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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

Strep-tag CAR-T cells can be enriched and exhibit potent anti-tumor activityin vivo(a) Enrichment of Strep-tag CAR-T cells containing 1, 2, or 3Strep-tag II sequences in the spacer region using StrepTactin coated beads onthe automated T-CATCH device. Flow cytometric analysis of the frequency ofStrep-tag CAR-T cells before and after enrichment using anti-Strep-tag IIstaining. Data is representative of six experiments using T cells from 3donors.(b) Yield of Strep-tag CAR-T cells after T-CATCH enrichment. Yieldwas determined by the absolute numbers of Strep-tag CAR-T cells in the enrichedfraction divided by the absolute numbers of Strep-tag CAR-T cells in thestarting population. Data is derived from four experiments and expressed asmeans ± SD. Statistical analysis was performed using theStudent’s t test. *P<0.05.(c) Experimental scheme for adoptive transfer of Strep-tag CAR-Tcells enriched by StrepTactin selection or using EGFR mAb.CD8+ and CD4+ T cells were stimulatedin independent cultures with anti-CD3/CD28 microbeads and transduced with theCD19-3ST/41BBζ CAR. Cultures were established at different times so thatT cell administration into tumor bearing mice occurred simultaneously.Anti-CD3/CD28 beads were removed at day 5 in all groups and CAR-T cells wereprepared for inoculation into mice either by selection on the T-CATCH at day 8,FACS sorting for EGFRt+ cells on day 10, or FACS sorting ofEGFRt+ cells followed by 8 days of culture on irradiatedCD19+ LCL cells with IL2 to remove residual bound antiEGFR mAb.(d) Flow cytometric analysis of CD19 CAR expression using Strep-tagII staining of CD8+ and CD4+ CAR-T cellsbefore enrichment, after T-CATCH purification, after EGFR mAb sorting, and afterEGFR mAb sorting followed by culture.(e) NSG mice engrafted 7 days earlier with0.5×106 Raji/ffluc were treated with a total dose of2.5×106 CAR-T cells selected by T-CATCH, EGFR sorting orEGFR sorting followed by culture and formulated in a CD4:CD8 ratio of 1:1. Tumorprogression and distribution were evaluated by serial bioluminescence imagingafter injection of luciferin substrate.(f) Persistence of CD19 CAR-T cells in each cohort of NSG/Raji mice.Flow cytometric analysis of CD4+ and CD8+CAR T cells in the peripheral blood of each group of mice after staining withCD45, CD8, CD4 and EGFR Ab at different time points after T cell infusion. Thefrequency of CAR-T cells is presented as percentage of live peripheral bloodcells.(g) Survival of mice treated with different CAR-T cell products orwith non-transduced T cells depicted as Kaplan-Meier curves. The data in d-g arerepresentative of two independent experiments.
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Figure 3: Strep-tag CAR-T cells can be enriched and exhibit potent anti-tumor activityin vivo(a) Enrichment of Strep-tag CAR-T cells containing 1, 2, or 3Strep-tag II sequences in the spacer region using StrepTactin coated beads onthe automated T-CATCH device. Flow cytometric analysis of the frequency ofStrep-tag CAR-T cells before and after enrichment using anti-Strep-tag IIstaining. Data is representative of six experiments using T cells from 3donors.(b) Yield of Strep-tag CAR-T cells after T-CATCH enrichment. Yieldwas determined by the absolute numbers of Strep-tag CAR-T cells in the enrichedfraction divided by the absolute numbers of Strep-tag CAR-T cells in thestarting population. Data is derived from four experiments and expressed asmeans ± SD. Statistical analysis was performed using theStudent’s t test. *P<0.05.(c) Experimental scheme for adoptive transfer of Strep-tag CAR-Tcells enriched by StrepTactin selection or using EGFR mAb.CD8+ and CD4+ T cells were stimulatedin independent cultures with anti-CD3/CD28 microbeads and transduced with theCD19-3ST/41BBζ CAR. Cultures were established at different times so thatT cell administration into tumor bearing mice occurred simultaneously.Anti-CD3/CD28 beads were removed at day 5 in all groups and CAR-T cells wereprepared for inoculation into mice either by selection on the T-CATCH at day 8,FACS sorting for EGFRt+ cells on day 10, or FACS sorting ofEGFRt+ cells followed by 8 days of culture on irradiatedCD19+ LCL cells with IL2 to remove residual bound antiEGFR mAb.(d) Flow cytometric analysis of CD19 CAR expression using Strep-tagII staining of CD8+ and CD4+ CAR-T cellsbefore enrichment, after T-CATCH purification, after EGFR mAb sorting, and afterEGFR mAb sorting followed by culture.(e) NSG mice engrafted 7 days earlier with0.5×106 Raji/ffluc were treated with a total dose of2.5×106 CAR-T cells selected by T-CATCH, EGFR sorting orEGFR sorting followed by culture and formulated in a CD4:CD8 ratio of 1:1. Tumorprogression and distribution were evaluated by serial bioluminescence imagingafter injection of luciferin substrate.(f) Persistence of CD19 CAR-T cells in each cohort of NSG/Raji mice.Flow cytometric analysis of CD4+ and CD8+CAR T cells in the peripheral blood of each group of mice after staining withCD45, CD8, CD4 and EGFR Ab at different time points after T cell infusion. Thefrequency of CAR-T cells is presented as percentage of live peripheral bloodcells.(g) Survival of mice treated with different CAR-T cell products orwith non-transduced T cells depicted as Kaplan-Meier curves. The data in d-g arerepresentative of two independent experiments.

Mentions: The ideal manufacturing process for T cell therapy would rapidly generatepure cell products for patient administration. Current procedures for obtainingCAR-T cells require 10 to 20 days of culture, and provide cell products with avariable proportion of transduced cells1,2,13. The affinity of Strep-tag II forStrepTactin and the ability to reverse binding with D-biotin suggested a simplestrategy to enrich Strep-tag CAR-T cells and shorten culture time. We examinedwhether Strep-tag CAR-T cells could be selected using an automated device(T-CATCH/IBA), in which agarose beads were functionalized with immobilizedStrepTactin and loaded into plastic tips. After washing to remove unbound T cells,Strep-tag CAR-T cells were released from the beads by the addition of D-biotin,which displaces bound Strep-tag II from StrepTactin. Strep-tag CAR-T cellscontaining two or three Strep-tag sequences were enriched from26–28% to >90% purity, with yields of 40 to 60%(Fig. 3a,b). T cells transduced with theStrep-tag NY-BR-1 TCR with two Strep-tags were also enriched to high purity usingStrepTactin beads (data not shown). CAR-T cells containing one Strep-tag II were notenriched, perhaps because the affinity of a single Strep-tag in the CAR wasinsufficient for stable binding to StrepTactin beads.


Inclusion of Strep-Tag II in design of antigen receptors for T cellimmunotherapy
Strep-tag CAR-T cells can be enriched and exhibit potent anti-tumor activityin vivo(a) Enrichment of Strep-tag CAR-T cells containing 1, 2, or 3Strep-tag II sequences in the spacer region using StrepTactin coated beads onthe automated T-CATCH device. Flow cytometric analysis of the frequency ofStrep-tag CAR-T cells before and after enrichment using anti-Strep-tag IIstaining. Data is representative of six experiments using T cells from 3donors.(b) Yield of Strep-tag CAR-T cells after T-CATCH enrichment. Yieldwas determined by the absolute numbers of Strep-tag CAR-T cells in the enrichedfraction divided by the absolute numbers of Strep-tag CAR-T cells in thestarting population. Data is derived from four experiments and expressed asmeans ± SD. Statistical analysis was performed using theStudent’s t test. *P<0.05.(c) Experimental scheme for adoptive transfer of Strep-tag CAR-Tcells enriched by StrepTactin selection or using EGFR mAb.CD8+ and CD4+ T cells were stimulatedin independent cultures with anti-CD3/CD28 microbeads and transduced with theCD19-3ST/41BBζ CAR. Cultures were established at different times so thatT cell administration into tumor bearing mice occurred simultaneously.Anti-CD3/CD28 beads were removed at day 5 in all groups and CAR-T cells wereprepared for inoculation into mice either by selection on the T-CATCH at day 8,FACS sorting for EGFRt+ cells on day 10, or FACS sorting ofEGFRt+ cells followed by 8 days of culture on irradiatedCD19+ LCL cells with IL2 to remove residual bound antiEGFR mAb.(d) Flow cytometric analysis of CD19 CAR expression using Strep-tagII staining of CD8+ and CD4+ CAR-T cellsbefore enrichment, after T-CATCH purification, after EGFR mAb sorting, and afterEGFR mAb sorting followed by culture.(e) NSG mice engrafted 7 days earlier with0.5×106 Raji/ffluc were treated with a total dose of2.5×106 CAR-T cells selected by T-CATCH, EGFR sorting orEGFR sorting followed by culture and formulated in a CD4:CD8 ratio of 1:1. Tumorprogression and distribution were evaluated by serial bioluminescence imagingafter injection of luciferin substrate.(f) Persistence of CD19 CAR-T cells in each cohort of NSG/Raji mice.Flow cytometric analysis of CD4+ and CD8+CAR T cells in the peripheral blood of each group of mice after staining withCD45, CD8, CD4 and EGFR Ab at different time points after T cell infusion. Thefrequency of CAR-T cells is presented as percentage of live peripheral bloodcells.(g) Survival of mice treated with different CAR-T cell products orwith non-transduced T cells depicted as Kaplan-Meier curves. The data in d-g arerepresentative of two independent experiments.
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Related In: Results  -  Collection

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Figure 3: Strep-tag CAR-T cells can be enriched and exhibit potent anti-tumor activityin vivo(a) Enrichment of Strep-tag CAR-T cells containing 1, 2, or 3Strep-tag II sequences in the spacer region using StrepTactin coated beads onthe automated T-CATCH device. Flow cytometric analysis of the frequency ofStrep-tag CAR-T cells before and after enrichment using anti-Strep-tag IIstaining. Data is representative of six experiments using T cells from 3donors.(b) Yield of Strep-tag CAR-T cells after T-CATCH enrichment. Yieldwas determined by the absolute numbers of Strep-tag CAR-T cells in the enrichedfraction divided by the absolute numbers of Strep-tag CAR-T cells in thestarting population. Data is derived from four experiments and expressed asmeans ± SD. Statistical analysis was performed using theStudent’s t test. *P<0.05.(c) Experimental scheme for adoptive transfer of Strep-tag CAR-Tcells enriched by StrepTactin selection or using EGFR mAb.CD8+ and CD4+ T cells were stimulatedin independent cultures with anti-CD3/CD28 microbeads and transduced with theCD19-3ST/41BBζ CAR. Cultures were established at different times so thatT cell administration into tumor bearing mice occurred simultaneously.Anti-CD3/CD28 beads were removed at day 5 in all groups and CAR-T cells wereprepared for inoculation into mice either by selection on the T-CATCH at day 8,FACS sorting for EGFRt+ cells on day 10, or FACS sorting ofEGFRt+ cells followed by 8 days of culture on irradiatedCD19+ LCL cells with IL2 to remove residual bound antiEGFR mAb.(d) Flow cytometric analysis of CD19 CAR expression using Strep-tagII staining of CD8+ and CD4+ CAR-T cellsbefore enrichment, after T-CATCH purification, after EGFR mAb sorting, and afterEGFR mAb sorting followed by culture.(e) NSG mice engrafted 7 days earlier with0.5×106 Raji/ffluc were treated with a total dose of2.5×106 CAR-T cells selected by T-CATCH, EGFR sorting orEGFR sorting followed by culture and formulated in a CD4:CD8 ratio of 1:1. Tumorprogression and distribution were evaluated by serial bioluminescence imagingafter injection of luciferin substrate.(f) Persistence of CD19 CAR-T cells in each cohort of NSG/Raji mice.Flow cytometric analysis of CD4+ and CD8+CAR T cells in the peripheral blood of each group of mice after staining withCD45, CD8, CD4 and EGFR Ab at different time points after T cell infusion. Thefrequency of CAR-T cells is presented as percentage of live peripheral bloodcells.(g) Survival of mice treated with different CAR-T cell products orwith non-transduced T cells depicted as Kaplan-Meier curves. The data in d-g arerepresentative of two independent experiments.
Mentions: The ideal manufacturing process for T cell therapy would rapidly generatepure cell products for patient administration. Current procedures for obtainingCAR-T cells require 10 to 20 days of culture, and provide cell products with avariable proportion of transduced cells1,2,13. The affinity of Strep-tag II forStrepTactin and the ability to reverse binding with D-biotin suggested a simplestrategy to enrich Strep-tag CAR-T cells and shorten culture time. We examinedwhether Strep-tag CAR-T cells could be selected using an automated device(T-CATCH/IBA), in which agarose beads were functionalized with immobilizedStrepTactin and loaded into plastic tips. After washing to remove unbound T cells,Strep-tag CAR-T cells were released from the beads by the addition of D-biotin,which displaces bound Strep-tag II from StrepTactin. Strep-tag CAR-T cellscontaining two or three Strep-tag sequences were enriched from26–28% to >90% purity, with yields of 40 to 60%(Fig. 3a,b). T cells transduced with theStrep-tag NY-BR-1 TCR with two Strep-tags were also enriched to high purity usingStrepTactin beads (data not shown). CAR-T cells containing one Strep-tag II were notenriched, perhaps because the affinity of a single Strep-tag in the CAR wasinsufficient for stable binding to StrepTactin beads.

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