, the perfusion rate is increased to, or is about, or is at least 290 ml/day. In certain embodiments, when the cells are incubated at a volume of, about, or at least 1000 mL, the cells are perfused at a rate of, about, or at least 290 ml/day when the density or concentration of the cells reaches a concentration of, about, or at least 0.61 x 10 6 cells/ml. In some embodiments, the perfusion rate is increased to, about, or at least 580 ml/day when the density or concentration of cells reaches a concentration of, about, or at least 0.81 x 10 6 cells/ml. In certain embodiments, when the density or concentration of cells reaches a concentration of, about, or at least 1.01 x 10 6 cells/ml, the perfusion rate is increased to, about, or at least 1160 ml/day. In some embodiments, when the density or concentration of cells reaches a concentration of, about, or at least 1.2 x 10 6 cells/ml, the perfusion rate is increased to, about, or at least 1160 ml/day.
In aspects of the provided embodiments, the perfusion rate is determined by assessing the density and/or concentration of cells or assessing the density and/or concentration of living cells during incubation, including timing at which perfusion is initiated or increased as described herein and above. In some embodiments, the density and/or concentration of cells may be determined using methods as described, including optical methods, including Digital Holographic Microscopy (DHM) or Differential Digital Holographic Microscopy (DDHM).
In some embodiments, the composition of enriched cells, such as engineered T cells (e.g., engineered cd4+ T cells or engineered cd8+ T cells), is incubated in the presence of a surfactant. In particular embodiments, incubating the cells of the composition reduces the amount of shear stress that may occur during incubation, for example, due to mixing, rocking, movement, and/or perfusion. In particular embodiments, the composition of enriched T cells (e.g., engineered T cells, e.g., engineered cd4+ T cells or engineered cd8+ T cells) is incubated with a surfactant, 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 are viable, e.g., are viable and/or do not undergo necrosis, apoptosis, or apoptosis, for a period of 1 day, 2 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 incubation. In particular embodiments, the composition of enriched T cells (e.g., engineered T cells, e.g., engineered cd4+ T cells or engineered cd8+ T cells) is incubated in the presence of a surfactant, 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 do not undergo cell death, e.g., apoptosis, and/or necrosis, as a result of shear or shear induced stress.
In particular embodiments, the composition of enriched T cells (e.g., engineered T cells, e.g., engineered CD4+ T cells or engineered CD8+ T cells) is incubated in the presence of an amount of surfactant between 0.1. Mu.l/ml and 10.0. Mu.l/ml, between 0.2. Mu.l/ml and 2.5. Mu.l/ml, between 0.5. Mu.l/ml and 5. Mu.l/ml, between 1. Mu.l/ml and 3. Mu.l/ml, or between 2. Mu.l/ml and 4. Mu.l/ml. In some embodiments, the composition of enriched T cells (e.g., engineered T cells, e.g., engineered CD4+ T cells or engineered CD8+ T cells) is incubated in the presence of an amount of surfactant of about, or at least 0.1 µl/ml、0.2 µl/ml、0.4 µl/ml、0.6 µl/ml、0.8 µl/ml、1 µl/ml、1.5 µl/ml、2 µl/ml、2.5 µl/ml、5 µl/ml、10 µl/ml、25 µl/ml or 50 μl/ml. In certain embodiments, the enriched T cell composition is incubated in the presence of a surfactant at or about 2 μl/ml.
In some embodiments, the surfactant is or includes an agent that reduces the surface tension of a liquid and/or solid. For example, surfactants include fatty alcohols (e.g., sterols), polyoxyethylene glycol octylphenol ether (e.g., triton X-100), or polyoxyethylene glycol sorbitan alkyl esters (e.g., polysorbate 20, 40, 60). In certain embodiments, the surfactant is selected from polysorbate 80 (PS 80), polysorbate 20 (PS 20), poloxamer 188 (P188). In exemplary embodiments, the concentration of surfactant in the chemically-defined feed medium is about 0.0025% to about 0.25% (v/v) PS80, about 0.0025% to about 0.25% (v/v) PS20, or about 0.1% to about 5.0% (w/v) P188.
In some embodiments, the surfactant is or includes an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, or a nonionic surfactant added thereto. Suitable anionic surfactants include, but are not limited to, alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, alkyl polyoxyethylene sulfates, sodium alginate, sodium dioctyl sulfosuccinate, phosphatidylglycerol, phosphatidylinosine, phosphatidylinositol, bis-phosphatidylglycerol, phosphatidylserine, phosphatidic acid and salts thereof, sodium carboxymethyl cellulose, cholic acid and other bile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid) and salts thereof (e.g., sodium deoxycholate).
In some embodiments, suitable nonionic surfactants include glycerides, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters (polysorbates), polyoxyethylene fatty acid esters, sorbitan esters, glyceryl monostearate, polyethylene glycols, polypropylene glycols, cetyl alcohol, cetylstearyl alcohol, stearyl alcohol, arylalkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxamers), poloxamers, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, amorphous cellulose, polysaccharides (including starch and starch derivatives, such as hydroxyethyl starch (HES)), polyvinyl alcohol, and polyvinylpyrrolidone. In certain embodiments, the nonionic surfactant is a polyoxyethylene and polyoxypropylene copolymer, and preferably is a block copolymer of propylene glycol and ethylene glycol. Such polymers are sold under the trade name poloxamer, sometimes also referred to as PLURONIC cube F68 or Kolliphor cube P188. Polyoxyethylene fatty acid esters include those having a short alkyl chain. An example of such a surfactant is SOLUTOL HS 15, polyethylene-660-hydroxystearate.
In some embodiments, suitable cationic surfactants may include, but are not limited to, natural phospholipids, synthetic phospholipids, quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitosan, lauryl dimethylbenzyl ammonium chloride, acyl carnitine hydrochloride, dimethyl Dioctadecyl Ammonium Bromide (DDAB), dioleoyl trimethylammonium propane (DOTAP), ditetradecyl trimethylammonium propane (dmtpap), dimethylaminoethane carbamoyl cholesterol (DC-Chol), 1, 2-diacylglycerol-3- (O-alkyl) phosphorylcholine, O-alkylphospholipid choline, alkylpyridinium halides, or long chain alkylamines (e.g., n-octylamine and oleamide).
Zwitterionic surfactants are electrically neutral, but have localized positive and negative charges within the same molecule. Suitable zwitterionic surfactants include, but are not limited to, zwitterionic phospholipids. Suitable phospholipids include phosphatidylcholine, phosphatidylethanolamine, diacyl-glycerol-phosphoethanolamine (e.g., ditetradecyl-glycerol-phosphoethanolamine (DMPE), dipalmitoyl-glycerol-phosphoethanolamine (DPPE), distearyl-glycerol-phosphoethanolamine (DSPE), and dioleoyl-glycerol-phosphoethanolamine (DOPE)). Mixtures of phospholipids (including anionic phospholipids and zwitterionic phospholipids) may be used in the present invention. Such mixtures include, but are not limited to, lysophospholipids, lecithins, or soybean phospholipids, or any combination thereof. Phospholipids (whether anionic, zwitterionic or mixtures of phospholipids) may be salted or desalted, hydrogenated or partially hydrogenated or be natural semisynthetic or synthetic.
In certain embodiments, the surfactant is a poloxamer, for example, poloxamer 188. In some embodiments, the T cell enriched composition is incubated in the presence of poloxamer in an amount between 0.1 and 10.0, between 0.2 and 2.5, between 0.5 and 5, between 1 and 3, or between 2 and 4. Mu.l/ml. In some embodiments, the T cell enriched composition is incubated in the presence of an amount of surfactant of about, or at least 0.1 µl/ml、0.2 µl/ml、0.4 µl/ml、0.6 µl/ml、0.8 µl/ml、1 µl/ml、1.5 µl/ml、2 µl/ml、2.5 µl/ml、5 µl/ml、10 µl/ml、25 µl/ml or 50 μl/ml. In certain embodiments, the enriched T cell composition is incubated in the presence of poloxamer at or about 2 μl/ml.
In certain embodiments, the incubation is ended when the cells achieve a threshold amount, concentration, and/or expansion, such as by harvesting the cells. In particular embodiments, incubation ends when, for example, the cells achieve or achieve about or at least 1.5-fold amplification, 2-fold amplification, 2.5-fold amplification, 3-fold amplification, 3.5-fold amplification, 4-fold amplification, 4.5-fold amplification, 5-fold amplification, 6-fold amplification, 7-fold amplification, 8-fold amplification, 9-fold amplification, 10-fold amplification, or greater than 10-fold amplification with respect to and/or relative to the amount of cell density at the beginning or start of incubation. In some embodiments, the threshold expansion is, for example, about and/or relative to 4-fold expansion of the amount or density of cells at the beginning or initial incubation.
In some embodiments, incubation is ended when the cells achieve a threshold total amount of cells, e.g., a threshold cell count, such as by harvesting the cells. In some embodiments, incubation is terminated when the cells achieve a threshold Total Nucleated Cell (TNC) count. In some embodiments, incubation is ended when the cells achieve a threshold living cell amount (e.g., a threshold living cell count). In some embodiments, the threshold cell count is or is about or at least 50 x10 6 cells, 100 x10 6 cells, 200 x10 6 cells, 300 x10 6 cells, 400 x10 6 cells, 600 x10 6 cells, 800 x10 6 cells, 1000 x10 6 cells, 1200 x10 6 cells, 1400 x10 6 cells, 1600 x10 6 cells, 1800 x10 6 cells, 2000 x10 6 cells, 2500 x10 6 cells, 3000 x10 6 cells, 4000 x10 6 cells, 5000 x10 6 cells, 10,000 x10 6 cells, 12,000 x10 6 cells, 15,000 x10 6 cells, or 20,000 x10 6 cells, or any of the preceding living cell thresholds. In certain embodiments, incubation is terminated when the cells achieve a threshold cell count. In some embodiments, incubation is completed at, about or within 6 hours, 12 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more after achieving the threshold cell count. In certain embodiments, incubation is terminated 1 day or about 1 day after achieving a threshold cell count. In certain embodiments, the threshold density is, is about, or at least 0.1 x10 6 cells/ml, 0.5 x10 6 cells/ml, 1 x10 6 cells/ml, 1.2 x10 6 cells/ml, 1.5 x10 6 cells/ml, 1.6x10 6 cells/ml, 1.8x10 6 cells/ml, 2.0x10 6 cells/ml, 2.5 x10 6 cells/ml, 3.0x10 6 cells/ml, 3.5x10 6 cells/ml, 4.0x10 6 cells/ml, 4.5 x10 6 cells/ml, 5.0x10 6 cells/ml, 6 x10 6 cells/ml, 8 x10 6 cells/ml, Or 10 x10 6 cells/ml, or any of the preceding viable cell thresholds. In certain embodiments, incubation is terminated when the cells achieve a threshold density. In some embodiments, the incubation is completed at, about or within 6 hours, 12 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more after the threshold density is achieved. In certain embodiments, incubation is complete 1 day or about 1 day after achieving the threshold density.
In some embodiments, the incubating step is performed for an amount of time required to achieve a threshold amount, density, and/or expansion for the cells. In some embodiments, the incubation is performed for an amount of time that is or is about or less than 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In particular embodiments, the average amount of time required to achieve a threshold density for cells of a plurality of separate compositions of isolated, enriched, and/or selected enriched T cells from different biological samples is about or less than 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the average amount of time required to achieve a threshold density for cells of a plurality of separate compositions of isolated, enriched, and/or selected enriched T cells from different biological samples is about or less than 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, or 4 weeks.
In certain embodiments, the incubation step is performed for a minimum of 4, 5, 6, 7, 8, 9, or 10 days, and/or until the cells achieve or are at a threshold cell count (or number) of 12 hours, 24 hours, 36 hours, 1 day, 2, or 3 days after a threshold live cell count (or number) of about 1000 x 10 6 cells, 1200 x 10 6 cells, 1400 x10 6 cells, 1600 x10 6 cells, 1800 x10 6 cells, 2000 x10 6 cells, 2500 x10 6 cells, 3000 x10 6 cells, 4000 x10 6 cells or 5000 x10 6 cells. In some embodiments, the culturing step is performed until the cells achieve a threshold cell count of or about 1200 x 10 6 cells and 1 day after culturing for a minimum of 10 days, and/or until the cells achieve a threshold cell count of or about 5000 x 10 6 cells for 1 day. In some embodiments, the culturing step is performed until the cells achieve a threshold cell count of at or about 1200 x 10 6 cells and 1 day after a minimum of 9 days of culture, and/or until the cells achieve a threshold cell count of at or about 5000 x 10 6 cells. In some embodiments, the culturing step is performed until the cells achieve a threshold cell count of or about 1000 x10 6 cells and 1 day after a minimum of 8 days of culture, and/or until the cells achieve a threshold cell count of or about 4000 x10 6 cells. In certain embodiments, the incubation is an expansion step and is performed for a minimum of 4, 5, 6, 7, 8, 9, or 10 days, and/or until the cells achieve or are after a threshold cell count (or number) of 12 hours, 24 hours, 36 hours, 1 day, 2 days, or 3 days of about 1000 x10 6 cells, 1200 x10 6 cells, 1400 x10 6 cells, 1600 x10 6 cells, 1800 x10 6 cells, 2000 x10 6 cells, 2500 x10 6 cells, 3000 x10 6 cells, 4000 x10 6 cells or 5000 x10 6 cells. In some embodiments, the expansion step is performed until the cells achieve a threshold cell count of or about 1200 x 10 6 cells and 1 day after a minimum of 10 days of expansion, and/or until the cells achieve a threshold cell count of or about 5000 x 10 6 cells. In some embodiments, the expansion step is performed until the cells achieve a threshold cell count of or about 1200 x 10 6 cells and 1 day after a minimum of 9 days of expansion, and/or until the cells achieve a threshold cell count of or about 5000 x 10 6 cells. In some embodiments, the expansion step is performed until the cells achieve a threshold cell count of or about 1000 x 10 6 cells and 1 day after a minimum of 8 days of expansion, and/or until the cells achieve a threshold cell count of or about 4000 x 10 6 cells. In some embodiments, the expansion step is performed until the cells achieve a threshold cell count of or about 1400 x 10 6 cells and 1 day after a minimum of 5 days of expansion, and/or until the cells achieve a threshold cell count of or about 4000 x 10 6 cells.
In some embodiments, incubating is performed for at least a minimum amount of time. In some embodiments, the incubation is performed for at least 14 days, at least 12 days, at least 10 days, at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, at least 36 hours, at least 24 hours, at least 12 hours, or at least 6 hours, even if the threshold is achieved before the minimum amount of time. In some embodiments, in some cases, increasing the minimum amount of time to incubate may reduce activation and/or reduce the level of one or more activation markers in the incubated cells, the formulated cells, and/or the cells outputting the composition. In some embodiments, the minimum incubation time is counted from the determined point (e.g., the selection step; the thawing step; and/or the activation step) of the exemplary process to the day the cells were harvested.
In aspects of the provided embodiments, the density and/or concentration of cells or living cells is monitored during or performed during incubation, such as until a threshold amount, density and/or expansion as described is achieved. In some embodiments, such methods include those as described, including optical methods, including Digital Holographic Microscopy (DHM) or Differential Digital Holographic Microscopy (DDHM).
In certain embodiments, the cultured cells are export cells. In some embodiments, the composition of enriched T cells (e.g., engineered T cells) that have been incubated is an output composition of enriched T cells. In certain embodiments, the cd4+ T cells and/or cd8+ T cells that have been incubated are export cd4+ and/or cd8+ T cells. In certain embodiments, the composition enriched for cd4+ T cells (e.g., engineered cd4+ T cells) that has been incubated is an output composition enriched for cd4+ T cells. In some embodiments, the composition enriched for cd8+ T cells (e.g., engineered cd8+ T cells) that has been incubated is an output composition enriched for cd8+ T cells.
In some embodiments, the cells are incubated in the presence of one or more cytokines under conditions that promote proliferation and/or expansion. In certain embodiments, at least a portion of the incubation is performed with constant mixing and/or perfusion (e.g., mixing or perfusion controlled by a bioreactor). In some embodiments, the cells are incubated in the presence of one or more cytokines and with a surfactant (e.g., a poloxamer such as poloxamer 188) to reduce shear and/or shear stress from constant mixing and/or infusion. In some embodiments, the composition enriched for cd4+ T cells (e.g., engineered cd4+ T cells) is incubated in the presence of recombinant IL-2, IL-7, IL-15, and poloxamer, wherein at least a portion of the incubation is performed with constant mixing and/or perfusion. In certain embodiments, a composition enriched for cd8+ T cells (e.g., engineered cd8+ T cells) is incubated in the presence of recombinant IL-2, IL-15, and poloxamer, wherein at least a portion of the incubation is performed with constant mixing and/or perfusion. In some embodiments, the incubation is performed until the cells reach a threshold expansion of at least 4 times, for example, compared to when the incubation was initiated.
E. compositions of recombinant receptor engineered cells
Also provided are compositions containing T cells engineered with recombinant receptors, such as CARs. In some embodiments, the compositions are made by the methods for making, generating or producing cell therapies and/or engineered cells as described, and may include formulating genetically engineered cells produced by the provided processing steps to produce therapeutic cell compositions containing cells expressing recombinant receptors. In some embodiments, provided methods relating to the formulation of cells include treating transduced cells in a closed system, such as cells transduced and/or expanded using the treatment steps described above.
In some cases, treating the cells in one or more steps (e.g., performed in a centrifugal chamber and/or closed system) for making, generating, or producing cell therapies and/or engineering the cells may include formulating the cells before or after culturing (e.g., incubating and expanding) and/or one or more other treatment steps as described, e.g., formulating genetically engineered cells produced by the provided transduction treatment steps. In some cases, the cells may be formulated in an amount for dosage administration (e.g., for single unit dosage administration or multi-dosage administration). In some embodiments, the unit dose and/or dose administration is determined using activation of cells of the therapeutic composition determined according to the methods provided in section I above. In some embodiments, the degree of activation of cells of the therapeutic composition is assessed according to the methods provided in section I for the purpose of determining a unit dose and/or dose administration. In some embodiments, provided methods relating to the formulation of cells include treating transduced cells in a closed system, such as cells transduced and/or expanded using the treatment steps described above.
In certain embodiments, one or more compositions of enriched T cells (e.g., engineered and cultured T cells, e.g., export T cells), therapeutic cell compositions, are formulated. In particular embodiments, one or more compositions enriched for T cells (e.g., engineered and cultured T cells, e.g., export T cells), therapeutic cell compositions, are formulated after the one or more compositions have been engineered and/or cultured. In certain embodiments, the one or more compositions are input compositions. In some embodiments, one or more of the input compositions have been previously frozen and stored at low temperature and thawed prior to incubation.
In certain embodiments, the one or more therapeutic compositions of enriched T cells (e.g., engineered and cultured T cells, e.g., export T cells) are or comprise two separate compositions of enriched T cells, e.g., separate engineered and/or cultured compositions. In particular embodiments, two separate therapeutic compositions enriched for T cells, e.g., two separate compositions enriched for cd4+ T cells and cd8+ T cells that are selected, isolated, and/or enriched, individually engineered, and individually cultured from the same biological sample, are formulated separately. In certain embodiments, the two separately therapeutic cell compositions include a composition enriched for cd4+ T cells, such as a composition of engineered and/or cultured cd4+ T cells. In particular embodiments, the two separately therapeutic cell compositions include compositions enriched for cd8+ T cells, such as compositions engineered and/or incubated for cd8+ T cells. In some embodiments, two separate therapeutic compositions enriched for cd4+ T cells and enriched for cd8+ T cells (e.g., a separate composition of engineered and cultured cd4+ T cells and engineered and cultured cd8+ T cells) are formulated separately. In some embodiments, the T cell enriched monotherapy composition is formulated. In certain embodiments, the monotherapy composition is a composition enriched for cd4+ T cells, such as a composition engineered and/or incubated for cd4+ T cells. In some embodiments, the monotherapy composition is a composition enriched for cd4+ and cd8+ T cells that have been combined from a separate composition prior to formulation.
In some embodiments, the separate therapeutic compositions enriched for cd4+ and cd8+ T cells (e.g., the separate compositions of engineered and incubated cd4+ and cd8+ T cells) are combined into a single therapeutic composition and formulated. In certain embodiments, the separately formulated therapeutic compositions enriched for cd4+ and enriched for cd8+ T cells are combined into a single therapeutic composition after the formulation has been performed and/or completed. In particular embodiments, the separate therapeutic compositions enriched for cd4+ and cd8+ T cells (e.g., the separate compositions of engineered and cultured cd4+ and cd8+ T cells) are formulated as separate compositions, respectively.
In some embodiments, the therapeutic composition of formulated enriched cd4+ T cells (e.g., engineered and cultured cd4+ T cells, e.g., exported 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 about 100% cd4+ T cells. In some embodiments, the composition comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or at or about 100% of cd4+ T cells that express the recombinant receptor and/or have been transduced or transfected with a recombinant polynucleotide. In certain embodiments, the therapeutic composition formulated enriched for cd4+ T cells (e.g., engineered and cultured cd4+ T cells, e.g., exported 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.
In some embodiments, the therapeutic composition of formulated enriched cd8+ T cells (e.g., engineered and cultured cd8+ T cells, e.g., exported 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 about 100% cd8+ T cells. In certain embodiments, the therapeutic composition comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or at least about 100% cd8+ T cells expressing a recombinant receptor and/or that have been transduced or transfected with a recombinant polynucleotide. In certain embodiments, a therapeutic composition enriched in cd8+ T cells (e.g., engineered and incubated cd8+ T cells, e.g., exported cd8+ T cells) incubated under stimulating 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.
In certain embodiments, the formulated cell is an export cell. In some embodiments, the formulated therapeutic composition of enriched T cells (e.g., the formulated composition of engineered and cultured T cells) is an output composition of enriched T cells. In particular embodiments, the formulated cd4+ T cells and/or formulated cd8+ T cells are export cd4+ and/or cd8+ T cells. In particular embodiments, the formulated composition enriched for cd4+ T cells is an output composition enriched for cd4+ T cells. In some embodiments, the formulated composition enriched for cd8+ T cells is an output composition enriched for cd8+ T cells.
In some embodiments, the cells may be formulated into a container, such as a bag or vial. In some embodiments, the cells are formulated after the cells have achieved a threshold cell count, density, and/or between 0 and 10 days, between 0 and 5 days, between 2 and 7 days, between 0.5 and 4 days, or between 1 and 3 days after expansion during the incubation period. In certain embodiments, the cells are formulated at or about 12 hours, 18 hours, 24 hours, 1 day, 2 days, or 3 days or within 12 hours, 18 hours, 24 hours, 1 day, 2 days, or 3 days after threshold cell count, density, and/or expansion have been achieved during the incubation period. In some embodiments, the cells are formulated within or about 1 day after threshold cell counts, densities, and/or expansion have been achieved during incubation.
In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer, which in some aspects may include a pharmaceutically acceptable carrier or excipient. In some embodiments, the treatment comprises exchanging the medium for a pharmaceutically acceptable or a medium or formulation buffer required for administration to the subject. In some embodiments, the treating step may involve washing the transduced and/or expanded cells to replace the cells in a pharmaceutically acceptable buffer, which may include one or more optional pharmaceutically acceptable carriers or excipients. Examples of such pharmaceutical forms, including pharmaceutically acceptable carriers or excipients, may be any of the forms described below in connection with the administration of the cells and compositions to a subject. In some embodiments, the pharmaceutical composition contains cells in an amount effective to treat or prevent a disease or disorder (e.g., a therapeutically effective amount or a prophylactically effective amount).
The term "pharmaceutical formulation" refers to a formulation which is in a form such that the biological activity of the active ingredient contained therein is effective, and which is free of additional components having unacceptable toxicity to the subject to whom the formulation is administered.
By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject in addition to the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
In some aspects, the choice of vector depends in part on the particular cell and/or method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. Vectors are described, for example, in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt forming counterions such as sodium, metal complexes (e.g., zinc-protein complexes), and/or non-ionic complexes such as PEG and/or non-surfactants such as PEG.
In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, remington: THE SCIENCE AND PRACTICE of Pharmacy, lippincott Williams & Wilkins; 21st ed. (5/1/2005).
In some embodiments, the pharmaceutical composition contains an amount (e.g., a therapeutically effective amount or a prophylactically effective amount) of the cells effective to treat or prevent the disease or disorder. In some embodiments, the treatment or prevention efficacy is monitored by periodic assessment of the subject being treated. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until inhibition of the desired disease symptoms occurs. However, other dosage regimens may be useful and may be determined. The desired dose may be delivered by administering the composition by a single bolus, by multiple bolus injections, or by continuous infusion.
The cells may be administered using standard administration techniques, formulations and/or devices. Formulations and devices (e.g., syringes and vials) for storing and administering the compositions are provided. The administration of the cells may be autologous or heterologous. For example, the immune response cells or progenitor cells can be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived immune response cells or their progeny (e.g., in vivo, ex vivo, or derived in vitro) may be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immune responsive cells) is administered, it is typically formulated in unit dose injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the population of cells is administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
In some embodiments, the compositions are provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, the liquid composition is somewhat more convenient to administer, particularly by injection. In another aspect, the adhesive composition may be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
Sterile injectable solutions may be prepared by incorporating the cells in a solvent, for example, with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose and the like. The composition may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, pigments and the like, depending on the route of administration and the desired formulation. In some aspects, reference may be made to standard text for the preparation of suitable formulations.
Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. The prevention of the action of microorganisms can be ensured by different antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents which delay absorption (for example, aluminum monostearate and gelatin).
Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.
In some embodiments, the formulation buffer contains a cryopreservative. In some embodiments, the cells are formulated with a cryopreservation agent solution comprising 1.0% to 30% DMSO solution, such as 5% to 20% DMSO solution or 5% to 10% DMSO solution. In some embodiments, the cryopreservative solution is or contains, for example, PBS containing 20% DMSO and 8% Human Serum Albumin (HSA), or other suitable cell freezing medium. In some embodiments, the cryopreservative solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the processing step may involve washing the transduced and/or expanded cells to exchange cells in a cryopreservative solution. In some embodiments, the cells are frozen, e.g., cryogenically frozen or cryopreserved, in a medium and/or solution having a final concentration of DMSO of or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0%, or DMSO between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8%. In particular embodiments, the cells are frozen, e.g., cryogenically frozen or cryopreserved, in a medium and/or solution having a final concentration of HSA of or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25%, or between 0.1% and-5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2%.
In particular embodiments, the therapeutic composition of enriched T cells (e.g., T cells that have been stimulated, engineered, and/or cultured) is formulated, cryogenically frozen, and then stored for a certain amount of time. In certain embodiments, the formulated cryogenically frozen cells are typically stored in multiple vials or containers until the cells are released for infusion. In some embodiments, vials or containers of a particular therapeutic composition may be used to assess activation of engineered T cells prior to infusion of the therapeutic cell composition. In particular embodiments, the formulated cryogenically frozen cells are stored for between 1 day and 6 months, between 1 month and 3 months, between 1 day and 14 days, between 1 day and 7 days, between 3 days and 6 days, between 6 months and 12 months, or longer than 12 months. In some embodiments, the cells are cryogenically frozen and stored for a duration of about, or for less than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In certain embodiments, after storage, the cells are thawed and administered to a subject. In certain embodiments, the cells are stored for or for about 5 days.
In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer (optionally including a cryoprotectant) having a volume of from 10 mL to 1000 mL or from about 10 mL to about 1000 mL, such as at least or about 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL. In some embodiments, the therapeutic cell composition is stored in a container (e.g., one or more vials or bags). In some embodiments, the vial or bag generally contains the cells to be administered, e.g., one or more unit doses thereof. The unit dose may be the amount or number of cells to be administered to the subject, or twice the number (or more) of cells to be administered. It may be the lowest dose or the lowest possible dose of cells to be administered to the subject.
In some embodiments, each container (e.g., bag or vial) individually contains a unit dose of cells. Thus, in some embodiments, each container contains the same or substantially the same number of cells. In some embodiments, each unit dose contains at least or about at least 1x 10 6、2 x 106、5 x 106、1 x 107、5 x 107 or 1x 10 8 engineered cells, total cells, T cells, or PBMCs. In some embodiments, the volume of the formulated cell composition in each container (e.g., bag or vial) is 10 mL to 100mL, such as at least or about 20mL, 30 mL, 40 mL, 50 mL, 60mL, 70 mL, 80 mL, 90 mL, or 100 mL. In some embodiments, the cells in the container (e.g., bag or vial) may be cryopreserved. In some embodiments, the container (e.g., vial) may be stored in liquid nitrogen until further use.
In some embodiments, the activation state of a composition comprising recombinant receptor-expressing cells (e.g., CAR-expressing cells) as produced by a method comprising one or more of the steps described herein is determined by measuring one or more activation markers described herein. In some embodiments, cells (e.g., CAR expressing cells) of a composition comprising recombinant receptor expressing cells (e.g., CAR expressing cells) can be administered to a subject (e.g., produced by a method comprising one or more of the steps and/or whose activation state has been determined) to treat a disease or disorder.
Recombinant receptors
In some embodiments, the provided methods for measuring or assessing T cell activation of a therapeutic cell composition are performed or carried out on cells from a composition containing or expressing or engineered to contain or express a recombinant receptor, such as a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). In certain embodiments, the methods provided herein produce and/or are capable of producing cells or populations or compositions containing and/or enriched for cells engineered to express or contain recombinant proteins and the activation of such produced engineered cells can be determined or measured. In various embodiments, provided methods are performed on T cell compositions that are cells engineered, transformed, transduced or transfected to express one or more recombinant receptors.
Receptors include antigen receptors and receptors containing one or more components thereof. Recombinant receptors can include chimeric receptors (such as those containing a ligand binding domain or binding fragment thereof and an intracellular signaling domain or region), functional non-TCR antigen receptors, chimeric Antigen Receptors (CARs), T Cell Receptors (TCRs) (such as recombinant or transgenic TCRs), chimeric autoantibody receptors (CAARs), and components of any of the foregoing. The recombinant receptor (e.g., CAR) typically includes an extracellular antigen (or ligand) binding domain linked (in some aspects via a linker and/or one or more transmembrane domains) to one or more intracellular signaling components. In some embodiments, the engineered cells express two or more receptors containing different components, domains, or regions. In some aspects, two or more receptors allow for spatial or temporal modulation or control of the specificity, activity, antigen (or ligand) binding, function, and/or expression of recombinant receptors.
A. chimeric Antigen Receptor (CAR)
In some embodiments of the provided methods, a chimeric receptor (e.g., chimeric antigen receptor) contains one or more domains that combine a ligand binding domain (e.g., an antibody or antibody fragment) that provides specificity for a desired target (e.g., an antigen (e.g., a tumor antigen)) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, thereby providing a primary activation signal. In some embodiments, the intracellular signaling domain contains or otherwise contains a costimulatory signaling domain to promote effector function. In some embodiments, the chimeric receptor, when genetically engineered into immune cells, can modulate T cell activity, and in some cases can modulate T cell differentiation or homeostasis, thereby producing genetically engineered cells with improved longevity, survival, and/or persistence in vivo, such as for adoptive cell therapy methods.
Exemplary antigen receptors, including CARs, and methods of engineering and introducing such receptors into cells include, for example, those described in International patent application publication No. WO 200014257、WO 2013126726、WO 2012/129514、WO 2014031687、WO 2013/166321、WO 2013/071154、WO 2013/123061、WO 2015/168613、WO 2016/030414;, U.S. patent application publication No. US 2002131960, U.S. patent 2013287748, U.S. 20130149337, U.S. 20190389925, U.S. patent No. ：6,451,995、7,446,190、8,252,592、8,339,645、8,398,282、7,446,179、6,410,319、7,070,995、7,265,209、7,354,762、7,446,191、8,324,353、8,479,118、10,266,580;, and European patent application No. EP2537416, and/or those described in Sadelain et al, cancer discover.2013, month 4, 3 (4): 388-398; davila et al (2013) PLoS ONE 8 (4): e61338; turtle et al, curr.Opin.Immunol, 2012, month 10, 24 (5): 633-39; wu et al, cancer, 2012, month 3, 18 (2): 160-75. In some aspects, antigen receptors include CARs as described in U.S. Pat. No. 7,446,190, and those described in international patent application publication No. WO/2014055668 A1. Examples of CARs include CARs as disclosed in any of the above publications, such as WO 2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US patent No. 7,446,190, US patent No. 8,389,282; kochenderfer et al, 2013, nature REVIEWS CLINICAL Oncology, 10, 267-276 (2013); wang et al (2012) J. Immunother.35 (9): 689-701; and Brentjens et al, SCI TRANSL Med.2013 (177). See also WO 2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Pat. No. 7,446,190 and U.S. Pat. No. 8,389,282.
Chimeric receptors, such as CARs, typically include an extracellular target binding domain (e.g., an antigen binding domain), such as, for example, a portion of an antibody molecule, typically a Variable Heavy (VH) chain region and/or a Variable Light (VL) chain region of an antibody, such as scFv antibody fragments.
In some embodiments, the antigen to which the receptor is targeted is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or disorder (e.g., tumor or pathogenic cells) as compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.
In some embodiments, the antigen is or includes αvβ6 integrin (avb 6 integrin), B Cell Maturation Antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA 9, also known as CAIX or G250), cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and rage-2), carcinoembryonic antigen (CEA), cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG 4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), hepataxin B2, hepataxin receptor A2 (EPHa 2), estrogen receptor, fc receptor-like protein 5 (FCRL 5; also known as Fc receptor homolog 5 or FCRH 5), and, Fetal Acholine receptor (fetal AchR), folate Binding Protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD 2), ganglioside GD3, glycoprotein 100 (gp 100), glypican-3 (GPC 3), G-protein coupled receptor 5D (GPRC 5D), her2/neu (receptor tyrosine kinase erb-B2), her3 (erb-B3), her4 (erb-B4), erbB dimer, human high molecular weight melanomA-Associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1 (HLa-A1), human leukocyte antigen A2 (HLa-A2), IL-22 receptor alpha (IL-22 Rα), IL-13 receptor alpha 2 (IL-13 Rα2), kinase insertion domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine rich repeat containing protein 8 family member A (LRRC 8A), lewis Y, melanomA-Associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine Cytomegalovirus (CMV), mucin 1 (MUC 1), MUC16, natural killer cell group 2 member D (NKG 2D) ligand, melanin A (MART-1), neural Cell Adhesion Molecule (NCAM), carcinoembryonic antigen, melanoma preferential expression antigen (PRAME), progesterone receptor, prostate specific antigen, prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR 1), survivin, trophoblast glycoprotein (TPBG, also known as 5T 4), tumor-associated glycoprotein 72 (TAG 72), tumor-associated glycoprotein 72, Tyrosinase-related protein 1 (TRP 1, also known as TYRP1 or gp 75), tyrosinase-related protein 2 (TRP 2, also known as dopachrome tautomerase, dopachrome delta isomerase, or DCT), vascular Endothelial Growth Factor Receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR 2), wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigen, or a molecule comprising or associated with a universal tag, and/or a biotinylated molecule, and/or expressed by HIV, HCV, HBV or other pathogens. In some embodiments, the receptor-targeted antigen comprises an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen is or comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30. In some embodiments, the antigen is or includes a pathogen-specific antigen or a pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and/or a parasitic antigen.
In some embodiments, the antibody is an antigen-binding fragment (e.g., scFv) comprising one or more linkers that connect two antibody domains or regions (e.g., a heavy chain Variable (VH) region and a light chain Variable (VL) region). The linker is typically a peptide linker, e.g., a flexible and/or soluble peptide linker. Linkers include those that are rich in glycine and serine and/or, in some cases, threonine. In some embodiments, the linker further comprises charged residues (e.g., lysine and/or glutamic acid), which may improve solubility. In some embodiments, the linker further comprises one or more prolines. In some aspects, a glycine and serine (and/or threonine) rich linker comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they comprise at least or at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the linker consists essentially entirely of glycine, serine, and/or threonine. The length of the linker is typically between about 5 and about 50 amino acids, typically between or about 10 and or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having a different number of repeats of sequences GGGGS (4 GS; SEQ ID NO: 13) or GGGS (3 GS; SEQ ID NO: 14), such as between 2, 3, 4 and 5 repeats of such sequences. Exemplary linkers include those having or consisting of the sequences set forth in SEQ ID NO. 15 (GGGGSGGGGSGGGGS), SEQ ID NO. 16 (GSTSGSGKPGSGEGSTKG) or SEQ ID NO. 17 (SRGGGGSGGGGSGGGGSLEMA).
In some embodiments, the receptor-targeted antigen comprises an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the receptor-targeted antigen is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30.
In some embodiments, the antigen or antigen binding domain is CD19. In some embodiments, the scFv comprises a VH and a VL derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds CD19 is a mouse-derived antibody, such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. patent publication No. US 2016/0152723.
The term "antibody" is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F (ab ') 2 fragments, fab' fragments, fv fragments, recombinant IgG (IgG) fragments, heavy chain Variable (VH) regions capable of specifically binding an antigen, single chain antibody fragments (including single chain variable fragments (scFv)), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific (e.g., bispecific or trispecific) antibodies, diabodies, triabodies and tetrabodies, tandem diavs, tandem triavs. Unless otherwise indicated, the term "antibody" should be understood to encompass functional antibody fragments thereof, also referred to herein as "antigen-binding fragments". The term also encompasses whole or full length antibodies, including antibodies of any class or subclass (including IgG and subclasses thereof, igM, igE, igA and IgD).
The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR," and are known in the art to refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and/or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3 and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3 and FR-L4) in each full-length light chain variable region.
The exact amino acid sequence boundaries for a given CDR or FR can be readily determined using any of a number of well known protocols, including those described in Kabat et Al (1991), "Sequences of Proteins of Immunological Interest," Public HEALTH SERVICE, national Institutes of Health, bethesda, MD ("Kabat" numbering scheme), "Al-Lazikani et Al, (1997) JMB 273,927-948 (" Chothia "numbering scheme); macCallum et Al , J. Mol.Biol.262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol.Biol.262, 732-745".（"Contact" numbering scheme); LEFRANC MP et Al, , "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003, 27 (1): 55-77 (" IMGT "numbering scheme); honyger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001, 8 th day (6) 309 (3): 657-70 (" Aho "numbering scheme); martin et Al," Modeling antibody hypervariable loops: a combined algorithm, "PNAS, 1989, 86 (23): 9268-9272 (" AbM "numbering scheme).
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering of both Kabat and Chothia protocols is based on the most common antibody region sequence length, with insertions provided by the insert letter (e.g. "30 a") and deletions in some antibodies. Both of these schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbers. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM protocol is a compromise between Kabat and Chothia definitions and is a protocol based on the use of Oxford Molecular's AbM antibody modeling software.
Table 1 lists exemplary location boundaries for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3 identified by Kabat, chothia, abM and Contact schemes, respectively. For CDR-H1, residue numbers are listed using the two numbering schemes of Kabat and Chothia. FR is located between the CDRs, e.g., FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, etc. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, when numbered using the Kabat numbering convention shown, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop.
Thus, unless otherwise specified, it is to be understood that a "CDR" or "complementarity determining region" or a separately specified CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., variable region thereof) encompasses one (or a particular) complementarity determining region as defined by any of the foregoing schemes or other known schemes. For example, where a particular CDR (e.g., CDR-H3) is stated to contain the amino acid sequence of the corresponding CDR in a given V H or V L region amino acid sequence, it is to be understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the foregoing schemes or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes, but it should be understood that the provided antibodies may include CDRs as described according to any other of the above-described numbering schemes or other numbering schemes known to the skilled artisan.
Likewise, unless otherwise specified, the FR of a given antibody or region thereof, such as its variable region, or a separately specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4) should be understood to encompass one (or a particular) framework region as defined by any known scheme. In some cases, an identification scheme for identifying a particular CDR, FR, or a plurality of particular FR or CDRs is specified, such as CDRs defined by Kabat, chothia, abM or Contact methods or other known schemes. In other cases, specific amino acid sequences of CDRs or FR are given.
The term "variable region" or "variable domain" refers to a domain of an antibody that is involved in the binding of the antibody to an antigen in the heavy or light chain of the antibody. The variable regions of the heavy and light chains of natural antibodies (V H and V L, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three CDRs. (see, e.g., kindt et al Kuby Immunology, 6 th edition, W.H. Freeman and Co., page 91 (2007); a single V H or V L domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies binding to the particular antigen may be isolated using the V H or V L domain from antibodies binding to the antigen to screen libraries of complementary V L or V H domains, respectively. See, e.g., portolano et al, J. Immunol.150:880-887 (1993); clarkson et al, nature 352:624-628 (1991).
Antibodies included in the provided CARs include antibody fragments. An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2, diabodies, linear antibodies, heavy chain variable (V H) regions, single chain antibody molecules (e.g., scFv) and single domain antibodies containing only the V H region, and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen binding domain in a provided CAR is or includes an antibody fragment comprising a variable heavy chain (V H) region and a variable light chain (V L) region. In particular embodiments, the antibody is a single chain antibody fragment (e.g., scFv) comprising a heavy chain variable (V H) region and/or a light chain variable (V L) region.
In some embodiments, the scFv is derived from FMC63.FMC63 is typically a mouse monoclonal IgG1 antibody raised against human-derived Nalm-1 and Nalm-16 expressing CD19 cells (Ling, N.R. et al (1987) Leucocyte typing III.302). In some embodiments, the FMC63 antibody comprises CDRH1 and H2 as set forth in SEQ ID NO. 18 and 19, respectively, and CDRH3 as set forth in SEQ ID NO. 20 or 21, and CDRL1 as set forth in SEQ ID NO. 40, and CDR L2 as set forth in SEQ ID NO. 23 or 24, and CDR L3 as set forth in SEQ ID NO. 25 or 26. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V H) comprising the amino acid sequence of SEQ ID NO. 27 and a light chain variable region (V L) comprising the amino acid sequence of SEQ ID NO. 28.
In some embodiments, the scFv comprises a variable light chain comprising the CDRL1 sequence of SEQ ID NO. 22, the CDRL2 sequence of SEQ ID NO. 23, and the CDRL3 sequence of SEQ ID NO. 25, and/or a variable heavy chain comprising the CDRH1 sequence of SEQ ID NO. 18, the CDRH2 sequence of SEQ ID NO. 19, and the CDRH3 sequence of SEQ ID NO. 20. In some embodiments, the scFv comprises a variable heavy chain region as set forth in SEQ ID NO. 27 and a variable light chain region as set forth in SEQ ID NO. 28. In some embodiments, the variable heavy chain and the variable light chain are linked by a linker. In some embodiments, the linker is shown in SEQ ID NO. 16. In some embodiments, the scFv comprises V H, a linker, and V L in that order. In some embodiments, the scFv comprises V L, a linker, and V H in that order. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO. 29 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 29. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO. 30 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 30.
In some embodiments, the scFv is derived from SJ25C1.SJ25C1 is a mouse monoclonal IgG1 antibody raised against human-derived Nalm-1 and Nalm-16 expressing CD19 (Ling, N.R. et al (1987) Leucocyte typing III.302). In some embodiments, the SJ25C1 antibody comprises the CDRH1, H2, and H3 sequences shown in SEQ ID NOS: 36-38, respectively, and CDRL1, L2, and L3 sequences shown in SEQ ID NOS: 33-35, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V H) comprising the amino acid sequence of SEQ ID NO. 31 and a light chain variable region (V L) comprising the amino acid sequence of SEQ ID NO. 32.
In some embodiments, the scFv comprises a variable light chain comprising the CDRL1 sequence of SEQ ID NO. 33, the CDRL2 sequence of SEQ ID NO. 33, and the CDRL3 sequence of SEQ ID NO. 35, and/or a variable heavy chain comprising the CDRH1 sequence of SEQ ID NO. 36, the CDRH2 sequence of SEQ ID NO. 37, and the CDRH3 sequence of SEQ ID NO. 38. In some embodiments, the scFv comprises a variable heavy chain region as set forth in SEQ ID NO. 31 and a variable light chain region as set forth in SEQ ID NO. 32. In some embodiments, the variable heavy chain and the variable light chain are linked by a linker. In some embodiments, the linker is shown in SEQ ID NO. 15. In some embodiments, the scFv comprises V H, a linker, and V L in that order. In some embodiments, the scFv comprises V L, a linker, and V H in that order. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 39 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 39.
In some embodiments, the antibody or antigen binding fragment (e.g., scFv or V H domain) specifically recognizes an antigen (e.g., BCMA). In some embodiments, the antibody or antigen binding fragment is derived from, or is a variant of, an antibody or antigen binding fragment that specifically binds BCMA.
In some embodiments, the CAR is an anti-BCMA CAR that is specific for BCMA (e.g., human BCMA). Chimeric antigen receptors containing anti-BCMA antibodies (including mouse anti-human BCMA antibodies and human anti-human antibodies) and cells expressing such chimeric receptors have been previously described. See Carpenter et al CLIN CANCER res, 2013, 19 (8): 2048-2060, WO 2016/090320,WO 2016090327,WO 2010104949A2 and WO 2017173256. In some embodiments, the antigen or antigen binding domain is BCMA. In some embodiments, the scFv comprises a VH and a VL derived from an antibody or antibody fragment specific for BCMA. In some embodiments, antibodies or antibody fragments that bind BCMA are or contain VH and VL from antibodies or antibody fragments described in international patent application publication nos. WO 2016/090327 and WO 2016/090320.
In some embodiments, an anti-BCMA CAR comprises an antigen binding domain (e.g., scFv) comprising a variable heavy (V H) region and/or a variable light (V L) region derived from an antibody described in WO 2016/090320 or WO 2016090327. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown as SEQ ID NO: 40 and V L shown as SEQ ID NO: 41. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown as SEQ ID NO: 42 and V L shown as SEQ ID NO: 43. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown as SEQ ID NO: 44 and V L shown as SEQ ID NO: 45. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown as SEQ ID NO: 46 and V L shown as SEQ ID NO: 47. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown as SEQ ID NO: 48 and V L shown as SEQ ID NO: 49. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown as SEQ ID NO: 50 and V L shown as SEQ ID NO: 51. In some embodiments, the antigen binding domain (e.g., scFv) comprises V H shown in SEQ ID NO: 52 and V L shown in SEQ ID NO: 53. In some embodiments, V H or V L has a sequence that exhibits at least 85%, a sequence that is at least as long as any of the foregoing V H or V L sequences, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and retains amino acid sequence binding to BCMA. In some embodiments, the V H region is located at the amino terminus of the V L region. In some embodiments, the V H region is located at the carboxy terminus of the V L region.
In some embodiments, the antigen or antigen binding domain is GPRC5D. In some embodiments, the scFv comprises a VH and a VL derived from an antibody or antibody fragment specific for GPRC5D. In some embodiments, the antibodies or antibody fragments that bind GPRC5D are or contain VH and VL from antibodies or antibody fragments described in International patent application publication Nos. WO 2016/090329 and WO 2016/090312.
In some aspects, the CAR contains a ligand (e.g., antigen) binding domain that binds or recognizes (e.g., specifically binds) a universal tag or universal epitope. In some aspects, the binding domain may bind a molecule, tag, polypeptide, and/or epitope, which may be linked to a different binding molecule (e.g., an antibody or antigen binding fragment) that recognizes an antigen associated with a disease or disorder. Exemplary tags or epitopes include dyes (e.g., fluorescein isothiocyanate) or biotin. In some aspects, a binding molecule (e.g., an antibody or antigen binding fragment) is linked to a tag that recognizes an antigen (e.g., a tumor antigen) associated with a disease or disorder, and an engineered cell expresses a CAR specific for the tag to effect cytotoxicity or other effector function of the engineered cell. In some aspects, the specificity of the CAR for an antigen associated with a disease or disorder is provided by a tagged binding molecule (e.g., an antibody), and different tagged binding molecules can be used to target different antigens. Exemplary CARs specific for a universal tag or universal epitope include, for example, those described in U.S. 9,233,125;WO 2016/030414; urbanska et al, (2012) CANCER RES 72:1844-1852, and Tamada et al, (2012) CLIN CANCER RES 18:6436-6445.
In some embodiments, the antigen is or includes a pathogen-specific antigen or a pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and/or a parasitic antigen. In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or antigen-binding fragment (e.g., scFv), that specifically recognizes an intracellular antigen (e.g., a tumor-associated antigen) presented on the surface of a cell as a Major Histocompatibility Complex (MHC) -peptide complex. In some embodiments, antibodies or antigen binding portions thereof that recognize MHC-peptide complexes may be expressed on cells as part of a recombinant receptor (e.g., antigen receptor). Antigen receptors include functional non-T Cell Receptor (TCR) antigen receptors, such as Chimeric Antigen Receptors (CARs). In some embodiments, a CAR containing an antibody or antigen binding fragment that exhibits TCR-like specificity for a peptide-MHC complex may also be referred to as a TCR-like CAR. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, that is recognized on the cell surface in the context of an MHC molecule as the TCR. In some embodiments, in some aspects, the extracellular antigen-binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular signaling components by a linker and/or one or more transmembrane domains. In some embodiments, such molecules may generally mimic or mimic the signal through a native antigen receptor (e.g., TCR), and optionally mimic or mimic the signal through a combination of such a receptor and a co-stimulatory receptor.
Reference to "major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, containing polymorphic peptide binding sites or grooves, which in some cases may be complexed with peptide antigens of a polypeptide, including peptide antigens processed by cellular machinery. In some cases, MHC molecules may be displayed or expressed on the cell surface, including as complexes with peptides, i.e., MHC-peptide complexes, for presenting antigens having a conformation recognizable by antigen receptors on T cells (e.g., TCR or TCR-like antibodies). Generally, MHC class I molecules are heterodimers with a transmembrane alpha chain (in some cases with three alpha domains) and non-covalently associated β2 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which typically cross the membrane. MHC molecules may include an effective portion of an MHC that contains an antigen binding site or sites for binding peptides and sequences required for recognition by appropriate antigen receptors. In some embodiments, MHC class I molecules deliver cytosolic derived peptides to the cell surface where the MHC-peptide complex is recognized by T cells (e.g., typically CD8 + T cells, but in some cases CD4 + T cells). In some embodiments, MHC class II molecules deliver peptides derived from the vesicle system to the cell surface, wherein the peptides are generally recognized by CD4 + T cells. Generally, MHC molecules are encoded by a set of linked loci, which are collectively referred to as H-2 in mice and Human Leukocyte Antigen (HLA) in humans. Thus, human MHC may also be referred to as Human Leukocyte Antigen (HLA) in general.
The term "MHC-peptide complex" or "peptide-MHC complex" or variants thereof refers to complexes or associations of peptide antigens with MHC molecules, e.g. typically formed by non-covalent interactions of the peptides in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor (e.g., a TCR-like CAR, or an antigen-binding portion thereof).
In some embodiments, peptides (e.g., peptide antigens or epitopes) of the polypeptides may be associated with MHC molecules, such as for recognition by antigen receptors. Typically, the peptide is derived from or based on a fragment of a longer biomolecule (e.g., a polypeptide or protein). In some embodiments, the peptide generally has a length of about 8 to about 24 amino acids. In some embodiments, the peptide is from or about 9 to 22 amino acids in length for recognition in MHC class II complexes. In some embodiments, the peptide is from or from about 8 to 13 amino acids in length for recognition in an MHC class I complex. In some embodiments, upon recognition of a peptide in the context of an MHC molecule (e.g., MHC-peptide complex), an antigen receptor (e.g., a TCR or TCR-like CAR) generates or triggers an activation signal to a T cell, inducing a T cell response, such as T cell proliferation, cytokine production, cytotoxic T cell response, or other response.
In some embodiments, TCR-like antibodies or antigen-binding portions are known or can be produced by known methods (see, e.g., U.S. published application number US 2002/0150914;US 2003/0223994;US 2004/0191260;US 2006/0034850;US 2007/00992530;US 20090226474;US 20090304679; and international application publication number WO 03/068201).
In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes may be produced by immunizing a host with an effective amount of an immunogen comprising the particular MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen capable of binding to MHC, such as a tumor antigen, such as a general tumor antigen, a myeloma antigen, or other antigen as described below. In some embodiments, an effective amount of an immunogen is then administered to the host for eliciting an immune response, wherein the immunogen remains in its three-dimensional form for a time sufficient to elicit an immune response directed against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine whether the desired antibodies recognizing the three-dimensional presentation of peptides in the binding groove of the MHC molecule are produced. In some embodiments, the antibodies produced can be evaluated to confirm that the antibodies can distinguish MHC-peptide complexes from MHC molecules alone, peptides of interest alone, and complexes of MHC with unrelated peptides. The desired antibody can then be isolated.
In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be generated by employing an antibody library display method (e.g., phage antibody library). In some embodiments, phage display libraries of mutant Fab, scFv, or other antibody forms can be produced, for example, wherein members of the library are mutated at one or more residues of one or more CDRs. See, for example, U.S. patent application publication Nos. US 20020150914, US 20140294841, and Cohen CJ. Et al (2003) J mol. Recog. 16:324-332.
In some embodiments, the antigen is CD20. In some embodiments, the scFv comprises VH and VL derived from antibodies or antibody fragments specific for CD20. In some embodiments, the antibody or antibody fragment that binds CD20 is an antibody that is rituximab or is derived from rituximab, such as rituximab scFv.
In some embodiments, the antigen is CD22. In some embodiments, the scFv comprises VH and VL derived from antibodies or antibody fragments specific for CD22. In some embodiments, the antibody or antibody fragment that binds CD22 is an antibody that is m971 or derived from m971, e.g., is an m971 scFv.
In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv.
In some embodiments, the antibody portion of the recombinant receptor (e.g., CAR) further comprises at least a portion of an immunoglobulin constant region, such as a hinge region (e.g., an IgG4 hinge region) and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is a human IgG such as IgG4 or IgG 1. In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition component (e.g., scFv) and the transmembrane domain. The length of the spacer may provide enhanced cellular reactivity following antigen binding compared to the absence of the spacer. Exemplary spacers include, but are not limited to, those described in Hudecek et al (2013) Clin.cancer Res., 19:3153, international patent application publication No. WO 2014031687, U.S. Pat. No. 8,822,647, or published application No. US 2014/0271635.
In some embodiments, the constant region or portion is a human IgG such as IgG4 or IgG 1. In some embodiments, the spacer has sequence ESKYGPPCPPCP (shown as SEQ ID NO: 54) and is encoded by the sequence shown as SEQ ID NO: 55. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 56. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 57. In some embodiments, the constant region or portion is IgD. In some embodiments, the spacer is part of an immunoglobulin constant region that is or comprises a hinge sequence. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 58. In some embodiments, the spacer has 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 any one of SEQ ID NOs 54, 55, 56, 57 or 58. In some embodiments, the spacer has the sequence shown in SEQ ID NOS 59-67. In some embodiments, the spacer has 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 any one of SEQ ID NOs 59-67.
In some embodiments, the antigen receptor comprises an intracellular domain directly or indirectly linked to an extracellular domain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that connects an extracellular domain and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises ITAM. For example, in some aspects, an antigen recognition domain (e.g., an extracellular domain) is typically linked to one or more intracellular signaling components (e.g., signaling components that mimic activation by an antigen receptor complex (e.g., a TCR complex) (in the case of a CAR) and/or signaling by another cell surface receptor). In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane domains and intracellular signaling domains.
In one embodiment, a transmembrane domain is used that naturally associates with one domain in a receptor (e.g., CAR). In some cases, the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane regions include those derived from (i.e., comprising at least one or more of) the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the connection is through a linker, spacer and/or one or more transmembrane domains. In some aspects, the transmembrane domain comprises a transmembrane portion of CD 28.
In some embodiments, the extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are connected by a spacer (such as any of the spacers described herein). In some embodiments, the receptor contains an extracellular portion of a molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.
Intracellular signaling domains include those that mimic or approximate signals through natural antigen receptors, signals through a combination of such receptors and co-stimulatory receptors, and/or signals through co-stimulatory receptors alone. In some embodiments, there is a short oligopeptide or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length (e.g., a glycine and serine containing linker, e.g., a glycine-serine duplex), and a linkage is formed between the transmembrane domain and cytoplasmic signaling domain of the CAR.
In some aspects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences, those that initiate antigen dependent primary activation by TCR (primary cytoplasmic signaling sequences), and those that act in an antigen independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
The receptor (e.g., CAR) typically includes at least one or more intracellular signaling components. In some aspects, the CAR comprises a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif (which is referred to as an immunoreceptor tyrosine activation motif or ITAM). Examples of primary cytoplasmic signaling sequences containing ITAM include those derived from the cd3ζ chain, fcrγ, cd3γ, cd3δ, and cd3ε. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain derived from cd3ζ, a portion or sequence thereof.
In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as a TCR CD3 chain, e.g., a cd3ζ chain, that mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen binding portion is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD3 transmembrane domain. In some embodiments, the receptor (e.g., CAR) further comprises a portion of one or more additional molecules (e.g., fc receptor gamma, CD8, CD4, CD25, or CD 16). For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD 3- ζ) or Fc receptor γ and CD8, CD4, CD25, or CD 16.
In some embodiments, upon attachment of a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune cell (e.g., a T cell engineered to express the CAR). For example, in some contexts, the CAR induces a function of a T cell, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of the antigen receptor component or co-stimulatory molecule (e.g., if it transduces an effector function signal) is used in place of the intact immunostimulatory chain. In some embodiments, one or more intracellular signaling domains include cytoplasmic sequences of T Cell Receptors (TCRs), and in some aspects also include those cytoplasmic sequences of co-receptors that cooperate with such receptors in the natural environment to initiate signal transduction upon antigen receptor engagement.
In the context of native TCRs, complete activation typically requires not only signaling through the TCR, but also a co-stimulatory signal. Thus, in some embodiments, to facilitate complete activation, components for generating secondary or co-stimulatory signals are also included in the CAR. In other embodiments, the CAR does not include a component for generating a co-stimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for generating a secondary or co-stimulatory signal.
In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR comprises a signaling domain and/or transmembrane portion of a co-stimulatory receptor (e.g., CD28, 4-1BB, OX40, DAP10, and ICOS). In some aspects, the same CAR includes both an activating component and a co-stimulatory component. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a T cell costimulatory molecule, or a functional variant thereof, such as located between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
In some embodiments, the activation domain is included within one CAR, and the co-stimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR comprises an activated or stimulated CAR, a co-stimulated CAR (see WO 2014/055668), all expressed on the same cell. In some aspects, the cell includes one or more stimulating or activating CARs and/or co-stimulating CARs. In some embodiments, the cells further comprise an inhibitory CAR (iCAR, see Fedorov et al, sci.tranl.medicine, 5 (215) (month 12 2013), such as a CAR that recognizes an antigen other than an antigen associated with and/or specific for a disease or disorder, wherein an activation signal delivered by a disease-targeted CAR is reduced or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In some embodiments, the two receptors induce activation and inhibitory signals to the cell, respectively, such that the attachment of one receptor to its antigen activates the cell or induces a response, but the attachment of the second inhibitory receptor to its antigen induces a signal that inhibits or attenuates the response. An example is the combination of an activating CAR with an Inhibitory CAR (iCAR). For example, such a strategy may be used, for example, to reduce the likelihood of off-target effects in the context in which activating a CAR binds to an antigen that is expressed in a disease or disorder but is also expressed on normal cells, and an inhibitory receptor binds to a separate antigen that is expressed on normal cells but is not expressed on cells of the disease or disorder.
In some aspects, the chimeric receptor is or includes an inhibitory CAR (e.g., iCAR) and includes an intracellular component that attenuates or inhibits an immune response (e.g., ITAM in a cell and/or co-stimulates a promoted response). Examples of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 adenosine receptors (including A2 AR). In some aspects, the engineered cells include an inhibitory CAR comprising or derived from a signaling domain of such an inhibitory molecule, such that it is used to attenuate cellular responses induced, for example, by activating and/or co-stimulating the CAR.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3- ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1 BB, TNFRSF9) co-stimulatory domain linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more (e.g., two or more) co-stimulatory domains and an activation domain (e.g., a primary activation domain) in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3 zeta, CD28, and 4-1 BB.
In some embodiments, the antigen receptor further comprises a marker, and/or the cell expressing the CAR or other antigen receptor further comprises a surrogate marker, such as a cell surface marker, which can be used to confirm that the cell is transduced or engineered to express the receptor. In some aspects, the marker comprises all or part (e.g., a truncated form) of CD34, NGFR, or an epidermal growth factor receptor, such as a truncated form of such a cell surface receptor (e.g., tgfr). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence (e.g., a cleavable linker sequence, such as T2A). For example, the tag and optionally the linker sequence may be any as disclosed in published patent application number WO 2014031687. For example, the marker may be truncated EGFR (tgfr), which is optionally linked to a linker sequence, such as a T2A cleavable linker sequence.
Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence shown in SEQ ID NO:2 or 3 or an amino acid sequence exhibiting 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. Exemplary T2A linker sequences comprise the amino acid sequence shown in SEQ ID NO. 1 or 4 or an amino acid sequence exhibiting 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. 1 or 4.
In some embodiments, the marker is a molecule (e.g., a cell surface protein) or portion thereof that is not found naturally on a T cell or is not found naturally on a T cell surface. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., a molecule that is not recognized as "self" by the host immune system of the adoptively transferred cell.
In some embodiments, the marker does not provide any therapeutic function and/or does not produce an effect other than use as a genetically engineered marker (e.g., for selection of successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts a desired effect, such as a ligand of a cell encountered in vivo, such as a costimulatory or immune checkpoint molecule, to enhance and/or attenuate the response of the cell upon adoptive transfer and upon encountering the ligand.
In some cases, the CAR is referred to as a first, second, and/or third generation CAR. In some aspects, the first generation CAR is a CAR that provides only CD3 chain-induced signaling upon antigen binding, in some aspects the second generation CAR is a CAR that provides such signaling and costimulatory signaling, e.g., a CAR that includes an intracellular signaling domain from a costimulatory receptor (e.g., CD28 or CD 137), and in some aspects the third generation CAR is a CAR that includes multiple costimulatory domains of different costimulatory receptors.
For example, in some embodiments, the CAR contains an antibody (e.g., an antibody fragment, such as scFv) that is specific for an antigen (including any of those described), a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains a signaling portion of CD28 or a functional variant thereof and a signaling portion of cd3ζ or a functional variant thereof. In some embodiments, the CAR contains an antibody (e.g., an antibody fragment, such as scFv) that is specific for an antigen (including any one as described), a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of cd3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer, such as a hinge-only spacer, comprising a portion of an Ig molecule (e.g., a human Ig molecule) (e.g., an Ig hinge, e.g., an IgG4 hinge).
In some embodiments, the transmembrane domain of a recombinant receptor (e.g., CAR) is or comprises the transmembrane domain of human CD28 (e.g., accession number P01747.1) or a variant thereof, such as a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 68 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: 68, and in some embodiments, the portion of the recombinant receptor that contains the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, one or more intracellular signaling components of a recombinant receptor (e.g., CAR) contain an intracellular co-stimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain having a LL replaced with a GG at positions 186-187 of the native CD28 protein. For example, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID No. 70 or 71 or an amino acid sequence exhibiting 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. 70 or 71. In some embodiments, the intracellular domain comprises the intracellular co-stimulatory signaling domain of 4-1BB (e.g., accession number Q07011.1), or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 72 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: 72.
In some embodiments, the intracellular signaling domain of a recombinant receptor (e.g., CAR) comprises a human CD3 zeta stimulating signaling domain or a functional variant thereof, such as the cytoplasmic domain of 112 AA of subtype 3 of human CD3 zeta (accession number: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID No. 73, 74, or 75 or an amino acid sequence exhibiting 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. 73, 74, or 75.
In some aspects, the spacer contains only hinge regions of IgG, such as only hinge of IgG4 or IgG1, such as the hinge-only spacer shown in SEQ ID NO: 54. In other embodiments, the spacer is or comprises an Ig hinge, such as an IgG 4-derived hinge, optionally linked to a CH2 and/or CH3 domain. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, as shown in SEQ ID NO: 57. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked only to the CH3 domain, as shown in SEQ ID NO: 56. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker, such as known flexible linkers.
For example, in some embodiments, the CAR comprises an antibody (e.g., an antibody fragment, including an scFv), a spacer (e.g., a spacer comprising a portion of an immunoglobulin molecule (e.g., a hinge region and/or one or more constant regions of a heavy chain molecule), such as a spacer comprising an Ig hinge, a transmembrane domain comprising all or a portion of a CD 28-derived transmembrane domain, a CD 28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment (e.g., scFv), a spacer (e.g., any spacer comprising an Ig hinge), a CD 28-derived transmembrane domain, a 4-1 BB-derived intracellular signaling domain, and a cd3ζ -derived signaling domain.
Exemplary surrogate markers may include truncated forms of a cell surface polypeptide, such as truncated forms that are nonfunctional and do not transduce or are incapable of transducing a signal or are generally transduced by a full length form of the cell surface polypeptide, and/or are not internalized or are incapable of internalization. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER), truncated epidermal growth factor receptor (tgfr, exemplary tgfr sequences shown in 2 or 3), or Prostate Specific Membrane Antigen (PSMA), or modified forms thereof. the tEGFR may contain epitopes recognized by antibodies cetuximab (Erbitux) or other therapeutic anti-EGFR antibodies or binding molecules, which epitopes may be used to identify or select cells that have been engineered to express the tEGFR construct and the encoded foreign protein, and/or to eliminate or isolate cells expressing the encoded foreign protein. See U.S. Pat. No.8,802,374 and Liu et al, nature Biotech.2016, month 4; 34 (4): 430-434). In some aspects, the marker (e.g., surrogate marker) comprises all or part (e.g., truncated form) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated non-human CD 19) or an epidermal growth factor receptor (e.g., tgfr). In some embodiments, the label is or comprises a fluorescent protein, such as Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP) (e.g., superfolder GFP (sfGFP)), red Fluorescent Protein (RFP) (e.g., tdTomato, mCherry, mStrawberry, asRed, dsRed, or DsRed 2), 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, monomer variants, and codon optimized and/or enhanced variants of fluorescent protein. In some embodiments, the label is or comprises an enzyme (e.g., luciferase), a lacZ gene from E.coli, alkaline phosphatase, secreted Embryonic Alkaline Phosphatase (SEAP), chloramphenicol Acetyl Transferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), beta-galactosidase, chloramphenicol Acetyl Transferase (CAT), beta-Glucuronidase (GUS), or variants thereof.
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 selection marker is an antibiotic resistance gene. In some embodiments, the resistance marker or selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the resistance marker or selectable marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene or a gecomycin resistance gene or modified form thereof.
In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence (e.g., a cleavable linker sequence, e.g., T2A). For example, the tag and optionally the linker sequence may be any one as disclosed in PCT publication No. WO 2014031687.
In some embodiments, the nucleic acid molecule encoding such CAR construct further comprises a sequence encoding a T2A ribosome-hopping element and/or a tgfr sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosome jump element as set forth in SEQ ID NO. 1 or 4, or an amino acid sequence exhibiting 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. 1 or 4.
In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be generated to express truncated EGFR (tgfr) as a non-immunogenic selection epitope (e.g., by introducing constructs encoding CAR and tgfr separated by a T2A ribosomal switch to express both proteins from the same construct), and such cells can then be detected using the truncated EGFR as a marker (see, e.g., U.S. patent No. 8,802,374). In some embodiments, the sequence encodes a tEGFR sequence shown in SEQ ID NO. 2 or 3 or an amino acid sequence exhibiting 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. In some cases, peptides such as T2A may result in ribosomes skipping synthesis of peptide bonds at the C-terminus of the 2A element (ribosome skipping), resulting in separation between the 2A sequence end and the next peptide downstream (see, e.g., de Felipe. Genetic VACCINES AND Ther.2:13 (2004) and deFelipe et al Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that may be used in the methods and nucleic acids disclosed herein include, but are not limited to, the 2A sequence from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 8), the 2A sequence from equine rhinitis A virus (E2A, e.g., SEQ ID NO: 7), the 2A sequence from Leptospira Minus beta tetrad virus (Thosea asigna virus) (T2A, e.g., SEQ ID NO: 1 or 4), and the 2A sequence from porcine tescens virus (porcine teschovirus) -1 (P2A, e.g., SEQ ID NO: 5 or 6), as described in U.S. patent publication No. 20070116690.
Recombinant receptors (e.g., CARs) expressed by cells administered to a subject typically recognize or specifically bind to molecules expressed in, associated with, and/or unique to the disease or disorder being treated or cells thereof. Upon specific binding to a molecule, e.g., an antigen, the receptor typically delivers an immunostimulatory signal (e.g., an ITAM-transduced signal) into the cell, thereby facilitating an immune response that targets the disease or disorder. For example, in some embodiments, the cell expresses a CAR that specifically binds to an antigen expressed by or associated with a cell or tissue of a disease or disorder.
B. Chimeric autoantibody receptors (CAAR)
In some embodiments, the recombinant receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR binds (e.g., specifically binds) or recognizes an autoantibody. In some embodiments, cells expressing CAAR (e.g., T cells engineered to express CAAR) may be used to bind to and kill cells expressing autoantibodies, rather than cells expressing normal antibodies. In some embodiments, the cells expressing the CAAR may be used to treat an autoimmune disease, such as an autoimmune disease, associated with the expression of an autoantigen. In some embodiments, cells expressing CAAR may target B cells that ultimately produce and display autoantibodies on their cell surfaces, which are labeled as disease-specific targets for therapeutic intervention. In some embodiments, CAAR expressing cells may be used to target disease-causing B cells by using antigen specific chimeric autoantibody receptors to effectively target and kill pathogenic B cells in autoimmune diseases. In some embodiments, the recombinant receptor is CAAR, as described in any one of U.S. patent application publication nos. US 2017/0051035.
In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling domains or domains (also interchangeably referred to as cytoplasmic signaling domains or regions). In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of stimulating and/or inducing a primary activation signal in a T cell, a signaling domain of a T Cell Receptor (TCR) component (e.g., an intracellular signaling domain or region of a CD3-Zeta (CD 3 Zeta) chain or a functional variant or signaling portion thereof), and/or a signaling domain comprising an immune receptor tyrosine activation motif (ITAM).
In some embodiments, the autoantibody binding domain comprises an autoantigen or fragment thereof. The choice of autoantigen may depend on the type of autoantibody targeted. For example, an autoantigen may be selected as a result of its recognition of an autoantibody on a target cell (e.g., B cell) associated with a particular disease state (e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease). In some embodiments, the autoimmune disease comprises Pemphigus Vulgaris (PV). Exemplary autoantigens include desmosomal mucin 1 (Dsg 1) and Dsg3.
C.T cell receptor (TCR)
In some embodiments, an engineered cell (e.g., a T cell) is provided that expresses a T Cell Receptor (TCR) or antigen binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide (e.g., an antigen of a tumor, virus, or autoimmune protein).
In some embodiments, a "T cell receptor" or "TCR" is a molecule or antigen-binding portion thereof that contains variable alpha and beta chains (also referred to as TCR alpha and TCR beta, respectively) or variable gamma and delta chains (also referred to as TCR alpha and TCR beta, respectively), and which is capable of specifically binding to peptides that bind to MHC molecules. In some embodiments, the TCR is in the αβ form. Generally, TCRs in the form of αβ and γδ are generally similar in structure, but T cells expressing them may have different anatomical locations or functions. TCRs can be found on the surface of cells or in soluble form. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to Major Histocompatibility Complex (MHC) molecules.
The term "TCR" is understood to encompass an intact TCR, as well as antigen-binding portions or antigen-binding fragments thereof, unless otherwise indicated. In some embodiments, the TCR is a complete or full length TCR, including TCRs in αβ form or γδ form. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but binds to a specific peptide that binds in an MHC molecule (e.g., to an MHC-peptide complex). In some cases, the antigen binding portion or fragment of the TCR may contain only a portion of the structural domain of the full length or complete TCR, but still be able to bind to a peptide epitope (e.g., MHC-peptide complex) that binds to the complete TCR. In some cases, the antigen binding portion contains the variable domains of the TCR (e.g., the variable alpha and beta chains of the TCR) sufficient to form a binding site for binding to a particular MHC-peptide complex. Typically, the variable chain of a TCR contains complementarity determining regions involved in the recognition of peptides, MHC and/or MHC-peptide complexes.
In some embodiments, the variable domain of the TCR contains hypervariable loops or Complementarity Determining Regions (CDRs), which are generally the major contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen binding sites of a given TCR molecule. The individual CDRs within the variable region of the TCR chain are typically separated by a Framework Region (FR) which generally exhibits less variability between TCR molecules than the CDRs (see, e.g., jores et al, proc.Nat' l Acad.Sci.U.S. A.87:9138, 1990; chothia et al, EMBO J.7:3745, 1988; see also Lefranc et al, dev.Comp.Immunol.27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or the most important of the three CDRs for antigen recognition and/or for interaction with the processed peptide portion of the peptide-MHC complex at a given TCR variable region. In some cases, CDR1 of the alpha chain may interact with the N-terminal portion of certain antigenic peptides. In some cases, CDR1 of the β chain may interact with the C-terminal portion of the peptide. In some cases, CDR2 has the strongest effect on interaction or recognition with the MHC portion of the MHC-peptide complex or is the predominant responsible CDR. In some embodiments, the variable region of the β chain may contain additional hypervariable regions (CDR 4 or HVR 4) that are normally involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).
In some embodiments, the TCR may also contain constant domains, transmembrane domains, and/or short cytoplasmic tails (see, e.g., janeway et al, immunology: the immunone SYSTEM IN HEALTH AND DISEASE, 3 rd edition, current Biology Publications, pages 4:33, 1997). In some aspects, each chain of the TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with a invariant protein of the CD3 complex involved in mediating signal transduction.
In some embodiments, the TCR chain comprises one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an alpha chain or a beta chain) may contain two immunoglobulin-like domains adjacent to the cell membrane, such as a variable domain (e.g., V.alpha. Or V.beta.; typically amino acids 1 through 116 based on Kabat numbering, kabat et al , "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, th edition) and a constant domain (e.g., an alpha chain constant domain or C.alpha., typically positions 117 through 259 of the chain based on Kabat numbering; or a beta chain constant domain or C.beta., typically positions 117 through 295 of the chain based on Kabat). For example, in some cases, the extracellular portion of a TCR formed by two chains contains two membrane proximal constant domains and two membrane distal variable domains, wherein the variable domains each contain CDRs. The constant domain of the TCR may contain a short linking sequence in which the cysteine residues form a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, the TCR may have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain.
In some embodiments, the TCR chain comprises a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules (e.g., CD3 and its subunits). For example, TCRs containing a constant domain and a transmembrane region may anchor the protein in the cell membrane and associate with a constant subunit of the CD3 signaling device or complex. The intracellular tail of the CD3 signaling subunits (e.g., the cd3γ, cd3δ, cd3ε, and cd3ζ chains) contain one or more immune receptor tyrosine activation motifs or ITAMs involved in the signaling capacity of the TCR complex.
In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ), or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (alpha and beta chains or gamma and delta chains) linked by, for example, one or more disulfide bonds.
In some embodiments, TCRs may be produced from one or more known TCR sequences (e.g., sequences of vα, β chains) whose substantially full-length coding sequences are readily available. Methods for obtaining full length TCR sequences (including V chain sequences) from cellular sources are well known. In some embodiments, the nucleic acid encoding the TCR may be obtained from a variety of sources, such as by Polymerase Chain Reaction (PCR) amplification of the nucleic acid encoding the TCR within or isolated from one or more given cells, or by synthesis of publicly available TCR DNA sequences.
In some embodiments, the TCR is obtained from a biological source, such as from a cell (e.g., from a T cell (e.g., a cytotoxic T cell)), a T cell hybridoma, or other publicly available source. In some embodiments, T cells may be obtained from cells isolated in vivo. In some embodiments, the TCR is a thymus-selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, the TCR, or an antigen-binding portion thereof, or an antigen-binding fragment thereof, may be synthetically produced based on knowledge of the TCR sequence.
In some embodiments, the TCR is produced from a TCR identified or selected by screening a candidate TCR library for a target polypeptide antigen or a target T cell epitope thereof. TCR libraries can be generated by expanding V alpha and V beta libraries from T cells isolated from a subject, including cells present in PBMCs, spleen, or other lymphoid organs. In some cases, T cells may be expanded from Tumor Infiltrating Lymphocytes (TILs). In some embodiments, TCR libraries may be generated from cd4+ or cd8+ T cells. In some embodiments, the TCR may be amplified from a T cell source in a normal or healthy subject, i.e., a normal TCR library. In some embodiments, TCRs may be amplified from a T cell source in a subject with disease, i.e., a library of diseased TCRs. In some embodiments, libraries of V.alpha.and V.beta.genes are amplified using degenerate primers, such as by performing RT-PCR in samples (e.g., T cells) obtained from humans. In some embodiments, scTv libraries can be assembled from natural vα and vβ libraries, wherein amplified products are cloned or assembled to be separated by linkers. Depending on the subject and the source of the cells, the library may be HLA allele specific. Alternatively, in some embodiments, a TCR library can be generated by mutagenesis or diversification of a parent or scaffold TCR molecule. In some aspects, the TCR is subjected to directed evolution, e.g., of the alpha or beta chain, such as by mutagenesis. In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, the selected TCR can be modified by affinity maturation. In some embodiments, antigen-specific T cells may be selected, such as by screening, to assess CTL activity against the peptide. In some aspects, TCRs present on antigen-specific T cells, for example, can be selected, such as by binding activity (e.g., a particular affinity or avidity) to an antigen.
In some embodiments, the TCR, or antigen-binding portion thereof, is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as having higher affinity for a particular MHC-peptide complex. In some embodiments, directed evolution is achieved by display methods including, but not limited to, yeast display (Holler et al (2003) Nat Immunol, 4, 55-62; holler et al (2000) Proc NATL ACAD SCI U S A, 97, 5387-92), phage display (Li et al (2005) Nat Biotechnol, 23, 349-54) or T cell display (Chervin et al (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display pathway involves engineering or modifying a known parent or reference TCR. For example, in some cases, a wild-type TCR may be used as a template for generating a mutagenized TCR in which one or more residues of the CDRs are mutated and mutants are selected that have the desired altered properties (e.g., higher affinity for the desired target antigen).
In some embodiments, the peptide used to produce or produce the target polypeptide of the TCR of interest is known or can be readily identified. In some embodiments, peptides suitable for use in generating a TCR or antigen-binding portion can be determined based on the presence of HLA restriction motifs in a target polypeptide of interest (target polypeptide as described below). In some embodiments, the peptides are identified using available in silico predictive models. In some embodiments, such models include, but are not limited to ProPred1 (Singh and Raghava (2001) Bioinformatics 17 (12): 1236-1237) and SYFPEITHI (see schulter et al (2007) Immunoinformatics Methods in Molecular Biology, 409 (1): 75-93 2007) for predicting MHC class I binding sites. In some embodiments, the MHC restriction epitope is HLA-A0201, which is expressed in about 39% -46% of all caucasians, and thus represents a suitable choice of MHC antigen for the preparation of TCRs or other MHC-peptide binding molecules.
The HLA-A0201 binding motifs and cleavage sites of proteasomes and immunoproteasome using computer predictive models are known. Such models for predicting MHC class I binding sites include, but are not limited to ProPred1 (described in more detail in Singh and Raghava, proPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17 (12): 1236-1237 2001) and SYFPEITHI (see Schulter et al SYFPEITHI Database for Searching and T-Cell Epitope Prediction., Immunoinformatics Methods in Molecular Biology, , volume 409 (1): 75-93 2007).
In some embodiments, the TCR, or antigen-binding portion thereof, can be a recombinantly produced native protein or a mutant form thereof (in which one or more characteristics (e.g., binding characteristics) have been altered). In some embodiments, the TCR may be derived from one of a variety of animal species, such as human, mouse, rat, or other mammal. TCRs may be cell-bound or in soluble form. In some embodiments, for the purposes of the provided methods, the TCR is in a cell-bound form expressed on the surface of a cell.
In some embodiments, the TCR is a full length TCR. In some embodiments, the TCR is an antigen-binding moiety. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has a structure as described in WO 03/020763, WO 04/033685, WO 2011/044186.
In some embodiments, the TCR comprises a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR does contain a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR is capable of forming a TCR complex with CD 3. In some embodiments, any TCR (including dTCR or scTCR) may be linked to a signaling domain that produces an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell.
In some embodiments, the dTCR comprises a first polypeptide (wherein the sequence corresponding to the TCR a chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR a chain constant region extracellular sequence) and a second polypeptide (wherein the sequence corresponding to the TCR β chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR β chain constant region extracellular sequence), the first and second polypeptides being linked by a disulfide bond. In some embodiments, the linkage may correspond to a native interchain disulfide linkage present in a native dimeric αβ TCR. In some embodiments, the interchain disulfide linkage is not present in a native TCR. For example, in some embodiments, one or more cysteines may be incorporated into the constant region extracellular sequence of a dTCR polypeptide pair. In some cases, natural and unnatural disulfide bonds may be desired. In some embodiments, the TCR contains a transmembrane sequence to anchor to a membrane.
In some embodiments, the dTCR comprises a TCR a chain (comprising a variable a domain, a constant a domain, and a first dimerization motif attached to the C-terminus of the constant a domain) and a TCR β chain (comprising a variable β domain, a constant β domain, and a first dimerization motif attached to the C-terminus of the constant β domain), wherein the first and second dimerization motifs readily interact to form a covalent bond between an amino acid of the first dimerization motif and an amino acid of the second dimerization motif, thereby linking the TCR a chain to the TCR β chain.
In some embodiments, the TCR is a scTCR. In general, scTCRs can be produced using known methods, see for example Soo Hoo, W.F. et al PNAS (USA) 89, 4759 (1992), W lfing, C. And Pluckthun, A., J.mol. Biol.242, 655 (1994), kurucz, I. et al PNAS (USA) 90 3830 (1993), international publications PCT Nos. WO 96/13593, WO 96/18105, WO 99/60120, WO 99/18129, WO 03/020763, WO 2011/044186, and Schlueter, C.J. et al J.mol. Biol.256, 859 (1996). In some embodiments, the scTCR contains an incorporated unnatural inter-chain disulfide bond to facilitate association of TCR chains (see, e.g., international publication PCT No. WO 03/020763). In some embodiments, the scTCR is a non-disulfide linked truncated TCR in which the heterologous leucine zipper fused to its C-terminus facilitates chain association (see, e.g., international publication PCT No. WO 99/60120). In some embodiments, the scTCR comprises a TCR a variable domain covalently linked to a TCR β variable domain via a peptide linker (see, e.g., international publication PCT No. WO 99/18129).
In some embodiments, the scTCR comprises a first segment comprising an amino acid sequence corresponding to a TCR α chain variable region, a second segment comprising an amino acid sequence corresponding to a TCR β chain variable region sequence (fused to the N-terminus of the amino acid sequence corresponding to a TCR β chain constant domain extracellular sequence), and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.
In some embodiments, the scTCR comprises a first segment (which consists of an a-chain variable region sequence fused to the N-terminus of an a-chain extracellular constant domain sequence) and a second segment (which consists of a β -chain variable region sequence fused to the N-terminus of a sequence β -chain extracellular constant and transmembrane sequence), and optionally a linker sequence (which connects the C-terminus of the first segment to the N-terminus of the second segment).
In some embodiments, the scTCR comprises a first segment (which consists of a TCR β chain variable region sequence fused to the N-terminus of a β chain extracellular constant domain sequence) and a second segment (which consists of an a chain variable region sequence fused to the N-terminus of a sequence a chain extracellular constant and transmembrane sequence), and optionally a linker sequence (which connects the C-terminus of the first segment to the N-terminus of the second segment).
In some embodiments, the linker of the scTCR that connects the first and second TCR segments may be any linker capable of forming a single polypeptide chain while preserving TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula-P-AA-P-, wherein P is proline and AA represents an amino acid sequence, wherein the amino acids are glycine and serine. In some embodiments, the first and second segments pair such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker is of sufficient length to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not so long as to block or reduce binding of the scTCR to the target ligand. In some embodiments, the linker may contain from 10 to 45 amino acids or from about 10 to about 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acid residues, e.g., 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula-PGGG- (SGGGG) 5-P-, wherein P is proline, G is glycine and S is serine (SEQ ID NO: 76). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 77)
In some embodiments, the scTCR contains a covalent disulfide bond that links a residue of an immunoglobulin region of a constant domain of an alpha chain to a residue of an immunoglobulin region of a constant domain of a beta chain. In some embodiments, there are no interchain disulfide bonds in the native TCR. For example, in some embodiments, one or more cysteines may be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, natural and unnatural disulfide bonds may be desired.
In some embodiments of dTCR or scTCR containing an introduced interchain disulfide bond, no native disulfide bond is present. In some embodiments, another residue is substituted with one or more native cysteines that form a native interchain disulfide bond, such as a serine or alanine substitution. In some embodiments, the introduced disulfide bond may be formed by mutating non-cysteine residues on the first and second segments to cysteines. Exemplary unnatural disulfide bonds for TCRs are described in published international PCT publication No. WO 2006/000830.
In some embodiments, the TCR, or antigen-binding fragment thereof, exhibits affinity for the target antigen with a equilibrium binding constant between or about 10 "5 and 10" 12M, and all individual values and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or ligand.
In some embodiments, one or more nucleic acids encoding a TCR (e.g., alpha and beta strands) can be amplified by PCR, cloning, or other suitable methods, and cloned into one or more suitable expression vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
In some embodiments, the vector may be a pUC series (FERMENTAS LIFE SCIENCES), pBluescript series (Stratagene, lajoba, california), pET series (Novagen, madison, wis.), pGEX series (PHARMACIA BIOTECH, uppsala, sweden) or pEX series (Clontech, palo Altuo, california). In some cases, phage vectors such as λg10, λgt11, λ ZapII (Stratagene), λembl4, and λnm1149 may also be used. In some embodiments, plant expression vectors may be used and include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, a viral vector, such as a retroviral vector, is used.
In some embodiments, the recombinant expression vector may be prepared using standard recombinant DNA techniques. In some embodiments, the vector may contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host (e.g., bacterial, fungal, plant, or animal) into which the vector is introduced, as appropriate and considering whether the vector is DNA-based or RNA-based. In some embodiments, the vector may contain a non-native promoter operably linked to a nucleotide sequence encoding a TCR or antigen-binding portion (or other MHC-peptide binding molecule). In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as the Cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and promoters found in the long terminal repeat of murine stem cell viruses. Other known promoters are also contemplated.
In some embodiments, to generate a vector encoding a TCR, the total cDNA of the α and β chains isolated from a T cell clone expressing the TCR of interest is PCR amplified and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the produced alpha and beta strands are incorporated into a retroviral (e.g., lentiviral) vector.
V. definition
Unless defined otherwise, all technical and scientific terms or nomenclature used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and such definitions contained herein should not be construed as indicating substantial differences from the commonly understood meanings in the art.
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is to be understood that the aspects and variations described herein include "consisting of" and/or "consisting essentially of" the aspects and variations.
The terms "at least one" and "one or more" are to be understood to include any integer greater than or equal to one, i.e., one, two, three, four, [. The term "plurality of" may be understood to include any integer greater than or equal to two, i.e., two, three, four, five, [..once again.
The phrase "at least one" with respect to a set of elements may be used herein to refer to at least one element from the group consisting of the elements. For example, the phrase "at least one" with respect to a set of elements may be used herein to refer to a selection of one of the listed elements, a plurality of individual listed elements, or a plurality of various listed elements.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as a inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose all possible sub-ranges as well as individual values within the range. For example, where a range of values is provided, it is to be understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term "about" as used herein refers to a general range of error for the corresponding value that is readily known. References herein to "about" a value or parameter include (and describe) implementations directed to the value or parameter itself. For example, a description relating to "about X" includes a description of "X".
As used herein, "event" or "cytometry event" refers to data measured from a single particle (e.g., a cell or synthetic particle) by a flow cytometer. Typically, the data measured from a single particle includes a plurality of parameters including one or more light scattering parameters and at least one fluorescence signal parameter. Thus, each event is represented as a vector of parameter measurements, where each measured parameter corresponds to one dimension of the data space.
"Fluorescent label (fluorescent marker)" or "fluorescent label (fluorescence marker)" refers to a fluorescent label comprising a fluorophore that is capable of absorbing energy over a range of wavelengths and releasing energy over a range of wavelengths different from the absorption range. Thus, it refers to a fluorescent compound that emits light when excited by light. It will be appreciated that the term "fluorescent label" or variants thereof may be used interchangeably with the term "fluorophore". The term "excitation wavelength" refers to the range of wavelengths over which the fluorophore absorbs energy. The term "emission wavelength" refers to the range of wavelengths over which a fluorophore emits energy or fluoresces.
As used herein, "fluorescence" or "fluorescence intensity" is an interchangeable term that refers to the output of a detection system that measures fluorescent radiation based on the intensity of a fluorogenic sample emitted by a particular fluorescent signal, such as emitted from a fluorescent label (e.g., fluorophore). Fluorescence intensity is the amount of light (photons) emitted by a fluorescent label after absorption of light or other electromagnetic radiation.
As used herein, "average fluorescence intensity" or "MFI" refers to the average value of fluorescence intensity in a particular fluorescent channel.
As used herein, "label" refers to a state in which a detectable label (e.g., a fluorescent label or stain) is attached. For example, cells of a population of cells may be labeled with one or more fluorescent markers such that one or more fluorescent signals may be measured by a flow cytometer.
As used herein, reference to an "enriched" cell population refers to one or more specific cell types or subsets or cell populations that have undergone an enrichment, isolation, or selection step to increase the number or percentage of the cell types or populations (e.g., as compared to their percentage in the starting cell population). Thus, it refers to increasing the percentage or frequency of such cells, or increasing the percentage or frequency of such cells relative to other cell types, such as by positive selection based on markers expressed by the population or cells, or by negative selection based on markers not present on the cell population or cells to be depleted. The term does not require complete removal of other cells, cell types or populations from the composition, and does not require that the so-enriched cells be present in the enriched composition at or even near 100%. In some embodiments, the enriched cell population contains greater than 50%, 60%, 70%, 80%, 90%, 95% or more of a particular cell type or subset. For example, the enriched population of cells can be an enriched population of T cells containing greater than 50%, 60%, 70%, 80%, 90%, 95% or more T cells (e.g., cd3+ cells) or a cd4+ or cd8+ subset thereof. Reference to positive selection or enrichment of cells of a particular type (such as those expressing a marker) refers to increasing the number or percentage of such cells, but does not require the complete absence of cells that do not express the marker. Likewise, negative selection, removal, or depletion of particular types of cells (such as those expressing a marker) refers to reducing the number or percentage of such cells, but need not result in complete removal of all such cells. For example, in some aspects, selection of one of the cd4+ or cd8+ populations enriches the population, i.e., the cd4+ or cd8+ population, but may also contain some residual or small percentage of other unselected cells, which in some cases may contain the other population of CD4 or CD8 that is still present in the enriched population.
As used herein, a statement that a cell or cell population is "positive" for a particular marker refers to the detectable presence of the particular marker (typically a surface marker) on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is substantially higher than that detected by the same procedure under otherwise identical conditions with an isotype-matched control, and/or that is substantially similar to that of a cell known to be positive for the marker, and/or that is substantially higher than that of a cell known to be negative for the marker.
As used herein, a statement that a cell or cell population is "negative" for a particular marker means that the particular marker (typically a surface marker) is not present on or in the cell in a substantially detectable manner. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is not detected by flow cytometry at a level that is substantially higher than that detected by the same procedure under otherwise identical conditions with an isotype-matched control, and/or that is substantially lower than that of cells known to be positive for the marker, and/or that is substantially similar to that of cells known to be negative for the marker.
As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human.
Exemplary embodiments VI
The provided embodiments include:
1. a method for assessing activation of T cells within a cellular composition, the method comprising:
(a) Detecting the surface expression level of or the 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:
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 strand), CD105 (endostatin), CD262 (DR 5, 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、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG 3k, 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, CD (IL-2 Rb), CD100, CD123, CD184 (CD 4), CD55, TIGIT (tm 3), CD196 (6), CD230 (CXCR) and CXCR 3, CD35 (rc) 230, CD261, CD35, CD9 (lrd 1, CD35, CD 24), felt (tsr 3), and felt (tsr 1, ct 3);
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 receptors, CD229 (Ly-9), CD84, and EGFR;
(b) Comparing the surface expression level or percentage of positive cells 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 percentage of positive cells of the marker in (i) is indicative of the T cells being activated, and a lower level or percentage of positive cells of the marker in (ii) is indicative of the T cells being activated, as compared to the reference.
2. The method of embodiment 1, wherein the reference consists of a non-stimulated control cell composition.
3. The method of embodiment 1, wherein 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.
4. The method of embodiment 1, wherein the reference is an average expression level or an average percentage of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.
5. The method of embodiment 1, wherein the reference is a median expression level or a median percentage of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.
6. A method for assessing surface expression of a T cell activating marker of a T cell, the method comprising detecting the level of surface expression of or the percentage of cells positive for one or more markers in cells comprising a composition of T cells, wherein the one or more markers are selected from group (i) and/or group (ii), wherein:
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 strand), CD105 (endostatin), CD262 (DR 5, 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、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG 3k, 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, CD (IL-2 Rb), CD100, CD123, CD184 (CD 4), CD55, TIGIT (tm 3), CD196 (6), CD230 (CXCR) and CXCR 3, CD35 (rc) 230, CD261, CD35, CD9 (lrd 1, CD35, CD 24), felt (tsr 3), and felt (tsr 1, ct 3); 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 receptors, CD229 (Ly-9), CD84, and EGFR.
7. The method of embodiment 6, wherein the surface expression level or percentage of positive cells of the one or more markers of (i) is positively correlated with T cell activation.
8. The method of embodiment 6 or 7, wherein the surface expression level or percentage of positive cells of the one or more markers in (ii) is inversely correlated with T cell activation.
9. A method of comparing activation of T cells in a donor, the method comprising:
(a) Detecting the level of surface expression of or the 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 the group (i) and/or the group (ii), wherein group (i) consists of chain ：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, CD105 (endostatin), CD262 (DR 5, 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、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD196 (CCR 6), CD230 (prion), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, DR3 (TRAIL), CD186 (CXCR 6), GARP (LRRC 32), CD116, CD334 (FGFR 4), 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 or percentage of positive cells of each of the one or more markers in an unstimulated cell composition, wherein a higher level or percentage of positive cells of the marker in (i) is indicative of the T cells being activated, and a lower level or percentage of positive cells of the marker in (ii) is indicative of the T cells being activated, as compared to the unstimulated cell composition.
10. The method of any one of embodiments 1-9, wherein prior to the detecting, the composition comprising T cells is incubated with a T cell stimulator under conditions that induce T cell activation.
11. The method of any one of embodiments 1-9, wherein the method comprises incubating the composition with a T cell stimulator prior to the detecting.
12. The method of any one of embodiments 1-9, wherein the method comprises incubating the composition with a T cell stimulator after the detecting.
13. The method of any one of embodiments 10-12, wherein the incubating with a T cell stimulating agent is performed in a subject.
14. The method of any one of embodiments 10-12, wherein the incubation with a T cell stimulator is performed in vitro or ex vivo.
15. The method according to any one of embodiments 10-14, wherein the incubation with the T cell stimulating agent is for 12-72 hours.
16. The method of any one of embodiments 10-15, wherein the incubation with the T cell stimulating agent is for about 24 hours.
17. The method of any one of embodiments 1-16, wherein the one or more markers of group (i) are selected from the group consisting of CD20, CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (FGFR 4), CD66a/c/e, and TSLPR (TSLP-R);
And the one or more markers of group (ii) are selected from the group consisting 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 receptors, CD229 (Ly-9), CD84, and EGFR.
18. The method of any one of embodiments 10-15, wherein the incubation with the T cell stimulating agent is for about 48 hours.
19. The method of any one of embodiments 1-15 and 18, wherein the one or more markers of group (i) are selected from the group consisting of CD20, CD105 (endostatin), 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 element), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD230 (prions), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropil-1), CD49d, CD73 (IL-g R) and CD 5' -rp 3 (lrrp) 2, MERTK 3 and (lrrp 32)
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.
20. The method according to any one of embodiments 1-19, 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、CD165、CD355（CRTAM） and GPR56,
And the 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 of any one of embodiments 1-20, wherein the one or more markers are (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.
22. A method of identifying activated T cells, the method comprising detecting cell surface expression of one or more markers in cells of a composition comprising T cells, wherein the one or more markers 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、CD165、CD355（CRTAM） and GPR56, and wherein cells expressing high levels of the one or more markers are activated T cells.
23. The method of any of embodiments 1-22, wherein the one or more markers is selected from the group (i) and consists of CD200 (OX 2), CD357 (GITR), CD120b, CD155 (PVR), CD107b (LAMP-2).
24. The method of any one of embodiments 1-20, wherein the one or more labels are of (ii) and are 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 of identifying activated T cells, the method comprising detecting cell surface expression of one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from the group consisting of 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 of any one of embodiments 1-25, wherein the detecting is detecting cd4+ or cd8+ T cells in the composition comprising T cells.
27. The method according to any one of embodiments 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,
And the one or more markers from group (ii) are selected from the group consisting of CD49f, CD124 (IL-4rα), CCRL2, CD217, CD192 (CCR 2), CD96 (TACTILE) and CD195 (CCR 5).
28. The method of any one of embodiments 1-25, wherein the detecting is detecting cd4+ T cells in the composition comprising T cells.
29. The method of embodiment 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 the group consisting of CD49f, CD124 (IL-4 ra), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7 ra).
30. A method for assessing surface expression of a T cell activation marker for a CD4+ T cell, the method comprising detecting the level of surface expression of or the percentage of cells positive for one or more markers in a CD4+ T cell 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-4Rα), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7Rα).
31. The method of embodiment 30, wherein the surface expression level or percentage of positive cells of the one or more markers in (i) is positively correlated with cd4+ T cell activation.
32. The method of embodiment 30 or 31, wherein the surface expression level or positive cell percentage of the one or more markers in (ii) is inversely correlated with cd4+ T cell activation.
33. A method for assessing activation of cd4+ T cells, the method comprising:
(a) Detecting the surface expression level of or the 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 the group consisting of 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-4Rα), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7Rα), and
(B) Comparing the surface expression level or the percentage of positive cells to the level or percentage of positive cells of each of the one or more markers in cells of an unstimulated control cell composition, wherein a higher level or percentage of positive cells of the marker in (i) is indicative of the cd4+ T cells being activated compared to the unstimulated control cell composition and a lower level or percentage of positive cells of the marker in (ii) is indicative of the cd4+ T cells being activated compared to the unstimulated control cell composition.
34. The method of any one of embodiments 1-25, wherein the detecting is detecting cd8+ T cells in the composition comprising T cells.
35. The method of embodiment 34, wherein the one or more markers selected from the group (i) is 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 the group consisting of CD96 (TACTILE), CD195 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1 and CD127 (IL-7rα).
36. A method for assessing surface expression of a T cell activation marker for a CD8+ T cell, the method comprising detecting the level of surface expression of or the percentage of cells positive for one or more markers in a CD8+ T cell 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1 and CD127 (IL-7Rα).
37. The method of embodiment 36, wherein the surface expression level or percentage of positive cells of the one or more markers of (i) is positively correlated with cd8+ T cell activation.
38. The method of embodiment 37, wherein the surface expression level or percentage of positive cells of the one or more markers of (ii) is inversely correlated with cd8+ T cell activation.
39. A method for assessing activation of cd8+ T cells, the method comprising:
(a) Detecting the surface expression level of or the 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 the group consisting of 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1 and CD127 (IL-7Rα), and
(B) Comparing the surface expression level or the percentage of positive cells to the level or percentage of positive cells of each of the one or more markers in cells of an unstimulated control cell composition, wherein a higher level or percentage of positive cells of the marker in (i) is indicative of the cd8+ T cells being activated, and a lower level or percentage of positive cells of the marker in (ii) is indicative of the cd8+ T cells being activated, as compared to the unstimulated control cell composition.
40. The method of any one of embodiments 1-39, wherein the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor.
41. The method of embodiment 27, wherein the one or more markers selected from the 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-4Rα), CD217, CD192 (CCR 2), CD195 (CCR 5) and CD96 (TACTILE).
42. A method for assessing activation of T cells, the method comprising:
(a) Detecting the surface expression level of or the 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 the group consisting of 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,
And set (ii) consists of ：CD120b、CD83、CD357（GITR）、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD355（CTRAM）、CD155（PVR）、CD74、CD170（Siglec-5） and GPR56.
43. The method of embodiment 41 or embodiment 42, wherein the one or more markers from group (i) are selected from the group consisting of CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74 and CD170 (Siglec-5),
And the one or more markers selected from group (ii) are selected from the group consisting of CD49f, CCRL2, CD124 (IL-4rα), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE).
44. The method of any one of embodiments 41-43, wherein the detecting is detecting a recombinant receptor expressing cd4+ T cell in the composition comprising a T cell.
45. The method of embodiment 44, wherein the one or more markers from group (i) are selected from the group consisting of CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR) and CD74,
And/or the one or more markers selected from group (ii) is selected from the group consisting of CD49f, CCRL2 and CD124 (IL-4 ra).
46. The method of any one of embodiments 41-43, wherein the detecting is detecting a recombinant receptor expressing cd8+ T cell in the composition comprising a T cell.
47. The method of embodiment 46, wherein the one or more markers from group (i) are selected from the group consisting of CD200 (OX 2), CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM) and GPR56,
And/or the one or more markers selected from group (ii) is selected from CCRL2, CD217, CD96 (TACTILE).
48. The method according to any one of embodiments 1-16, wherein the one or more markers of group (i) are selected from the group consisting of CD36L (SCARB 1, SR-BI), CD262 (DR 5, trail-R2), CD105 (endostatin), CD73 (extracellular-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).
49. A method for assessing activation of T cells, the method comprising:
(a) Detecting the surface expression level of or the 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 the group (i) and/or the group (ii), wherein the group (i) consists of CD36L (SCARB 1, SR-BI), CD262 (DR 5, trail-R2), CD105 (endostatin), CD73 (extracellular-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).
50. The method of embodiment 48, wherein the composition comprising T cells comprises cells that express a recombinant receptor.
51. The method of embodiment 49 or embodiment 50, wherein the detecting is detecting recombinant receptor-expressing cells of the composition of T cells.
52. The method of any one of embodiments 49-51, wherein the surface expression of the one or more markers of group (i) is increased on cells expressing the recombinant receptor as compared to cells not expressing the recombinant receptor.
53. The method of any one of embodiments 49-52, wherein the surface expression of the one or more markers of group (ii) is reduced on cells expressing the recombinant receptor as compared to cells not expressing the recombinant receptor.
54. The method of any one of embodiments 3-47, wherein 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 activation of recombinant receptor-dependent T cells.
55. The method of any one of embodiments 27-54, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR).
56. The method of embodiment 54 or embodiment 55, wherein the recombinant receptor stimulant comprises a recombinant target antigen recognized by the recombinant receptor.
57. The method of embodiment 54 or embodiment 55, wherein the recombinant receptor stimulant is an antibody specific for an extracellular antigen-binding domain of the recombinant receptor.
58. The method of embodiment 54 or embodiment 55, wherein the recombinant receptor stimulant is an anti-idiotype antibody specific for an extracellular antigen-binding domain of the recombinant receptor.
59. The method of any one of embodiments 54-58, wherein the recombinant receptor stimulant is immobilized or attached to a solid support.
60. The method of embodiment 59, wherein the solid support is a surface of a vessel, optionally a well or flask of a microplate.
61. The method of embodiment 59, wherein the solid support is a bead.
62. The method of embodiment 54 or embodiment 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 a subject.
63. The method of embodiment 62, wherein the antigen expressing cell is a cell line.
64. The method of embodiment 63, wherein the cell line is a tumor cell line.
65. The method of embodiment 63, wherein the antigen-expressing cell is a cell that has been engineered to express an antigen of the recombinant receptor.
66. The method of any one of embodiments 27-65, wherein the detecting is detecting recombinant receptor expressing T cells in the composition comprising T cells.
67. The method of any one of embodiments 1-26, wherein 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、CD120b、CD83、CD357（GITR）、CD200（OX2） and CD134 (OX 40),
And/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD192 (CCR 2), CD11b and CX3CR1.
68. The method of any one of embodiments 1-26, wherein the one or more markers from group (i) are selected from the group consisting of CD71, notch 1, CD107a (LAMP-1), CD166, CD245 (p 220/240), CD154, notch 2, CD165, and CD83,
And/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD11b and CX3CR1.
69. The method of embodiment 67 or 68, wherein said detecting is detecting cd4+ T cells in said composition comprising T cells.
70. The method of embodiment 69, wherein the one or more markers from group (i) are selected from the group consisting of CD71, notch 1, CD107a (LAMP-1), CD166, CD245 (p 220/240), CD154, notch 2, and CD165
And/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE) and CD127 (IL-7 Ra).
71. The method of embodiment 67 or 68, wherein said detecting is detecting cd8+ T cells in said composition comprising T cells.
72. The method of embodiment 71, wherein 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 CD11b, CX3CR1 and CD127 (IL-7 Ra).
73. The method of any one of embodiments 67-72, wherein said T cell stimulating agent is a pan T cell activator.
74. The method of embodiment 73, wherein the pan-T cell activating agent comprises an anti-CD 3 antibody and an anti-CD 28 antibody, optionally wherein the pan-T cell activating agent comprises an anti-CD 3 Fab and an anti-CD 28 Fab.
75. The method of embodiment 73 or embodiment 74, wherein the pan-T cell activating agent comprises anti-CD 3/anti-CD 28 beads.
76. The method of embodiment 73 or embodiment 74, wherein the pan-T cell activating reagent comprises a soluble anti-CD 3/anti-CD 28 streptavidin oligomerization reagent.
77. The method of any one of embodiments 1-76, wherein prior to the detecting of step (a), the method comprises contacting cells of the composition comprising T cells with one or more binding agents that bind the one or more labels.
78. The method of any one of embodiments 1-76, wherein prior to the detecting of step (a), the method comprises contacting cells of the composition comprising T cells with one or more binding agents comprising a substance for binding the one or more markers.
79. The method of embodiment 77 or embodiment 78, wherein the one or more binding agents are one or more antibodies or antigen binding fragments.
80. The method of any one of embodiments 77-79, wherein the one or more binding agents are detectably labeled.
81. The method of embodiment 80, wherein the one or more binding agents are fluorescently labeled.
82. The method of any one of embodiments 1-81, wherein the detecting is performed by flow cytometry.
83. The method of any one of embodiments 1-81, wherein the detecting of step (a) is performed in combination with a CITE-Seq or REAP-Seq.
84. The method according to any one of embodiments 1-81, wherein the detection of step (a) is performed by immunohistochemistry, optionally immunohistochemical fluorescence.
85. The method of any one of embodiments 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 labels 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 to one or more markers of the method of any one of embodiments 1-85.
87. The kit of embodiment 86, wherein the substance for detecting each of the one or more markers is an antibody.
88. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from the group (i) and/or group (ii), wherein group (i) consists of chain ：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, CD105 (endostatin), CD262 (DR 5, 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、 binder), CD323 (JAM 3), CD45RA, CD49c (integrin a 3), 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, CD (IL-2 Rb), CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD196 (CCR 6), CD230 (prion), CD235ab, CD261 (DR 4, TRAIL-R1), CD304 (neuropilin-1), CD49d, DR3 (TRAIL), CD186 (CXCR 6), GARP (LRRC 32), CD116, CD334 (FGFR 4), CD66a/c/e and TSLPR (TSLP-R),
And group (ii) consisted 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 receptors, CD229 (Ly-9), CD84 and EGFR.
89. A kit for determining T cell activation comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of CD20, CD100, CD123, CD184 (CXCR 4), CD55, TIGIT (VSTM 3), CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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 (FGFR 4), CD66a/c/e and TSLPR (TSLP-R),
And group (ii) consisted 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 receptors, CD229 (Ly-9), CD84 and EGFR.
90. A kit for determining T cell activation comprising a binding agent comprising a substance for binding to one or more markers selected from the group (i) and/or group (ii), wherein group (i) consists of CD20, CD105 (endostatin), 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、 binder), CD323 (JAM 3), CD357 (GITR), CD45RA, CD49c (integrin a 3), 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-2 Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR 4), CD55, TRAGIT (VSTM 3), CD230 (prion), CD235, CD261, CD4, CD49c (integrin a 3), CD3, 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, CD (IL-2 Rb), CD360, CD35, TRAGIT (CD 3), CD235, CD3, CD35 (RC 4, CD1, CD 3),
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）、CD11b、CX3CR1、NKp80、CD127（IL-7Ra） and CD49f.
91. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of 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-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b and CX3CR1 as follows.
92. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of 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-4Rα), CCRL2, CD217, CD192 (CCR 2), CD96 (TACTILE) and CD195 (CCR 5).
93. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of 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-4Rα), CCRL2, CD217, CD192 (CCR 2), CD195 (CCR 5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7Rα).
94. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of 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 (CCR 5), CD217, CCRL2, CD192 (CCR 2), CD11b, CX3CR1 and CD127 (IL-7Rα).
95. A kit for determining T cell activation comprising a binding agent comprising a substance for binding to one or more markers selected from the group (i) and/or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX 2), CD134 (OX 40), CD107b (LAMP-2), CD155 (PVR), CD74,
And the one or more markers selected from group (ii) consists of CD49f, CCRL2, CD124 (IL-4Rα), CD217, CD192 (CCR 2), CD355 (CRTAM), GPR56, CD195 (CCR 5) and CD96 (TACTILE).
96. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of 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 (OX 40),
And/or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR 5), CD96 (TACTILE), CD127 (IL-7 Ra), CD192 (CCR 2), CD11b and CX3CR1.
97. A kit for determining T cell activation comprising a binding agent comprising a substance for binding one or more markers selected from the group consisting of group (i) and/or group (ii), wherein group (i) consists of CD36L (SCARB 1, SR-BI), CD262 (DR 5, trail-R2), CD105 (endostatin), CD73 (extracellular-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).
98. The kit of any one of embodiments 86-97, wherein the binding agent is one or more antibodies or antigen-binding fragments.
99. The kit of any one of embodiments 86-98, wherein the binding agent is detectably labeled.
100. The kit of embodiment 99, wherein the binding agent is fluorescently labeled.
101. A method of isolating activated T cells, the method comprising identifying a population of activated T cells according to the method of any one of embodiments 1-85 and isolating the population.
102. A method of enriching for activated T cells, the method comprising identifying a population of activated T cells according to the method of any one of embodiments 1-85 and selecting the population, thereby obtaining a population of cells enriched for activated T cells.
103. A method of 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 the method of any one of embodiments 1-85.
104. A population of T cells produced according to the method of any one of embodiments 101-103.
Examples VII. Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1:T identification of cell-activated markers
A non-limiting assay was performed to evaluate T cell surface markers differentially expressed by T cell activation. Healthy donor T cells or T cells engineered with Chimeric Antigen Receptor (CAR) were stimulated and the change in percentage of positive cells was calculated based on a comparison of the signal to a blank sample without staining antibody added.
A.T cell stimulation and staining
1. T cell sample for analysis
T cell compositions containing anti-CD 19 CAR expressing T cells were generated from a leukapheresis sample from three healthy human adult donors by a process comprising selecting T cells (including cd4+ and cd8+ cells) from the sample based on immune affinity, thereby generating two compositions enriched for cd8+ and cd4+ cells, respectively. Cells of enriched cd4+ and cd8+ compositions were individually activated with anti-CD 3/anti-CD 28 beads and lentivirally transduced with vectors encoding anti-CD 19 Chimeric Antigen Receptor (CAR). The anti-CD 19 CAR contains an anti-CD 19 scFv derived from a murine antibody (variable region derived from FMC 63), an immunoglobulin derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD 3-zeta intracellular signaling domain. The transduced populations are then separately incubated in the presence of a stimulating agent for cell expansion. Amplified cd8+ and cd4+ cells were formulated separately and cryopreserved and stored.
Peripheral Blood Mononuclear Cells (PBMCs) from healthy donor apheresis were also obtained and cryopreserved.
2. T cell stimulation
To stimulate CAR engineered T cell samples, thawed cd4+ and cd8+ compositions that had been engineered with anti-CD 19 CAR alone were combined at a 1:1 ratio of CD8 and CD 4T cells. The cells are then CAR-dependent stimulated by transferring 100 x 10 6 cells in a sample of a CAR-expressing CD4/CD 8T cell composition into a culture flask pre-coated with an anti-idiotype antibody directed against the CD19 CAR (see, e.g., published PCT application No. WO 2018023100). The flask was coated with 50 mL of 11.8 μg/mL anti-idiotype solution.
To stimulate PBMC samples, PBMCs from apheresis samples were thawed and 100 x 10 6 cells were subjected to pan T cell stimulation by transferring them at a bead to cell ratio of 1:1 into a culture flask containing beads conjugated to anti-CD 3 and anti-CD 28 antibodies (e.g., dynabeads).
Both samples were incubated in medium containing 5% human serum supplemented with 110, 600 and 20 IU of IL-2, IL-7 and IL-15, respectively. A fresh set of identical samples was thawed as a non-stimulated control, and the cell control samples were also evaluated in each experimental run.
3. Non-limiting dyeing
After 24 hours or 48 hours of stimulation, 50x10 6 cells from each sample were transferred into 15 mL conical tubes and washed with Dulbecco Phosphate Buffered Saline (DPBS) followed by Live/read Blue staining. The samples were then blocked with a surface blocking mixture containing 10% normal mouse serum and 10 μg/mL human IgG in DPBS.
Next, samples were fluorescent barcoded by staining each sample with a unique combination of CD 45-fluorophore conjugated antibodies shown in table E1. The samples were then washed, combined into a 50mL conical tube, and stained with the 25-color framework antibody mixture shown in table E2. The multiplexed samples are then dispensed into wells (e.g., about 300) of one or more 96-well plates. Each well of the plate contains a unique antibody conjugated to PE such that during collection, each well is stained with a unique label on the backbone mixture plus PE, except for one well that does not have PE antibody and serves as a blank well. All antibodies used were pre-titrated to obtain the optimal concentration. Single-stained reference controls were generated using compensation beads. The reference control was matched to the antibody batch used for staining and collected using the same experimental conditions.
Erythrocytes were lysed and the samples were fixed and stored overnight at 4o protected from light, then analyzed by flow cytometry on a 5-laser Cytek Aurora.
Table E1 CD45 bar code
Table E2:25 color backbone stack
Cell samples were stained for analysis by non-flow restriction, using massively parallel cytometry processing data with machine learning. After acquisition, the unmixed correction is performed manually. Viable single cells were gated on the samples using OMIQ software and exported as purified Flow Cytometry Standard (FCS) files. The cleaned data is then gated to a subset of interest (CD 4T cells, CD 8T cells, B cells, etc.). Within each subset, PE positive percentages were calculated using PE blank wells as Fluorescence Minus One (FMO) controls. Within each subset, the Median Fluorescence Intensity (MFI) of the PE channels is also output. The PE positive percentage statistic is output and the data is transmitted to the R Studio for analysis using R packet unrestricted flow (R package infinityFlow). The difference in PE positive percentages for each marker/subset/sample/time point combination of interest was calculated.
B. Results
The data is interrogated in several different ways to analyze differential expression of T cell activation markers on cells of the stimulated sample.
1. Examination of markers across samples
The data were filtered to focus on the differences between the control time points of 48 hours (-48 hours) prior to stimulation and 0 hours without stimulation from the four chambers cd4+ PBMC cells, cd8+ PBMC cells, cd4+ anti-cd19car+ cells and cd8+ anti-cd19car+ cells. The top 15 up-regulation markers (based on percent increase in PE positive%) and the top 5 down-regulation markers (based on percent decrease in PE positive cells) were determined in various cell subsets in the 48 hour stimulated sample compared to the unstimulated sample. The top 15 upregulated T cell activation markers and top 5 downregulated T cell activation markers for cd8+ anti-cd19 car+ T cells compared between the-48 hour stimulated and 0 hour unstimulated samples are shown in fig. 1A, and the top 15 upregulated T cell activation markers and bottom 5 downregulated T cell activation markers for cd4+ anti-cd19 car+ T cells compared between the-48 hour stimulated and 0 hour unstimulated samples are shown in fig. 1B. The top 15 upregulated T cell activation markers and top 5 downregulated T cell activation markers for cd8+ PBMC T cells compared between the-48 hour stimulated and 0 hour unstimulated samples are shown in fig. 2A, and the top 15 upregulated T cell activation markers and top 5 downregulated T cell activation markers for cd4+ PBMC T cells compared between the-48 hour stimulated and 0 hour unstimulated samples are shown in fig. 2B. Among the top 15 upregulated T cell activation markers in all groups are highly increased non-canonical markers (e.g., markers that are not known to be classically associated with activation status), indicating their utility as activation markers in T cells.
The markers identified above are analyzed to determine if upregulation of the markers is specific for PBMC T cells or anti-cd19 car+ T cells or both, or is specific for cd4+ or cd8+ T cells or both. As shown in fig. 3A (cd4+ T cells) and fig. 3B (cd8+ T cells), certain T cell activation markers were upregulated either after pan-T cell activation of PBMCs or after CAR-dependent stimulation of CAR-expressing T cells. In some cases, the marker is upregulated after stimulation of both PBMCs and CAR-expressing T cells. The absence of specific bars indicating the% PE positive cells of certain markers does not mean that those markers are identical between unstimulated and stimulated samples, only that they are not present in the top 15 up-regulated markers or the bottom 5 down-regulated markers of cd4+ or cd8+ T cells when PBMC or CAR expressing T cell composition samples are stimulated.
2. Non-canonical markers for activating enrichment in cells
The presence of anti-cd19car+ T cell activation markers on activated T cells upregulated by stimulation was further assessed by comparison to standard canonical markers. In R Studio, boolean OR gates, HLA-DR, CD25 and 4-1BB, are created on the 3 canonical activation markers in the framework stack, and NOT gates are created based on the HLA-DR or CD25 or 41BB gates. T cell activation was assessed using three canonical markers as standard markers. The non-canonical tags are gated in an "active or" gate and an "active not" gate. From this analysis, a subset of five non-canonical markers (CD 200, CD357, CD120b, CD155, and CD107 b) were identified, with a much higher percentage of positives for non-canonical markers in the "activated or" gates compared to the "activated not" gates, as shown in fig. 4. Because each of these non-canonical markers increased on cells that showed activation using canonical markers, the results indicate that these non-canonical markers can be used to evaluate activation.
3. Relationship between non-canonical markers and canonical markers
A scatter plot was generated for each of the 40 markers shown in fig. 1A-1B and fig. 2A-2B, where each marker was compared to the three standard markers (HLA-DR, CD25 and 41 BB) used to generate fig. 4. FIGS. 5A-5S show the results of finding non-canonical markers including CD71、CD165、CD107a（LAMP-1）、CD107b（LAMP-2）、CD200（OX2）、CD245（p220/240）、CD74、CD83、GPR56、CD134、CD154、CD170（Siglec-5）、Notch 1、CD155（PVR）、CD357（GITR）、CD166、CD355（CRTAM）、Notch 2 and CD120b that are up-regulated in activated T cells. FIGS. 6A-6J show the results of the discovery of canonical markers up-regulated in activated T cells, including CD70, CD109, CD223 (LAG-3), CD25, CD62L, CD, CD137 (4-1 BB), CD152 (CTLA-4), CD274 (B7-H1, PD-L1), and CD179 (PD-1). FIGS. 7A-7K show the results of an observation of a non-canonical marker down-regulated in activated T cells, including CD11b、CD96（TACTILE）、CD127（IL-7Ra）、CCRL2、CD49f、CD195（CCR5）、CX3CR1、CD217、CD124（IL-4Ra）、CD192（CCR2）、KLRG1（MAFA）. that there is a linear relationship between a canonical marker and some non-canonical and canonical markers up-regulated upon T cell activation, indicating that there is a relationship between the non-canonical marker and the canonical marker. There was no linear relationship between non-canonical markers down-regulated by T cell activation and standard markers (fig. 7A-5K), where low biscationic populations could be seen in the scatter plots.
4. Examination of up-regulation of time-point specific markers
To determine if the markers were specifically up-regulated at different time points, the difference in up-regulation of the markers at different stimulation time points was examined for different cell populations (cd4+ PBMC cells, cd8+ PBMC cells, cd4+ anti-cd19car+ cells and cd8+ anti-cd19car+ cells). The time points assessed included (1) a control of 48 hours stimulation with 0 hours unstimulated (T0-T48), (2) 48 hours stimulation with 24 hours stimulation (T24-T48), and (3) a0 hour sample of 24 hours stimulation with unstimulated (T0-T24). All markers having a 10% difference between the compared time points are plotted in the venn plot shown in fig. 8, wherein the number of markers present in each zone is listed along with a representative marker from each group. A total of 145 markers were identified, some of which were up-regulated in all three comparisons, as indicated by the center, while others were specific for the different comparisons, indicating that some were time-sequence specific markers for detecting cell activation after stimulation.
Example 2 identification of T cell surface markers mediated by CAR mediated T cell activation
The data generated as part of example 1 was re-analyzed to find whether there was any activation marker differential expression between the car+ and CAR-compartments. A description of the analysis strategy is provided below.
A. Results
After acquisition, the unmixed correction is performed manually. Viable single cells were obtained using OMIQ software to gate the samples. The purified data is then demultiplexed into individual samples according to the planned fluorescence barcoding used. After sample de-multiplexing, four main subsets of interest were gated, cd4+car+, cd4+car-, cd8+car+ and cd8+car-. Within each subset, PE positive percentages were calculated using blank wells as Fluorescence Minus One (FMO) control within each subset, outputting PE positive percentage statistics. The differences between CAR-and car+ chambers for each marker were calculated for each cd4+ and cd8+ subset at each time point. Markers with the greatest difference between CAR-and car+ chambers were pooled and cross-referenced against the flow data to identify fourteen markers with the greatest difference between car+ and CAR-cells 48 hours post-stimulation.
Histograms for each of the fourteen markers were generated for cd4+car+, cd4+car-, cd8+car+ and cd8+car-cells in the unstimulated, 24-hour (t=24 h) stimulated and 48-hour (t=48 h) stimulated groups (fig. 9A-9N). Table E3 lists fourteen markers, and whether they were up-or down-regulated in the t=48 h car+ group compared to the unstimulated (t=0) control, and in which graph each marker is shown. Of the 14 markers, all markers increased over time during stimulation, except for CD 96. CD96 decreases over time during stimulation. Most markers show a change in expression in cd8+ T cells prior to cd4+ T cells, and the difference in expression between car+ and CAR-cells is typically greater in cd8+ T cells for each marker. Of the 14 markers identified, only CD137L (4-1 BB), CD178 (Fas-L) and CD152 (CTLA-4) are canonical markers of T cell activation.
TABLE E3 markers with the greatest difference between CAR-and CAR+ cells
Example 3 comparison of T cell surface markers modulated by CAR-mediated T cell activation in different CAR-engineered T cell compositions
Conventional flow cytometry assays were performed to evaluate the expression of the 14 markers identified in example 2 in 72 hour stimulated, 48 hour stimulated, 24 hour stimulated and unstimulated cells in different CAR engineered T cell compositions engineered with different CARs and/or produced using different manufacturing processes.
A.T cell stimulation and staining
For each manufacturing process, T cell compositions containing CAR expressing T cells were generated from white blood cell apheresis samples from three different donors. In this example, four different manufacturing processes (process 1, process 2, process 3, and process 4) were compared. Process 3 is the same as that used to generate anti-CD 19 CAR expressing T cells used in examples 1 and 2. Process 1 differs from process 3 in that PBMCs are the starting source of cells, PBMCs are not enriched for cd4+ and cd8+ T cells prior to transduction, and cells are engineered with different CARs for BCMA. Process 2 differs from process 3 in that after selection of cd4+ and cd8+ T cells, the selected T cells are combined at a ratio of 1:1, and then the combined cd4+ and cd8+ T cells are transduced with a viral vector encoding a bispecific CAR against CD19 (containing VH and VL variable regions of murine FMC63 antibody) and then expanded. Process 4 is similar to process 2, in that cd4+ and cd8+ T cells are selected, combined at a ratio of 1:1, and then transduced with a viral vector encoding a CAR (in this case an anti-CD 19 CAR similar to the CAR described in example 1), however, in this process the cells are incubated with minimal expansion.
In summary, each manufacturing process was used to generate engineered compositions from 3 donor samples. All samples were from healthy donors, except that the sample of procedure 1 was from a multiple myeloma patient.
T cell compositions from each of four different treatments were thawed and counted. For procedure 3, individual thawed CD4+ and CD8+ T cells were combined in a 1:1 ratio, similar to the methods described in examples 1 and 2. For all other processes, the thawed composition contains cd4+ and cd8+ T cells.
After thawing and counting, CAR-dependent stimulation was performed on each T cell composition. For cell compositions produced from all processes except process 1, cells from the composition were exposed to an anti-idiotype antibody (directed against FMC63 variable domain of an anti-CD 19 CAR substantially as described in examples 1 and 2) coated on wells of a microplate at 11.84 ug/mL. For the procedure 1 samples, cells from the composition were exposed to human Fc BCMA conjugates coated on wells of microwells (to stimulate anti-BCMA CARs). All samples were incubated in medium containing 5% human serum supplemented with 200, 1200 and 200 IU of IL-2, IL-7 and IL-15, respectively.
After 72 hours, 48 hours and 24 hours of stimulation, between 0.5x10 6 and 2x10 6 viable cells from each sample were transferred into 96-well plates and washed with DPBS before staining with Live/read Blue. Samples were blocked with a surface blocking mixture containing 10% normal mouse serum and 10 μg/mL human IgG in DPBS.
Next, the samples were fluorescent barcoded by staining each sample with a unique combination of CD 45-fluorophore conjugated antibodies, such that each sample within the treatment method had a unique CD45 barcode. CD45 bar codes are shown in example 1 Table E1. The samples were then washed and combined with other samples from the same treatment process in a 15 mL conical tube. Next, all tubes were stained with gamma-delta T cell receptor (gdTCR) antibodies for 10 minutes, followed by staining with the 24-color scaffold antibody mixture described in table E4. Multiple samples from each procedure were then dispensed into 15 wells of a 96-well plate. Fourteen of the 15 wells contained unique PE conjugated activation markers as described in example 2. The fifteenth well contained no PE antibody as a Fluorescence Minus One (FMO) control. All antibodies used were pre-titrated to obtain the optimal concentration. Single-stained reference controls were generated using Slingshot Spectracomp and Viacomp beads. The reference control was matched to the antibody batch used for staining and collected using the same experimental conditions.
Table E4. 24 color frame stack of example 3.
Samples were fixed and immediately analyzed by flow cytometry on a 5-laser Cytek Aurora.
After acquisition, the unmixed correction is performed manually. Viable single cells were obtained using OMIQ software to gate the samples. The purified data is then demultiplexed into individual samples according to the fluorescent barcoding scheme used. After demultiplexing, samples were gated on car+ and CAR-. Within each subset, gate PE positive percentages (pe+%) were set individually for each barcode and marker, and pe+% metrics were output. The output data is then transmitted to the R studio for subsequent analysis.
B. Results
The data are plotted as area under the simulated curve (AUC), where the AUC values for each sample within each manufacturing process for both car+ and CAR-cells are calculated by summing the percent PE positive (PE%) for each marker across all time points.
The data are shown in fig. 10A-10B, which show AUC for each marker for car+ and CAR-cells grouped per manufacturing process. Significance testing was performed using rstatix packets, using paired t-test, and Benjamini-Hochberg correction for multiple hypothesis testing. In fig. 10A-10B, for each marker, the significance of each process and marker is shown above the bars as asterisks for car+ cells in each manufacturing process. In this figure, a default significance indicator of rstatix packets is applied, where a p value > 0.05 has no indicator, a p value <0.05 is indicated as "×", and a p value <0.01 is indicated as "×". The p-values of the AUC comparison between car+ and CAR-room for each marker within each manufacturing process are listed in table E5.
In summary, the results demonstrate similar modulation of T cell surface markers following CAR-dependent stimulation in different processes. Among these, certain markers such as CD83, notch, CD26L1, CD262, CD170, CD154 and CD73, and canonical markers FasL and 4-1BB, exhibited statistically significant upregulation in marker surface expression following CAR-dependent stimulation. Table E5 p-values for AUC comparison
To visualize when each marker reached peak expression in the car+ chamber, the expression of each marker over time was plotted, separated by manufacturing process, sample ID, and marker. Fig. 11A-11B show the data and percent positivity for each marker in the car+ chamber plotted on the y-axis, as well as the time plotted on the x-axis (in hours after stimulation). Each sample is represented as a separate line for each label and each process. Table E6 shows the point in time at which each marker reached its peak for each manufacturing process.
TABLE E6 time points of peak expression for each marker
The markers used in this experiment demonstrate enrichment or depletion in the car+ chamber following antigen-specific stimulation. Overall, trends were consistent within and between projects, indicating that they were used as markers of T cell activation and proliferation, regardless of manufacturing process and CAR used. Without wishing to be bound by theory, these results support the use of these markers to assess the functional quality of T cells produced by different manufacturing processes.
The present invention is not intended to be limited in scope by the specific disclosed embodiments, examples being provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods will be apparent from the description and teachings herein. Such changes may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.
Sequence(s)

Claims (104)

1.一种用于评估细胞组合物内的T细胞的激活的方法，所述方法包括：1. A method for evaluating T cell activation within a cellular composition, the method comprising: (a) 检测包含T细胞的组合物的细胞中一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中：(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: 组 (i) 的一种或多种标记物选自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链）、CD105（内皮联蛋白）、CD262（DR5、TRAIL-R2）、CD170（Siglec-5）、CD73（胞外-5'-核苷酸酶）、CD360（IL-21R）、CD20、CD107a（LAMP-1）、CD109、CD132（共同g链）、CD148、CD150（SLAM）、CD151（PETA-3）、CD154、CD165、CD166、CD200 R、CD217、CD218a（IL-18Ra）、CD227（MUC-1）、CD245（p220/240）、CD30、CD317（BST2、束缚素）、CD323（JAM3）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD97、整合素b7、小鼠IgG3 k、Notch 2、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、Ig轻链k、Ig轻链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）、神经节苷脂GD2、MERTK、TMEM8A、CD122（IL-2Rb）、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD196（CCR6）、CD230（朊病毒）、CD235ab、CD261（DR4、TRAIL-R1）、CD304（神经毡蛋白-1）、CD49d、DR3（TRAMP）、CD186（CXCR6）、GARP（LRRC32）、CD116、CD334（FGFR4）、CD66a/c/e和TSLPR（TSLP-R）；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); 并且组 (ii) 的一种或多种标记物选自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细胞受体、CD229（Ly-9）、CD84和EGFR；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) 将所述细胞组合物中的表面表达水平或阳性细胞百分比与参考物中的所述一种或多种标记物中的每一种的表面表达水平或阳性细胞百分比进行比较，其中与所述参考物相比，(i) 中的标记物的更高水平或更高阳性细胞百分比指示所述T细胞被激活，并且与所述参考物相比，(ii)中的标记物的更低水平或更低阳性细胞百分比指示所述T细胞被激活。(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.根据权利要求1所述的方法，其中所述参考物由未经刺激的对照细胞组合物构成。2. The method of claim 1, wherein the reference is composed of an unstimulated control cell composition. 3.根据权利要求1所述的方法，其中所述参考物是跨多种细胞组合物的表达水平或阳性细胞百分比，其中每种细胞组合物来自不同的患者、受试者或供体。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.根据权利要求1所述的方法，其中所述参考物是跨多种细胞组合物的平均表达水平或平均阳性细胞百分比，其中每种细胞组合物来自不同的患者、受试者或供体。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.根据权利要求1所述的方法，其中所述参考物是跨多种细胞组合物的中值表达水平或中值阳性细胞百分比，其中每种细胞组合物来自不同的患者、受试者或供体。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. 6.一种用于评估T细胞的T细胞激活标记物的表面表达的方法，所述方法包括检测包含T细胞的组合物的细胞中一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中：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: 组 (i) 的一种或多种标记物选自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链）、CD105（内皮联蛋白）、CD262（DR5、TRAIL-R2）、CD170（Siglec-5）、CD73（胞外-5'-核苷酸酶）、CD360（IL-21R）、CD20、CD107a（LAMP-1）、CD109、CD132（共同g链）、CD148、CD150（SLAM）、CD151（PETA-3）、CD154、CD165、CD166、CD200 R、CD217、CD218a（IL-18Ra）、CD227（MUC-1）、CD245（p220/240）、CD30、CD317（BST2、束缚素）、CD323（JAM3）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD97、整合素b7、小鼠IgG3 k、Notch 2、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、Ig轻链k、Ig轻链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）、神经节苷脂GD2、MERTK、TMEM8A、CD122（IL-2Rb）、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD196（CCR6）、CD230（朊病毒）、CD235ab、CD261（DR4、TRAIL-R1）、CD304（神经毡蛋白-1）、CD49d、DR3（TRAMP）、CD186（CXCR6）、GARP（LRRC32）、CD116、CD334（FGFR4）、CD66a/c/e和TSLPR（TSLP-R）；并且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 组 (ii) 的一种或多种标记物选自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细胞受体、CD229（Ly-9）、CD84和EGFR。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.根据权利要求6所述的方法，其中所述 (i) 中的一种或多种标记物的表面表达水平或阳性细胞百分比与T细胞激活呈正相关。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.根据权利要求6或7所述的方法，其中所述 (ii) 中的一种或多种标记物的表面表达水平或阳性细胞百分比与T细胞激活呈负相关。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. 9.一种比较供体内的T细胞的激活的方法，所述方法包括：9. A method for comparing the activation of T cells in vivo, the method comprising: (a) 检测在包含T细胞的组合物的细胞中一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组(ii)，其中组 (i) 由以下组成：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链）、CD105（内皮联蛋白）、CD262（DR5、TRAIL-R2）、CD170（Siglec-5）、CD73（胞外-5'-核苷酸酶）、CD360（IL-21R）、CD20、CD107a（LAMP-1）、CD109、CD132（共同g链）、CD148、CD150（SLAM）、CD151（PETA-3）、CD154、CD165、CD166、CD200 R、CD217、CD218a（IL-18Ra）、CD227（MUC-1）、CD245（p220/240）、CD30、CD317（BST2、束缚素）、CD323（JAM3）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD97、整合素b7、小鼠IgG3 k、Notch 2、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、Ig轻链k、Ig轻链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）、神经节苷脂GD2、MERTK、TMEM8A、CD122（IL-2Rb）、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD196（CCR6）、CD230（朊病毒）、CD235ab、CD261（DR4、TRAIL-R1）、CD304（神经毡蛋白-1）、CD49d、DR3（TRAMP）、CD186（CXCR6）、GARP（LRRC32）、CD116、CD334（FGFR4）、CD66a/c/e和TSLPR（TSLP-R）；(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) 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-gR) 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, CD200R, 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 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-10R), 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); 并且组 (ii) 由以下组成：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细胞受体、CD229（Ly-9）、CD84和EGFR；Furthermore, 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) 将所述表面表达水平或所述阳性细胞百分比与未经刺激的细胞组合物中的所述一种或多种标记物中的每一种的水平或阳性细胞百分比进行比较，其中与所述未经刺激的细胞组合物相比，(i) 中的标记物的更高水平或更高阳性细胞百分比指示所述T细胞被激活，并且与所述未经刺激的细胞组合物相比，(ii)中的标记物的更低水平或更低阳性细胞百分比指示所述T细胞被激活。(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.根据权利要求1-9中任一项所述的方法，其中在所述检测之前，将所述包含T细胞的组合物与T细胞刺激剂在诱导T细胞激活的条件下孵育。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.根据权利要求1-9中任一项所述的方法，其中所述方法包括在所述检测之前将所述组合物与T细胞刺激剂孵育。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.根据权利要求1-9中任一项所述的方法，其中所述方法包括在所述检测之后将所述组合物与T细胞刺激剂孵育。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.根据权利要求10-12中任一项所述的方法，其中所述与T细胞刺激剂的孵育在受试者体内进行。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.根据权利要求10-12中任一项所述的方法，其中所述与T细胞刺激剂的孵育在体外或离体进行。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.根据权利要求10-14中任一项所述的方法，其中所述与T细胞刺激剂的孵育进行12-72小时。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.根据权利要求10-15中任一项所述的方法，其中所述与T细胞刺激剂的孵育进行约24小时。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.根据权利要求1-16中任一项所述的方法，其中所述组 (i) 的一种或多种标记物选自CD20、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD105（内皮联蛋白）、CD107a（LAMP-1）、CD109、CD120b、CD132（共同g链）、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、束缚素）、CD323（JAM3）、CD357（GITR）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD83、CD97、整合素b7、小鼠IgG3 k、Notch 1、Notch 2、CD107b（LAMP-2）、CD155（PVR）、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、CD74、GPR56、Ig轻链k、Ig轻链l、IgM、CD116、CD334（FGFR4）、CD66a/c/e和TSLPR（TSLP-R）；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); 并且所述组 (ii) 的一种或多种标记物选自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细胞受体、CD229（Ly-9）、CD84和EGFR。Furthermore, 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. 18.根据权利要求10-15中任一项所述的方法，其中所述与T细胞刺激剂的孵育进行约48小时。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.根据权利要求1-15和18中任一项所述的方法，其中所述组 (i) 的一种或多种标记物选自CD20、CD105（内皮联蛋白）、CD107a（LAMP-1）、CD109、CD120b、CD132（共同g链）、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、束缚素）、CD323（JAM3）、CD357（GITR）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD83、CD97、整合素b7、小鼠IgG3 k、Notch 1、Notch 2、CD107b（LAMP-2）、CD155（PVR）、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、CD74、GPR56、Ig轻链k、Ig轻链l、IgM、CD119（IFN-g R a链）、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）、神经节苷脂GD2、MERTK、TMEM8A、CD122（IL-2Rb）、CD360（IL-21R）、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD230（朊病毒）、CD235ab、CD261（DR4、TRAIL-R1）、CD304（神经毡蛋白-1）、CD49d、CD73（胞外-5'-核苷酸酶）、DR3（TRAMP）、CD186（CXCR6）和GARP（LRRC32）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). 并且所述组 (ii) 的一种或多种标记物选自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）和CD49f。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.根据权利要求1-19中任一项所述的方法，其中所述组 (i) 的一种或多种标记物选自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）和GPR56，20. 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. 并且所述组 (ii) 的一种或多种标记物选自CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b和CX3CR1。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.根据权利要求1-20中任一项所述的方法，其中所述一种或多种标记物是 (i) 的，并且选自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）和GPR56。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. 22.一种鉴定激活的T细胞的方法，所述方法包括检测包含T细胞的组合物的细胞中的一种或多种标记物的细胞表面表达，其中所述一种或多种标记物选自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）和GPR56，并且其中表达高水平的所述一种或多种标记物的细胞是激活的T细胞。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.根据权利要求1-22中任一项所述的方法，其中所述一种或多种标记物选自组 (i)，并且由以下组成：CD200（OX2）、CD357（GITR）、CD120b、CD155（PVR）、CD107b（LAMP-2）。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.根据权利要求1-20中任一项所述的方法，其中所述一种或多种标记物是 (ii) 的，并且选自CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b和CX3CR1。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. 25.一种鉴定激活的T细胞的方法，所述方法包括检测包含T细胞的组合物的细胞中的一种或多种标记物的细胞表面表达，其中所述一种或多种标记物选自CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b和CX3CR1，并且其中表达低水平的所述一种或多种标记物的细胞是激活的T细胞。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.根据权利要求1-25中任一项所述的方法，其中所述检测是检测所述包含T细胞的组合物中的CD4+或CD8+ T细胞。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.根据权利要求1-26中任一项所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD107b（LAMP-2）、CD120b、CD357（GITR）、CD83、CD200（OX2）、CD134（OX40）、CD155（PVR）、CD74、CD170（Siglec-5）、CD355（CRTAM）和GPR56，27. 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. 并且所述来自组 (ii) 的一种或多种标记物选自CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD96（TACTILE）和CD195（CCR5）。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.根据权利要求1-25中任一项所述的方法，其中所述检测是检测所述包含T细胞的组合物中的CD4+ T细胞。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.根据权利要求28所述的方法，其中所述来自组 (i) 的一种或多种标记物选自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和CD165，29. 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. 并且所述来自组 (ii) 的一种或多种标记物选自CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）和CD127（IL-7Rα）。And 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). 30.一种用于评估CD4+ T细胞的T细胞激活标记物的表面表达的方法，所述方法包括检测包含T细胞的组合物的CD4+ T细胞中的一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中组 (i) 由以下组成：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和CD165，30. 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. 并且组 (ii) 由以下组成：CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）和CD127（IL-7Rα）。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). 31.根据权利要求30所述的方法，其中所述 (i) 中的一种或多种标记物的表面表达水平或阳性细胞百分比与CD4+ T细胞激活呈正相关。31. The method of claim 30, wherein the surface expression level of one or more markers or the percentage of positive cells in (i) is positively correlated with CD4+ T cell activation. 32.根据权利要求30或31所述的方法，其中所述 (ii) 中的一种或多种标记物的表面表达水平或阳性细胞百分比与CD4+ T细胞激活呈负相关。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 (ii) is negatively correlated with CD4+ T cell activation. 33.一种用于评估CD4+ T细胞的激活的方法，所述方法包括：33. A method for assessing the activation of CD4+ T cells, the method comprising: (a) 检测包含T细胞的组合物的CD4+ T细胞中的一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中组 (i) 由以下组成：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和CD165，(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 of 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. 并且组 (ii) 由以下组成：CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）和CD127（IL-7Rα）；以及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) 将所述表面表达水平或所述阳性细胞百分比与未经刺激的对照细胞组合物的细胞中的所述一种或多种标记物中的每一种的水平或阳性细胞百分比进行比较，其中与所述未经刺激的对照细胞组合物相比，(i) 中的标记物的更高水平或更高阳性细胞百分比指示所述CD4+ T细胞被激活，并且与所述未经刺激的对照细胞组合物相比，(ii) 中的标记物的更低水平或更低阳性细胞百分比指示所述CD4+ T细胞被激活。(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.根据权利要求1-25中任一项所述的方法，其中所述检测是检测所述包含T细胞的组合物中的CD8+ T细胞。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.根据权利要求34所述的方法，其中所述选自组 (i) 的一种或多种标记物选自CD120b、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD357（GITR）、CD155（PVR）、CD355（CRTAM）、GPR56、CD71、CD107a（LAMP1）、Notch 1、CD166、CD165、CD83和Notch 2，35. 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. 并且所述来自组 (ii) 的一种或多种标记物选自CD96（TACTILE）、CD195（CCR5）、CD217、CCRL2、CD192（CCR2）、CD11b、CX3CR1和CD127（IL-7Rα）。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). 36.一种用于评估CD8+ T细胞的T细胞激活标记物的表面表达的方法，所述方法包括检测包含T细胞的组合物的CD8+ T细胞中的一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中组 (i) 由以下组成：CD120b、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD357（GITR）、CD155（PVR）、CD355（CRTAM）、GPR56、CD71、CD107a（LAMP1）、Notch 1、CD166、CD165、CD83和Notch 2，36. 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. 并且组 (ii) 由以下组成：CD96（TACTILE）、CD195（CCR5）、CD217、CCRL2、CD192（CCR2）、CD11b、CX3CR1和CD127（IL-7Rα）。And group (ii) consists of the following: CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1 and CD127 (IL-7Rα). 37.根据权利要求36所述的方法，其中所述 (i) 中的一种或多种标记物的表面表达水平或阳性细胞百分比与CD8+ T细胞激活呈正相关。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.根据权利要求37所述的方法，其中所述 (ii) 中的一种或多种标记物的表面表达水平或阳性细胞百分比与CD8+ T细胞激活呈负相关。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. 39.一种用于评估CD8+ T细胞的激活的方法，所述方法包括：39. A method for assessing the activation of CD8+ T cells, the method comprising: (a) 检测包含T细胞的组合物的CD8+ T细胞中的一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中组 (i) 由以下组成：CD120b、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD357（GITR）、CD155（PVR）、CD355（CRTAM）、GPR56、CD71、CD107a（LAMP1）、Notch 1、CD166、CD165、CD83和Notch 2，(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. 并且组 (ii) 由以下组成：CD96（TACTILE）、CD195（CCR5）、CD217、CCRL2、CD192（CCR2）、CD11b、CX3CR1和CD127（IL-7Rα）；以及And group (ii) consists of the following: CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Rα); and (b) 将所述表面表达水平或所述阳性细胞百分比与未经刺激的对照细胞组合物的细胞中的所述一种或多种标记物中的每一种的水平或阳性细胞百分比进行比较，其中与所述未经刺激的对照细胞组合物相比，(i) 中的标记物的更高水平或更高阳性细胞百分比指示所述CD8+ T细胞被激活，并且与所述未经刺激的对照细胞组合物相比，(ii) 中的标记物的更低水平或更低阳性细胞百分比指示所述CD8+ T细胞被激活。(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.根据权利要求1-39中任一项所述的方法，其中所述包含T细胞的组合物包含经基因工程化以表达重组受体的T细胞。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.根据权利要求27所述的方法，其中所述一种或多种选自组 (i) 的标记物由以下组成：CD120b、CD83、CD357（GITR）、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD355（CTRAM）、CD155（PVR）、CD74、CD170（Siglec-5）和GPR56，41. The method of claim 27, wherein the one or more markers selected from group (i) comprise: CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56. 并且所述选自组 (ii) 的一种或多种标记物由以下组成：CD49f、CCRL2、CD124（IL-4Rα）、CD217、CD192（CCR2）、CD195（CCR5）和CD96（TACTILE）。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). 42.一种用于评估T细胞的激活的方法，所述方法包括：42. A method for assessing T cell activation, the method comprising: (a) 检测包含T细胞的组合物的CD8+ T细胞中的一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中组 (i) 由以下组成：CD120b、CD83、CD357（GITR）、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD355（CTRAM）、CD155（PVR）、CD74、CD170（Siglec-5）和GPR56，(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. 并且组 (ii) 由以下组成：CD120b、CD83、CD357（GITR）、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD355（CTRAM）、CD155（PVR）、CD74、CD170（Siglec-5）和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.根据权利要求41或权利要求42所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD134（OX40）、CD107b（LAMP-2）、CD155（PVR）、CD74和CD170（Siglec-5），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). 并且所述选自组 (ii) 的一种或多种标记物选自CD49f、CCRL2、CD124（IL-4Rα）、CD217、CD355（CRTAM）、GPR56和CD96（TACTILE）。Furthermore, one or more markers selected from group (ii) are selected from CD49f, CCRL2, CD124 (IL-4Rα), CD217, CD355 (CRTAM), GPR56 and CD96 (TACTILE). 44.根据权利要求41-43中任一项所述的方法，其中所述检测是检测所述包含T细胞的组合物中的重组受体表达CD4+ T细胞。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.根据权利要求44所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD134（OX40）、CD107b（LAMP-2）、CD155（PVR）和CD74，45. 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. 和/或所述选自组 (ii) 的一种或多种标记物选自CD49f、CCRL2和CD124（IL-4Rα）。And/or one or more markers selected from group (ii) are selected from CD49f, CCRL2 and CD124 (IL-4Rα). 46.根据权利要求41-43中任一项所述的方法，其中所述检测是检测所述包含T细胞的组合物中的重组受体表达CD8+ T细胞。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.根据权利要求46所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD200（OX2）、CD107b（LAMP-2）、CD155（PVR）、CD355（CRTAM）和GPR56，47. 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. 和/或所述选自组 (ii) 的一种或多种标记物选自CCRL2、CD217、CD96（TACTILE）。And/or one or more markers selected from group (ii) are selected from CCRL2, CD217, CD96 (TACTILE). 48.根据权利要求1-16中任一项所述的方法，其中所述组 (i) 的一种或多种标记物选自CD36L（SCARB1、SR-BI）、CD262（DR5、Trail-R2）、CD105（内皮联蛋白）、CD73（胞外-5’-核苷酸酶）、CD83、CD119（IFN-g R a链）、CD154、CD170（Siglec-5）、Notch 1和CD360（IL-21R），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). 并且所述组 (i) 的一种或多种标记物选自CD96（TACTILE）。Furthermore, one or more markers of group (i) are selected from CD96 (TACTILE). 49.一种用于评估T细胞的激活的方法，所述方法包括：49. A method for assessing T cell activation, the method comprising: (a) 检测T细胞组合物中的一种或多种标记物的表面表达水平或对一种或多种标记物呈阳性的细胞百分比，所述T细胞组合物包含表达重组受体的T细胞，其中所述一种或多种标记物选自组 (i) 和/或组 (ii)，其中组 (i) 由以下组成：CD36L（SCARB1、SR-BI）、CD262（DR5、Trail-R2）、CD105（内皮联蛋白）、CD73（胞外-5’-核苷酸酶）、CD83、CD119（IFN-g R a链）、CD154、CD170（Siglec-5）、Notch 1和CD360（IL-21R），(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-gRa chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R). 并且组 (ii) 由CD96（TACTILE）组成。Furthermore, group (ii) consists of CD96 (TACTILE). 50.根据权利要求48所述的方法，其中所述包含T细胞的组合物包含表达重组受体的细胞。50. The method of claim 48, wherein the composition comprising T cells comprises cells expressing a recombinant receptor. 51.根据权利要求49或权利要求50所述的方法，其中所述检测是检测所述T细胞组合物的重组受体表达细胞。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.根据权利要求49-51中任一项所述的方法，其中与不表达所述重组受体的细胞相比，在表达所述重组受体的细胞上，所述组 (i) 的一种或多种标记物的表面表达增加。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.根据权利要求49-52中任一项所述的方法，其中与不表达所述重组受体的细胞相比，在表达所述重组受体的细胞上，所述组 (ii) 的一种或多种标记物的表面表达降低。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 to cells not expressing the recombinant receptor. 54.根据权利要求3-47中任一项所述的方法，其中所述包含T细胞的组合物包含经基因工程化以表达重组受体的T细胞，并且其中所述T细胞刺激剂是诱导重组受体依赖性T细胞激活的重组受体刺激剂。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.根据权利要求27-54中任一项所述的方法，其中所述重组受体是嵌合抗原受体（CAR）。55. The method according to any one of claims 27-54, wherein the recombinant receptor is a chimeric antigen receptor (CAR). 56.根据权利要求54或权利要求55所述的方法，其中所述重组受体刺激剂包含由所述重组受体识别的重组靶抗原。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.根据权利要求54或权利要求55所述的方法，其中所述重组受体刺激剂是对所述重组受体的细胞外抗原结合结构域具有特异性的抗体。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.根据权利要求54或权利要求55所述的方法，其中所述重组受体刺激剂是对所述重组受体的细胞外抗原结合结构域具有特异性的抗独特型抗体。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.根据权利要求54-58中任一项所述的方法，其中所述重组受体刺激剂固定或附接至固体支持物。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.根据权利要求59所述的方法，其中所述固体支持物是器皿的表面，任选地是微孔板的孔或烧瓶。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.根据权利要求59所述的方法，其中所述固体支持物是珠。61. The method of claim 59, wherein the solid support is a bead. 62.根据权利要求54或权利要求55所述的方法，其中所述重组受体刺激剂是抗原表达细胞，任选地其中所述细胞是克隆、来自细胞系、或取自受试者的原代细胞。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.根据权利要求62所述的方法，其中所述抗原表达细胞是细胞系。63. The method of claim 62, wherein the antigen-expressing cell is a cell line. 64.根据权利要求63所述的方法，其中所述细胞系是肿瘤细胞系。64. The method of claim 63, wherein the cell line is a tumor cell line. 65.根据权利要求63所述的方法，其中所述抗原表达细胞是已经被工程化以表达所述重组受体的抗原的细胞。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.根据权利要求27-65中任一项所述的方法，其中所述检测是检测所述包含T细胞的组合物中的重组受体表达T细胞。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.根据权利要求1-26中任一项所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD71、Notch 1、CD107a（LAMP-1）、CD166、CD245（p220/240）、CD154、Notch 2、CD165、CD120b、CD83、CD357（GITR）、CD200（OX2）和CD134（OX40）,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). 和/或所述来自组 (ii) 的一种或多种标记物选自KLRG1（MAFA）、CD195（CCR5）、CD96（TACTILE）、CD127（IL-7Ra）、CD192（CCR2）、CD11b和CX3CR1。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.根据权利要求1-26中任一项所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD71、Notch 1、CD107a（LAMP-1）、CD166、CD245（p220/240）、CD154、Notch 2、CD165和CD83，68. 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. 和/或所述来自组 (ii) 的一种或多种标记物选自KLRG1（MAFA）、CD195（CCR5）、CD96（TACTILE）、CD127（IL-7Ra）、CD11b和CX3CR1。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.根据权利要求67或68所述的方法，其中所述检测是检测所述包含T细胞的组合物中的CD4+ T细胞。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.根据权利要求69所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD71、Notch 1、CD107a（LAMP-1）、CD166、CD245（p220/240）、CD154、Notch 2和CD16570. 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. 和/或所述来自组 (ii) 的一种或多种标记物选自KLRG1（MAFA）、CD195（CCR5）、CD96（TACTILE）和CD127（IL-7Ra）。And/or one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE) and CD127 (IL-7Ra). 71.根据权利要求67或68所述的方法，其中所述检测是检测所述包含T细胞的组合物中的CD8+ T细胞。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.根据权利要求71所述的方法，其中所述来自组 (i) 的一种或多种标记物选自CD71、Notch 1、CD107a（LAMP-1）、CD166、Notch 2、CD165、CD83，72. 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. 和/或所述来自组 (ii) 的一种或多种标记物选自CD11b、CX3CR1和CD127（IL-7Ra）。And/or one or more markers from group (ii) are selected from CD11b, CX3CR1 and CD127 (IL-7Ra). 73.根据权利要求67-72中任一项所述的方法，其中所述T细胞刺激剂是泛T细胞激活剂。73. The method according to any one of claims 67-72, wherein the T cell stimulant is a pan-T cell activator. 74.根据权利要求73所述的方法，其中所述泛T细胞激活试剂包含抗CD3抗体和抗CD28抗体，任选地其中所述泛T细胞激活试剂包含抗CD3 Fab和抗CD28 Fab。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.根据权利要求73或权利要求74所述的方法，其中所述泛T细胞激活试剂包含抗CD3/抗CD28珠。75. The method of claim 73 or claim 74, wherein the pan-T cell activating agent comprises anti-CD3/anti-CD28 beads. 76.根据权利要求73或权利要求74所述的方法，其中所述泛T细胞激活试剂包含可溶性抗CD3/抗CD28链霉亲和素寡聚试剂。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.根据权利要求1-76中任一项所述的方法，其中在步骤 (a) 的所述检测之前，所述方法包括使所述包含T细胞的组合物的细胞与结合所述一种或多种标记物的一种或多种结合剂接触。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.根据权利要求1-76中任一项所述的方法，其中在步骤 (a) 的所述检测之前，所述方法包括使所述包含T细胞的组合物的细胞与一种或多种结合剂接触，所述一种或多种结合剂包含用于结合所述一种或多种标记物的物质。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.根据权利要求77或权利要求78所述的方法，其中所述一种或多种结合剂是一种或多种抗体或抗原结合片段。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.根据权利要求77-79中任一项所述的方法，其中所述一种或多种结合剂被可检测地标记。80. The method according to any one of claims 77-79, wherein the one or more binders are detectably labeled. 81.根据权利要求80所述的方法，其中所述一种或多种结合剂是荧光标记的。81. The method of claim 80, wherein one or more binders are fluorescently labeled. 82.根据权利要求1-81中任一项所述的方法，其中通过流式细胞术进行所述检测。82. The method according to any one of claims 1-81, wherein the detection is performed by flow cytometry. 83.根据权利要求1-81中任一项所述的方法，其中步骤 (a) 的所述检测与CITE-Seq或REAP-seq结合进行。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.根据权利要求1-81中任一项所述的方法，其中步骤 (a) 的所述检测通过免疫组织化学，任选地通过免疫组织化学荧光进行。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.根据权利要求1-84中任一项所述的方法，其中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或30种不同的标记物用于步骤 (a)中的检测。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). 86.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合根据权利要求1-85中任一项所述的方法中的一种或多种标记物的物质。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.根据权利要求86所述的试剂盒，其中用于检测所述一种或多种标记物中的每一种的物质是抗体。87. The kit according to claim 86, wherein the substance used to detect each of the one or more markers is an antibody. 88.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：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链）、CD105（内皮联蛋白）、CD262（DR5、TRAIL-R2）、CD170（Siglec-5）、CD73（胞外-5'-核苷酸酶）、CD360（IL-21R）、CD20、CD107a（LAMP-1）、CD109、CD132（共同g链）、CD148、CD150（SLAM）、CD151（PETA-3）、CD154、CD165、CD166、CD200 R、CD217、CD218a（IL-18Ra）、CD227（MUC-1）、CD245（p220/240）、CD30、CD317（BST2、束缚素）、CD323（JAM3）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD97、整合素b7、小鼠IgG3 k、Notch 2、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、Ig轻链k、Ig轻链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）、神经节苷脂GD2、MERTK、TMEM8A、CD122（IL-2Rb）、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD196（CCR6）、CD230（朊病毒）、CD235ab、CD261（DR4、TRAIL-R1）、CD304（神经毡蛋白-1）、CD49d、DR3（TRAMP）、CD186（CXCR6）、GARP（LRRC32）、CD116、CD334（FGFR4）、CD66a/c/e和TSLPR（TSLP-R），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-γ). 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, CD200R, 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 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-10R), 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). 并且组 (ii) 由以下组成：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细胞受体、CD229（Ly-9）、CD84和EGFR。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. 89.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD20、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD105（内皮联蛋白）、CD107a（LAMP-1）、CD109、CD120b、CD132（共同g链）、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、束缚素）、CD323（JAM3）、CD357（GITR）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD83、CD97、整合素b7、小鼠IgG3 k、Notch 1、Notch 2、CD107b（LAMP-2）、CD155（PVR）、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、CD74、GPR56、Ig轻链k、Ig轻链l、IgM、CD116、CD334（FGFR4）、CD66a/c/e和TSLPR（TSLP-R），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). 并且组 (ii) 由以下组成：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细胞受体、CD229（Ly-9）、CD84和EGFR。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. 90.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD20、CD105（内皮联蛋白）、CD107a（LAMP-1）、CD109、CD120b、CD132（共同g链）、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、束缚素）、CD323（JAM3）、CD357（GITR）、CD45RA、CD49c（整合素a3）、CD54、CD63、CD71、CD8、CD83、CD97、整合素b7、小鼠IgG3 k、Notch 1、Notch 2、CD107b（LAMP-2）、CD155（PVR）、CD164、CD319（CRACC）、Cd355（CRTAM）、CD4、CD49a、CD49b、CD74、GPR56、Ig轻链k、Ig轻链l、IgM、CD119（IFN-g R a链）、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）、神经节苷脂GD2、MERTK、TMEM8A、CD122（IL-2Rb）、CD360（IL-21R）、CD100、CD123、CD184（CXCR4）、CD55、TIGIT（VSTM3）、CD230（朊病毒）、CD235ab、CD261（DR4、TRAIL-R1）、CD304（神经毡蛋白-1）、CD49d、CD73（胞外-5'-核苷酸酶）、DR3（TRAMP）、CD186（CXCR6）、GARP（LRRC32），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, 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 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). 并且组 (ii) 由以下组成：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）和CD49f。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. 91.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：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）和GPR56，91. 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. 并且组 (ii) 由以下组成：CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）、CD127（IL-7Rα）、CD11b和CX3CR1。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. 92.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD107b（LAMP-2）、CD120b、CD357（GITR）、CD83、CD200（OX2）、CD134（OX40）、CD155（PVR）、CD74、CD170（Siglec-5）、CD355（CRTAM）和GPR56，92. 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. 并且组 (ii) 由以下组成：CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD96（TACTILE）和CD195（CCR5）。And group (ii) consists of the following: CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE) and CD195 (CCR5). 93.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：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和CD165，93. 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. 并且组 (ii) 由以下组成：CD49f、CD124（IL-4Rα）、CCRL2、CD217、CD192（CCR2）、CD195（CCR5）、KLRG1（MAFA）、CD96（TACTILE）和CD127（IL-7Rα）。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). 94.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD120b、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD357（GITR）、CD155（PVR）、CD355（CRTAM）、GPR56、CD71、CD107a（LAMP1）、Notch 1、CD166、CD165、CD83和Notch 2，94. 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. 并且组 (ii) 由以下组成：CD96（TACTILE）、CD195（CCR5）、CD217、CCRL2、CD192（CCR2）、CD11b、CX3CR1和CD127（IL-7Rα）。And group (ii) consists of the following: CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1 and CD127 (IL-7Rα). 95.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD120b、CD83、CD357（GITR）、CD200（OX2）、CD134（OX40）、CD107b（LAMP-2）、CD155（PVR）、CD74，95. 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, 并且所述选自组 (ii) 的一种或多种标记物由以下组成：CD49f、CCRL2、CD124（IL-4Rα）、CD217、CD192（CCR2）、CD355（CRTAM）、GPR56、CD195（CCR5）和CD96（TACTILE）。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). 96.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD71、Notch 1、CD107a（LAMP-1）、CD166、CD245（p220/240）、CD154、Notch 2、CD165、CD120b、CD83、CD357（GITR）、CD200（OX2）和CD134（OX40），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). 和/或所述来自组 (ii) 的一种或多种标记物选自KLRG1（MAFA）、CD195（CCR5）、CD96（TACTILE）、CD127（IL-7Ra）、CD192（CCR2）、CD11b和CX3CR1。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. 97.一种用于确定T细胞激活的试剂盒，所述试剂盒包含结合剂，所述结合剂包含用于结合选自组 (i) 和/或组 (ii) 的一种或多种标记物的物质，其中组 (i) 由以下组成：CD36L（SCARB1、SR-BI）、CD262（DR5、Trail-R2）、CD105（内皮联蛋白）、CD73（胞外-5’-核苷酸酶）、CD83、CD119（IFN-g R a链）、CD154、CD170（Siglec-5）、Notch 1和CD360（IL-21R），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-gRa chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R). 和/或所述来自组 (ii) 的一种或多种标记物选自CD96（TACTILE）。And/or one or more markers from group (ii) are selected from CD96 (TACTILE). 98.根据权利要求86-97中任一项所述的试剂盒，其中所述结合剂是一种或多种抗体或抗原结合片段。98. The kit according to any one of claims 86-97, wherein the binder is one or more antibody or antigen-binding fragments. 99.根据权利要求86-98中任一项所述的试剂盒，其中所述结合剂被可检测地标记。99. The kit according to any one of claims 86-98, wherein the binder is detectably labeled. 100.根据权利要求99所述的试剂盒，其中所述结合剂是荧光标记的。100. The kit of claim 99, wherein the binder is fluorescently labeled. 101.一种分离激活T细胞的方法，所述方法包括根据权利要求1-85中任一项所述的方法鉴定激活T细胞的群体并分离所述群体。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. 102.一种富集激活T细胞的方法，所述方法包括根据权利要求1-85中任一项所述的方法鉴定激活T细胞的群体并选择所述群体，从而获得富含激活T细胞的细胞群。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. 103.一种耗尽激活T细胞的细胞群的方法，所述方法包括根据权利要求1-85中任一项所述的方法鉴定激活T细胞的群体并耗尽所述激活T细胞的群体。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. 104.一种根据权利要求101-103中任一项所述的方法产生的T细胞群。104. A population of T cells generated by the method according to any one of claims 101-103.
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