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Inclusion of Strep-Tag II in design of antigen receptors for T cellimmunotherapy

View Article: PubMed Central - PubMed

ABSTRACT

The tactical introduction of Strep-tag II into synthetic antigenreceptors provides engineered T cells with a marker for identification and rapidpurification, and a functional element for selective antibody coatedmicrobead-driven large-scale expansion. Such receptor designs can be applied tochimeric antigen receptors of different ligand specificities and costimulatorydomains, and to T cell receptors to facilitate cGMP manufacturing of adoptive Tcell therapies to treat cancer and other diseases.

No MeSH data available.


Related in: MedlinePlus

Expression and function of CD19 CARs that contain Strep-tag II(a) Analysis of CD19 CAR expression. Primary humanCD8+ T cells were transduced with epHIV7 lentiviralvectors encoding a CD19-Hi CAR or CD19 CARs with Strep-tag in variousextracellular locations. Each CAR contained a 4-1BB/CD3ζ intracellularsignaling domain and EGFRt downstream of a T2A element. Transduced cells weresorted for EGFRt+ cells by fluorescence-activated cellsorting (FACS), and purity confirmed by staining with anti-EGFR (grey –top panels). Cell surface expression of the Strep-tag CARs was evaluated bystaining with anti-Strep-tag II antibodies (grey - bottom panels).Non-transduced cells (white) served as controls for staining. (b)Cytolytic activity of CD19-Hi and Strep-tag 4-1BBζ CAR-T cells. Aftersorting for EGFRt expression, CD8+ T cells transduced witheach of the CARs were tested for lysis of CD19+ Raji lymphomaand K562 leukemia transduced with CD19 (K562/CD19) or ROR1 (K562/ROR1) atvarious effector/target (E:T) ratios.(c) IFN-γ and IL2 production by CD19-Hi and Strep-tag4-1BBζ CAR-T cells 24h after stimulation with K562/CD19 and K562/ROR1.PMA/Ionomycin treated T cells were used as a positive control. The data ina–c is representative of three experiments with CD8+T cells from different donors.(d) Cohorts of NSG mice were inoculated with0.5×106 firefly luciferase expressingCD19+ Raji lymphoma cells (Raji-ffluc) via tail veininjection and treated 7 days after tumor inoculation with2.5×106 CD19 4-1BBζ CAR-T cells with Hi, 1ST, and3ST spacers, respectively. CAR-T cells were formulated in a CD8:CD4 ratio of1:1. Tumor progression and distribution were evaluated by serial bioluminescenceimaging.(e) Tracking CAR-expressing T cells in vivo by staining withanti-Strep-tag II mAb. Blood obtained from mice 8 days after the T cellinfusions was stained with anti-human CD45, CD8, CD4, anti-Strep-tag II andanti-EGFR mAbs, and analyzed by flow cytometry. Expression of Strep-tag II andEGFRt on CD45+ CD8+ andCD45+ CD4+ T cells is shown.(f) Kinetics of expansion and contraction of CD19 CAR-T cells in theblood after adoptive transfer to NSG mice bearing Raji tumors. The meanfrequency of CD45+CD8+EGFRt+ andCD45+CD4+ EGFRt+human T cells in blood of the mice (n=5) of each group at various timesafter the T cell infusion is shown. The data in d–f are representativeof three experiments.(g) Fold-change in expression of selected cytokine genes inCD19-1ST/4-1BBζ CAR-T cells after infusion to Raji tumor bearing andnon-tumor bearing NSG mice. CAR-T cells were sorted 2 days after infusion fromblood, bone marrow and spleen after staining with anti-EGFR or anti-Strep-tagmAb. Gene expression was analyzed using a human common cytokine PCR array. Themean fold change values of cytokine genes in the sorted CAR-T cells fromNSG/Raji vs sorted CAR-T cells from non-tumor bearing NSG mice were calculated.Samples were run in triplicate and data are presented as the mean fold increase± SD.(h) Cytokine production by CD8+ T cellsexpressing CD19-1ST/4-1BBζ CARs 2 days after stimulation with Raji cellsin vitro. Supernatants of CAR-T cells co-cultured withCD19+ Raji cells (intra-assay triplicates) for 48 h werepooled together and analyzed using the Luminex Multiplex platform to validatethe production of cytokines that were upregulated in CAR-T cells in NSG micebearing Raji tumors. PMA/Ionomycin treated and non-treated T cells were used aspositive and negative control, respectively. The results are representative ofbiological replicates.
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Figure 1: Expression and function of CD19 CARs that contain Strep-tag II(a) Analysis of CD19 CAR expression. Primary humanCD8+ T cells were transduced with epHIV7 lentiviralvectors encoding a CD19-Hi CAR or CD19 CARs with Strep-tag in variousextracellular locations. Each CAR contained a 4-1BB/CD3ζ intracellularsignaling domain and EGFRt downstream of a T2A element. Transduced cells weresorted for EGFRt+ cells by fluorescence-activated cellsorting (FACS), and purity confirmed by staining with anti-EGFR (grey –top panels). Cell surface expression of the Strep-tag CARs was evaluated bystaining with anti-Strep-tag II antibodies (grey - bottom panels).Non-transduced cells (white) served as controls for staining. (b)Cytolytic activity of CD19-Hi and Strep-tag 4-1BBζ CAR-T cells. Aftersorting for EGFRt expression, CD8+ T cells transduced witheach of the CARs were tested for lysis of CD19+ Raji lymphomaand K562 leukemia transduced with CD19 (K562/CD19) or ROR1 (K562/ROR1) atvarious effector/target (E:T) ratios.(c) IFN-γ and IL2 production by CD19-Hi and Strep-tag4-1BBζ CAR-T cells 24h after stimulation with K562/CD19 and K562/ROR1.PMA/Ionomycin treated T cells were used as a positive control. The data ina–c is representative of three experiments with CD8+T cells from different donors.(d) Cohorts of NSG mice were inoculated with0.5×106 firefly luciferase expressingCD19+ Raji lymphoma cells (Raji-ffluc) via tail veininjection and treated 7 days after tumor inoculation with2.5×106 CD19 4-1BBζ CAR-T cells with Hi, 1ST, and3ST spacers, respectively. CAR-T cells were formulated in a CD8:CD4 ratio of1:1. Tumor progression and distribution were evaluated by serial bioluminescenceimaging.(e) Tracking CAR-expressing T cells in vivo by staining withanti-Strep-tag II mAb. Blood obtained from mice 8 days after the T cellinfusions was stained with anti-human CD45, CD8, CD4, anti-Strep-tag II andanti-EGFR mAbs, and analyzed by flow cytometry. Expression of Strep-tag II andEGFRt on CD45+ CD8+ andCD45+ CD4+ T cells is shown.(f) Kinetics of expansion and contraction of CD19 CAR-T cells in theblood after adoptive transfer to NSG mice bearing Raji tumors. The meanfrequency of CD45+CD8+EGFRt+ andCD45+CD4+ EGFRt+human T cells in blood of the mice (n=5) of each group at various timesafter the T cell infusion is shown. The data in d–f are representativeof three experiments.(g) Fold-change in expression of selected cytokine genes inCD19-1ST/4-1BBζ CAR-T cells after infusion to Raji tumor bearing andnon-tumor bearing NSG mice. CAR-T cells were sorted 2 days after infusion fromblood, bone marrow and spleen after staining with anti-EGFR or anti-Strep-tagmAb. Gene expression was analyzed using a human common cytokine PCR array. Themean fold change values of cytokine genes in the sorted CAR-T cells fromNSG/Raji vs sorted CAR-T cells from non-tumor bearing NSG mice were calculated.Samples were run in triplicate and data are presented as the mean fold increase± SD.(h) Cytokine production by CD8+ T cellsexpressing CD19-1ST/4-1BBζ CARs 2 days after stimulation with Raji cellsin vitro. Supernatants of CAR-T cells co-cultured withCD19+ Raji cells (intra-assay triplicates) for 48 h werepooled together and analyzed using the Luminex Multiplex platform to validatethe production of cytokines that were upregulated in CAR-T cells in NSG micebearing Raji tumors. PMA/Ionomycin treated and non-treated T cells were used aspositive and negative control, respectively. The results are representative ofbiological replicates.

Mentions: We introduced one or more Strep-tag II sequences with Gly/Ser linkers at theNH2 terminus, between the VL and VH, or between the scFv and the hinge of CD19 CARswith 4-1BB/CD3ζ or CD28/CD3ζ signaling domains (Supplementary Fig. 1a)8. The constructs were encoded in alentiviral vector with truncated epidermal growth factor receptor (EGFRt) downstreamof a T2A sequence to provide an independent transduction marker9. A conventional CD19–CAR (CD19-Hi)without Strep-tag II served as a control for functional assays (Supplementary Fig. 1a). We transducedhuman CD8+ T cells, sorted for EGFRt expression and evaluated CARsurface expression by staining with anti-Strep-tag II monoclonal antibody (mAb). AllStrep-tag CAR-T cells were stained with anti-Strep-tag II mAb, independent of theposition or number of Strep-tag II sequences, and staining intensity was highest forCAR-T cells that contained three Strep-tag II sequences (Fig. 1a). All the Strep-tag CAR-T cells lysed K562/CD19and CD19+ Raji cells as efficiently as T cells expressing theCD19-Hi CAR and did not recognize control K562/ROR1 cells (Fig. 1b). CD19-specific recognition by Strep-tag CAR-Tcells was confirmed by the production of interleukin 2 (IL-2) and interferon(IFN)-γ after co-culture with CD19+ tumor cells (Fig. 1c). We then examined if Strep-tag could beintroduced into a TCR specific for the breast cancer antigen, NY-BR-110. Strep-tag TCRs were expressed inprimary CD8+ T cells as determined by staining withanti-Strep-tag mAb or HLA tetramer, and conferred equivalent function asintroduction of the wild-type NY-BR-1 TCR (Supplementary Fig. 1b–f). Thesedata indicate that inclusion of Strep-tag II did not interfere with CAR or TCRexpression or in vitro function.


Inclusion of Strep-Tag II in design of antigen receptors for T cellimmunotherapy
Expression and function of CD19 CARs that contain Strep-tag II(a) Analysis of CD19 CAR expression. Primary humanCD8+ T cells were transduced with epHIV7 lentiviralvectors encoding a CD19-Hi CAR or CD19 CARs with Strep-tag in variousextracellular locations. Each CAR contained a 4-1BB/CD3ζ intracellularsignaling domain and EGFRt downstream of a T2A element. Transduced cells weresorted for EGFRt+ cells by fluorescence-activated cellsorting (FACS), and purity confirmed by staining with anti-EGFR (grey –top panels). Cell surface expression of the Strep-tag CARs was evaluated bystaining with anti-Strep-tag II antibodies (grey - bottom panels).Non-transduced cells (white) served as controls for staining. (b)Cytolytic activity of CD19-Hi and Strep-tag 4-1BBζ CAR-T cells. Aftersorting for EGFRt expression, CD8+ T cells transduced witheach of the CARs were tested for lysis of CD19+ Raji lymphomaand K562 leukemia transduced with CD19 (K562/CD19) or ROR1 (K562/ROR1) atvarious effector/target (E:T) ratios.(c) IFN-γ and IL2 production by CD19-Hi and Strep-tag4-1BBζ CAR-T cells 24h after stimulation with K562/CD19 and K562/ROR1.PMA/Ionomycin treated T cells were used as a positive control. The data ina–c is representative of three experiments with CD8+T cells from different donors.(d) Cohorts of NSG mice were inoculated with0.5×106 firefly luciferase expressingCD19+ Raji lymphoma cells (Raji-ffluc) via tail veininjection and treated 7 days after tumor inoculation with2.5×106 CD19 4-1BBζ CAR-T cells with Hi, 1ST, and3ST spacers, respectively. CAR-T cells were formulated in a CD8:CD4 ratio of1:1. Tumor progression and distribution were evaluated by serial bioluminescenceimaging.(e) Tracking CAR-expressing T cells in vivo by staining withanti-Strep-tag II mAb. Blood obtained from mice 8 days after the T cellinfusions was stained with anti-human CD45, CD8, CD4, anti-Strep-tag II andanti-EGFR mAbs, and analyzed by flow cytometry. Expression of Strep-tag II andEGFRt on CD45+ CD8+ andCD45+ CD4+ T cells is shown.(f) Kinetics of expansion and contraction of CD19 CAR-T cells in theblood after adoptive transfer to NSG mice bearing Raji tumors. The meanfrequency of CD45+CD8+EGFRt+ andCD45+CD4+ EGFRt+human T cells in blood of the mice (n=5) of each group at various timesafter the T cell infusion is shown. The data in d–f are representativeof three experiments.(g) Fold-change in expression of selected cytokine genes inCD19-1ST/4-1BBζ CAR-T cells after infusion to Raji tumor bearing andnon-tumor bearing NSG mice. CAR-T cells were sorted 2 days after infusion fromblood, bone marrow and spleen after staining with anti-EGFR or anti-Strep-tagmAb. Gene expression was analyzed using a human common cytokine PCR array. Themean fold change values of cytokine genes in the sorted CAR-T cells fromNSG/Raji vs sorted CAR-T cells from non-tumor bearing NSG mice were calculated.Samples were run in triplicate and data are presented as the mean fold increase± SD.(h) Cytokine production by CD8+ T cellsexpressing CD19-1ST/4-1BBζ CARs 2 days after stimulation with Raji cellsin vitro. Supernatants of CAR-T cells co-cultured withCD19+ Raji cells (intra-assay triplicates) for 48 h werepooled together and analyzed using the Luminex Multiplex platform to validatethe production of cytokines that were upregulated in CAR-T cells in NSG micebearing Raji tumors. PMA/Ionomycin treated and non-treated T cells were used aspositive and negative control, respectively. The results are representative ofbiological replicates.
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Related In: Results  -  Collection

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Figure 1: Expression and function of CD19 CARs that contain Strep-tag II(a) Analysis of CD19 CAR expression. Primary humanCD8+ T cells were transduced with epHIV7 lentiviralvectors encoding a CD19-Hi CAR or CD19 CARs with Strep-tag in variousextracellular locations. Each CAR contained a 4-1BB/CD3ζ intracellularsignaling domain and EGFRt downstream of a T2A element. Transduced cells weresorted for EGFRt+ cells by fluorescence-activated cellsorting (FACS), and purity confirmed by staining with anti-EGFR (grey –top panels). Cell surface expression of the Strep-tag CARs was evaluated bystaining with anti-Strep-tag II antibodies (grey - bottom panels).Non-transduced cells (white) served as controls for staining. (b)Cytolytic activity of CD19-Hi and Strep-tag 4-1BBζ CAR-T cells. Aftersorting for EGFRt expression, CD8+ T cells transduced witheach of the CARs were tested for lysis of CD19+ Raji lymphomaand K562 leukemia transduced with CD19 (K562/CD19) or ROR1 (K562/ROR1) atvarious effector/target (E:T) ratios.(c) IFN-γ and IL2 production by CD19-Hi and Strep-tag4-1BBζ CAR-T cells 24h after stimulation with K562/CD19 and K562/ROR1.PMA/Ionomycin treated T cells were used as a positive control. The data ina–c is representative of three experiments with CD8+T cells from different donors.(d) Cohorts of NSG mice were inoculated with0.5×106 firefly luciferase expressingCD19+ Raji lymphoma cells (Raji-ffluc) via tail veininjection and treated 7 days after tumor inoculation with2.5×106 CD19 4-1BBζ CAR-T cells with Hi, 1ST, and3ST spacers, respectively. CAR-T cells were formulated in a CD8:CD4 ratio of1:1. Tumor progression and distribution were evaluated by serial bioluminescenceimaging.(e) Tracking CAR-expressing T cells in vivo by staining withanti-Strep-tag II mAb. Blood obtained from mice 8 days after the T cellinfusions was stained with anti-human CD45, CD8, CD4, anti-Strep-tag II andanti-EGFR mAbs, and analyzed by flow cytometry. Expression of Strep-tag II andEGFRt on CD45+ CD8+ andCD45+ CD4+ T cells is shown.(f) Kinetics of expansion and contraction of CD19 CAR-T cells in theblood after adoptive transfer to NSG mice bearing Raji tumors. The meanfrequency of CD45+CD8+EGFRt+ andCD45+CD4+ EGFRt+human T cells in blood of the mice (n=5) of each group at various timesafter the T cell infusion is shown. The data in d–f are representativeof three experiments.(g) Fold-change in expression of selected cytokine genes inCD19-1ST/4-1BBζ CAR-T cells after infusion to Raji tumor bearing andnon-tumor bearing NSG mice. CAR-T cells were sorted 2 days after infusion fromblood, bone marrow and spleen after staining with anti-EGFR or anti-Strep-tagmAb. Gene expression was analyzed using a human common cytokine PCR array. Themean fold change values of cytokine genes in the sorted CAR-T cells fromNSG/Raji vs sorted CAR-T cells from non-tumor bearing NSG mice were calculated.Samples were run in triplicate and data are presented as the mean fold increase± SD.(h) Cytokine production by CD8+ T cellsexpressing CD19-1ST/4-1BBζ CARs 2 days after stimulation with Raji cellsin vitro. Supernatants of CAR-T cells co-cultured withCD19+ Raji cells (intra-assay triplicates) for 48 h werepooled together and analyzed using the Luminex Multiplex platform to validatethe production of cytokines that were upregulated in CAR-T cells in NSG micebearing Raji tumors. PMA/Ionomycin treated and non-treated T cells were used aspositive and negative control, respectively. The results are representative ofbiological replicates.
Mentions: We introduced one or more Strep-tag II sequences with Gly/Ser linkers at theNH2 terminus, between the VL and VH, or between the scFv and the hinge of CD19 CARswith 4-1BB/CD3ζ or CD28/CD3ζ signaling domains (Supplementary Fig. 1a)8. The constructs were encoded in alentiviral vector with truncated epidermal growth factor receptor (EGFRt) downstreamof a T2A sequence to provide an independent transduction marker9. A conventional CD19–CAR (CD19-Hi)without Strep-tag II served as a control for functional assays (Supplementary Fig. 1a). We transducedhuman CD8+ T cells, sorted for EGFRt expression and evaluated CARsurface expression by staining with anti-Strep-tag II monoclonal antibody (mAb). AllStrep-tag CAR-T cells were stained with anti-Strep-tag II mAb, independent of theposition or number of Strep-tag II sequences, and staining intensity was highest forCAR-T cells that contained three Strep-tag II sequences (Fig. 1a). All the Strep-tag CAR-T cells lysed K562/CD19and CD19+ Raji cells as efficiently as T cells expressing theCD19-Hi CAR and did not recognize control K562/ROR1 cells (Fig. 1b). CD19-specific recognition by Strep-tag CAR-Tcells was confirmed by the production of interleukin 2 (IL-2) and interferon(IFN)-γ after co-culture with CD19+ tumor cells (Fig. 1c). We then examined if Strep-tag could beintroduced into a TCR specific for the breast cancer antigen, NY-BR-110. Strep-tag TCRs were expressed inprimary CD8+ T cells as determined by staining withanti-Strep-tag mAb or HLA tetramer, and conferred equivalent function asintroduction of the wild-type NY-BR-1 TCR (Supplementary Fig. 1b–f). Thesedata indicate that inclusion of Strep-tag II did not interfere with CAR or TCRexpression or in vitro function.

View Article: PubMed Central - PubMed

ABSTRACT

The tactical introduction of Strep-tag II into synthetic antigenreceptors provides engineered T cells with a marker for identification and rapidpurification, and a functional element for selective antibody coatedmicrobead-driven large-scale expansion. Such receptor designs can be applied tochimeric antigen receptors of different ligand specificities and costimulatorydomains, and to T cell receptors to facilitate cGMP manufacturing of adoptive Tcell therapies to treat cancer and other diseases.

No MeSH data available.


Related in: MedlinePlus