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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

Activation, proliferation and function of Strep-tag CAR-T cells afterstimulation with anti-Strep tag II mAb(a) CD4+ and CD8+ T cellsexpressing each of the CD19 CARs were sorted for EGFRt expression and stimulatedwith anti-Strep-tag II or anti-Strep-tag II/CD28 mAb–coated microbeads.After 48 h of stimulation, expression of the CD25 activation marker wasdetermined by flow cytometry. Unstimulated cells (medium) were used ascontrols.(b) Growth curves of Strep-tag CAR-T cells. FACS sortedEGFRt+ CD19 CAR-T cells (CD8+ andCD4+) were cultured with anti-Strep-tag II oranti-Strep-tag II/CD28 mAb coated microbeads in CTL media containing IL-2(30–50 U/ml) and IL-15 (2 ng/ml) for 9 days. Aliquots of T cells wereremoved from the cultures for counting on days 3, 6, and 9 and the fold-increasein cell number determined. The data show the mean fold expansion obtained inthree experiments with T cells from different donors.(c) Stimulation of Strep-tag CAR-T cells with anti-Strep-tag II/CD28beads induces selective outgrowth of transduced cells. CD8+and CD4+ T cells were transduced with CD19 1ST/4-1BBζand 1ST/CD28ζ CARs and 10 days later stimulated with anti-Strep-tagII/CD28 microbeads plus IL-2 or cultured with IL-2 alone for 9 additional days.The percentage of Strep-tag II positive cells at day 0 (before stimulation) andday 9 (after stimulation) were measured by flow cytometry. The results arerepresentative of three experiments.(d) Anti-tumor activity of CD19 1ST/4-1BBζ or1ST/CD28ζ CAR-T cells in Raji-ffluc-bearing NSG mice.2.5×106 CD19 CAR-T cells expanded with anti-CD3/CD28beads or with anti-Strep-tag II/CD28 microbeads, and control non-transduced Tcells were formulated in a CD8:CD4 ratio of 1:1 and infused into cohorts of NSGmice 7 days after inoculation with 0.5×x106 Raji/ffluc tumorcells. Tumor progression and distribution were evaluated by serialbioluminescence imaging.
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Figure 2: Activation, proliferation and function of Strep-tag CAR-T cells afterstimulation with anti-Strep tag II mAb(a) CD4+ and CD8+ T cellsexpressing each of the CD19 CARs were sorted for EGFRt expression and stimulatedwith anti-Strep-tag II or anti-Strep-tag II/CD28 mAb–coated microbeads.After 48 h of stimulation, expression of the CD25 activation marker wasdetermined by flow cytometry. Unstimulated cells (medium) were used ascontrols.(b) Growth curves of Strep-tag CAR-T cells. FACS sortedEGFRt+ CD19 CAR-T cells (CD8+ andCD4+) were cultured with anti-Strep-tag II oranti-Strep-tag II/CD28 mAb coated microbeads in CTL media containing IL-2(30–50 U/ml) and IL-15 (2 ng/ml) for 9 days. Aliquots of T cells wereremoved from the cultures for counting on days 3, 6, and 9 and the fold-increasein cell number determined. The data show the mean fold expansion obtained inthree experiments with T cells from different donors.(c) Stimulation of Strep-tag CAR-T cells with anti-Strep-tag II/CD28beads induces selective outgrowth of transduced cells. CD8+and CD4+ T cells were transduced with CD19 1ST/4-1BBζand 1ST/CD28ζ CARs and 10 days later stimulated with anti-Strep-tagII/CD28 microbeads plus IL-2 or cultured with IL-2 alone for 9 additional days.The percentage of Strep-tag II positive cells at day 0 (before stimulation) andday 9 (after stimulation) were measured by flow cytometry. The results arerepresentative of three experiments.(d) Anti-tumor activity of CD19 1ST/4-1BBζ or1ST/CD28ζ CAR-T cells in Raji-ffluc-bearing NSG mice.2.5×106 CD19 CAR-T cells expanded with anti-CD3/CD28beads or with anti-Strep-tag II/CD28 microbeads, and control non-transduced Tcells were formulated in a CD8:CD4 ratio of 1:1 and infused into cohorts of NSGmice 7 days after inoculation with 0.5×x106 Raji/ffluc tumorcells. Tumor progression and distribution were evaluated by serialbioluminescence imaging.

Mentions: Anti-CD3/CD28 mAb coated beads are used to non-selectively activate T cellsand facilitate transduction with viral vectors, but induce proliferation of bothtransduced and non-transduced T cells 11,12. We hypothesizedthat multivalent binding of Strep-tag would selectively activate CAR signaling andinduce proliferation only of transduced T cells. Co-culture of Strep-tag CAR andcontrol CAR-T cells with microbeads coated with anti-Strep-tag II mAb alone orcombined with anti-CD28 mAb induced CD25 upregulation only in Strep-tag CAR-T cells(Fig. 2a), and expanded CD4 and CD8Strep-tag CAR-T cells >100-fold in 9 days of culture (Fig. 2b). Activation and proliferation was independent ofscFv specificity and costimulatory domains in the CAR (Supplementary Fig. 4a–c). Thefrequency of Strep-tag CAR-T cells increased from ~26–33% to84–92% in cultures stimulated with anti-Strep-tag II/CD28 beads, butdid not increase in cultures with IL-2 alone (Fig.2c). Analysis of TCR diversity of CAR-T cells before and after expansionwith anti-Strep-tag II/CD28 showed that a diverse repertoire was retained, and theexpanded T cells expressed CD62L, CD28, and CD27, maintained CAR-directed cytolyticfunction, and proliferated in response to tumor cells (Supplementary Fig. 5a–d). CAR-Tcells expanded with anti-Strep-tag II/CD28 stimulation were as effective ineliminating Raji lymphoma in NSG mice as those expanded with anti-CD3/CD28 (Fig. 2d). Receptor tyrosine kinase-like orphanreceptor-1 (ROR1)–specific CAR-T cells expanded with anti-Strep-tag II/CD28also retained function in vitro, and in vivoagainst ROR1+ MDA-MB-231 breast cancer xenografts in NSG mice,demonstrating that this approach can be applied broadly in CAR-T cell therapy (Supplementary Fig.6a–c).


Inclusion of Strep-Tag II in design of antigen receptors for T cellimmunotherapy
Activation, proliferation and function of Strep-tag CAR-T cells afterstimulation with anti-Strep tag II mAb(a) CD4+ and CD8+ T cellsexpressing each of the CD19 CARs were sorted for EGFRt expression and stimulatedwith anti-Strep-tag II or anti-Strep-tag II/CD28 mAb–coated microbeads.After 48 h of stimulation, expression of the CD25 activation marker wasdetermined by flow cytometry. Unstimulated cells (medium) were used ascontrols.(b) Growth curves of Strep-tag CAR-T cells. FACS sortedEGFRt+ CD19 CAR-T cells (CD8+ andCD4+) were cultured with anti-Strep-tag II oranti-Strep-tag II/CD28 mAb coated microbeads in CTL media containing IL-2(30–50 U/ml) and IL-15 (2 ng/ml) for 9 days. Aliquots of T cells wereremoved from the cultures for counting on days 3, 6, and 9 and the fold-increasein cell number determined. The data show the mean fold expansion obtained inthree experiments with T cells from different donors.(c) Stimulation of Strep-tag CAR-T cells with anti-Strep-tag II/CD28beads induces selective outgrowth of transduced cells. CD8+and CD4+ T cells were transduced with CD19 1ST/4-1BBζand 1ST/CD28ζ CARs and 10 days later stimulated with anti-Strep-tagII/CD28 microbeads plus IL-2 or cultured with IL-2 alone for 9 additional days.The percentage of Strep-tag II positive cells at day 0 (before stimulation) andday 9 (after stimulation) were measured by flow cytometry. The results arerepresentative of three experiments.(d) Anti-tumor activity of CD19 1ST/4-1BBζ or1ST/CD28ζ CAR-T cells in Raji-ffluc-bearing NSG mice.2.5×106 CD19 CAR-T cells expanded with anti-CD3/CD28beads or with anti-Strep-tag II/CD28 microbeads, and control non-transduced Tcells were formulated in a CD8:CD4 ratio of 1:1 and infused into cohorts of NSGmice 7 days after inoculation with 0.5×x106 Raji/ffluc tumorcells. Tumor progression and distribution were evaluated by serialbioluminescence imaging.
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Figure 2: Activation, proliferation and function of Strep-tag CAR-T cells afterstimulation with anti-Strep tag II mAb(a) CD4+ and CD8+ T cellsexpressing each of the CD19 CARs were sorted for EGFRt expression and stimulatedwith anti-Strep-tag II or anti-Strep-tag II/CD28 mAb–coated microbeads.After 48 h of stimulation, expression of the CD25 activation marker wasdetermined by flow cytometry. Unstimulated cells (medium) were used ascontrols.(b) Growth curves of Strep-tag CAR-T cells. FACS sortedEGFRt+ CD19 CAR-T cells (CD8+ andCD4+) were cultured with anti-Strep-tag II oranti-Strep-tag II/CD28 mAb coated microbeads in CTL media containing IL-2(30–50 U/ml) and IL-15 (2 ng/ml) for 9 days. Aliquots of T cells wereremoved from the cultures for counting on days 3, 6, and 9 and the fold-increasein cell number determined. The data show the mean fold expansion obtained inthree experiments with T cells from different donors.(c) Stimulation of Strep-tag CAR-T cells with anti-Strep-tag II/CD28beads induces selective outgrowth of transduced cells. CD8+and CD4+ T cells were transduced with CD19 1ST/4-1BBζand 1ST/CD28ζ CARs and 10 days later stimulated with anti-Strep-tagII/CD28 microbeads plus IL-2 or cultured with IL-2 alone for 9 additional days.The percentage of Strep-tag II positive cells at day 0 (before stimulation) andday 9 (after stimulation) were measured by flow cytometry. The results arerepresentative of three experiments.(d) Anti-tumor activity of CD19 1ST/4-1BBζ or1ST/CD28ζ CAR-T cells in Raji-ffluc-bearing NSG mice.2.5×106 CD19 CAR-T cells expanded with anti-CD3/CD28beads or with anti-Strep-tag II/CD28 microbeads, and control non-transduced Tcells were formulated in a CD8:CD4 ratio of 1:1 and infused into cohorts of NSGmice 7 days after inoculation with 0.5×x106 Raji/ffluc tumorcells. Tumor progression and distribution were evaluated by serialbioluminescence imaging.
Mentions: Anti-CD3/CD28 mAb coated beads are used to non-selectively activate T cellsand facilitate transduction with viral vectors, but induce proliferation of bothtransduced and non-transduced T cells 11,12. We hypothesizedthat multivalent binding of Strep-tag would selectively activate CAR signaling andinduce proliferation only of transduced T cells. Co-culture of Strep-tag CAR andcontrol CAR-T cells with microbeads coated with anti-Strep-tag II mAb alone orcombined with anti-CD28 mAb induced CD25 upregulation only in Strep-tag CAR-T cells(Fig. 2a), and expanded CD4 and CD8Strep-tag CAR-T cells >100-fold in 9 days of culture (Fig. 2b). Activation and proliferation was independent ofscFv specificity and costimulatory domains in the CAR (Supplementary Fig. 4a–c). Thefrequency of Strep-tag CAR-T cells increased from ~26–33% to84–92% in cultures stimulated with anti-Strep-tag II/CD28 beads, butdid not increase in cultures with IL-2 alone (Fig.2c). Analysis of TCR diversity of CAR-T cells before and after expansionwith anti-Strep-tag II/CD28 showed that a diverse repertoire was retained, and theexpanded T cells expressed CD62L, CD28, and CD27, maintained CAR-directed cytolyticfunction, and proliferated in response to tumor cells (Supplementary Fig. 5a–d). CAR-Tcells expanded with anti-Strep-tag II/CD28 stimulation were as effective ineliminating Raji lymphoma in NSG mice as those expanded with anti-CD3/CD28 (Fig. 2d). Receptor tyrosine kinase-like orphanreceptor-1 (ROR1)–specific CAR-T cells expanded with anti-Strep-tag II/CD28also retained function in vitro, and in vivoagainst ROR1+ MDA-MB-231 breast cancer xenografts in NSG mice,demonstrating that this approach can be applied broadly in CAR-T cell therapy (Supplementary Fig.6a–c).

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