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One-step enzymatic modification of the cell surface redirects cellular cytotoxicity and parasite tropism.

Swee LK, Lourido S, Bell GW, Ingram JR, Ploegh HL - ACS Chem. Biol. (2014)

Bottom Line: Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen.Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies.This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.

ABSTRACT
Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.

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Installation of VHHson mouse lymphocytes. (a) In vitro activated CD8T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and with or without 20 μM of sortase A. Controlor sortagged cells were incubated with purified GFP. Binding of GFPwas analyzed by flow cytometry. (b) Control or sortagged cells wereincubated with purified GFP. The amount of bound GFP was estimatedby analysis of cell lysates by SDS-PAGE and immunoblotting againstGFP and comparing the resultant signal to a GFP standard (right lanes).(c) Erythrocyte-depleted splenocytes were incubated with or without500 μM enhancer-LPETG and 20 μM sortase A. After 60 min,500 μM biotin-LPETG was added to reactions where indicated fora further 15 min. Dot plots show the binding of APC-conjugated streptavidinand GFP by sortagged cells after washing. (d) In vitro activated CD8 T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and 20 μM of sortase A. Sortagged cells were incubatedwith splenocytes from WT mice for 20 h. Histograms show the percentageof propidium iodide-negative CD4 and CD19 cells, compared to cellsincubated with control activated OTI CD8 T cells. Error bars: standarddeviation (n = 3). (e) CAR T cells are geneticallyengineered to express a synthetic receptor composed of an extracellularsingle-chain variable fragment and one or several cytoplasmic activatingmotifs that mediate signal transduction and T cell activation uponantigen binding. (f) Sortase-mediated conjugation of VHHs on activatedT cells affords redirection of cytotoxicity toward cells expressingthe targeted antigen.
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fig2: Installation of VHHson mouse lymphocytes. (a) In vitro activated CD8T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and with or without 20 μM of sortase A. Controlor sortagged cells were incubated with purified GFP. Binding of GFPwas analyzed by flow cytometry. (b) Control or sortagged cells wereincubated with purified GFP. The amount of bound GFP was estimatedby analysis of cell lysates by SDS-PAGE and immunoblotting againstGFP and comparing the resultant signal to a GFP standard (right lanes).(c) Erythrocyte-depleted splenocytes were incubated with or without500 μM enhancer-LPETG and 20 μM sortase A. After 60 min,500 μM biotin-LPETG was added to reactions where indicated fora further 15 min. Dot plots show the binding of APC-conjugated streptavidinand GFP by sortagged cells after washing. (d) In vitro activated CD8 T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and 20 μM of sortase A. Sortagged cells were incubatedwith splenocytes from WT mice for 20 h. Histograms show the percentageof propidium iodide-negative CD4 and CD19 cells, compared to cellsincubated with control activated OTI CD8 T cells. Error bars: standarddeviation (n = 3). (e) CAR T cells are geneticallyengineered to express a synthetic receptor composed of an extracellularsingle-chain variable fragment and one or several cytoplasmic activatingmotifs that mediate signal transduction and T cell activation uponantigen binding. (f) Sortase-mediated conjugation of VHHs on activatedT cells affords redirection of cytotoxicity toward cells expressingthe targeted antigen.

Mentions: Expression of tumor-specificchimeric antigen receptors at the surface of CD8 T cells is emergingas practical approach to eradicate leukemic cells.13 To investigate whether LPETG-tagged single domain antibodies(VHH) could be attached to the surface of activated CD8 T cells bymeans of sortagging, we incubated cytotoxic OTI CD8 T cells with increasingconcentrations of a GFP-specific (“enhancer”)33 or a mouse class II MHC-specific (“VHH7”)34 single domain antibody in the presence or absenceof sortase A. To monitor installation of VHHs as well as assess theirspecificity in a cell-bound format, we measured the ability of cellsto bind GFP by flow cytometry. Only cells incubated with enhancer-LPETGand sortase A bound recombinant GFP in a dose-dependent fashion (Figure 2a). The concentration of VHHs used for the reactionis proportional to the number of VHHs installed. We estimated thenumber of VHHs installed per cell by measuring the number of boundGFP molecules by SDS-PAGE and immunoblotting, using a solution ofGFP of known concentration as standard (Figure 2b). Sortagging of T cells in the presence of 500 μM of VHHsand sortase A resulted in the conjugation of ∼1 million VHHsper cell. To investigate whether two different probes could be installedon lymphocytes, we incubated erythrocytes-cell depleted splenocyteswith or without enhancer-LPETG for 60 min, followed by the additionof biotin-LPETG to the reaction for 15 min (Figure 2c). Cells incubated with enhancer-LPETG prior to biotin-LPETGhad similar amounts of surface-conjugated biotin compared to cellsincubated with biotin-LPTEG alone (Figure 2c). These data suggest that sortagging of VHHs to cells only minimallyaffect subsequent conjugation of biotin-LPETG. The smaller LPETG-taggedprobes may have more ready access to surface-displayed nucleophilesleft unoccupied by larger LPETG-tagged proteins.


One-step enzymatic modification of the cell surface redirects cellular cytotoxicity and parasite tropism.

Swee LK, Lourido S, Bell GW, Ingram JR, Ploegh HL - ACS Chem. Biol. (2014)

Installation of VHHson mouse lymphocytes. (a) In vitro activated CD8T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and with or without 20 μM of sortase A. Controlor sortagged cells were incubated with purified GFP. Binding of GFPwas analyzed by flow cytometry. (b) Control or sortagged cells wereincubated with purified GFP. The amount of bound GFP was estimatedby analysis of cell lysates by SDS-PAGE and immunoblotting againstGFP and comparing the resultant signal to a GFP standard (right lanes).(c) Erythrocyte-depleted splenocytes were incubated with or without500 μM enhancer-LPETG and 20 μM sortase A. After 60 min,500 μM biotin-LPETG was added to reactions where indicated fora further 15 min. Dot plots show the binding of APC-conjugated streptavidinand GFP by sortagged cells after washing. (d) In vitro activated CD8 T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and 20 μM of sortase A. Sortagged cells were incubatedwith splenocytes from WT mice for 20 h. Histograms show the percentageof propidium iodide-negative CD4 and CD19 cells, compared to cellsincubated with control activated OTI CD8 T cells. Error bars: standarddeviation (n = 3). (e) CAR T cells are geneticallyengineered to express a synthetic receptor composed of an extracellularsingle-chain variable fragment and one or several cytoplasmic activatingmotifs that mediate signal transduction and T cell activation uponantigen binding. (f) Sortase-mediated conjugation of VHHs on activatedT cells affords redirection of cytotoxicity toward cells expressingthe targeted antigen.
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fig2: Installation of VHHson mouse lymphocytes. (a) In vitro activated CD8T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and with or without 20 μM of sortase A. Controlor sortagged cells were incubated with purified GFP. Binding of GFPwas analyzed by flow cytometry. (b) Control or sortagged cells wereincubated with purified GFP. The amount of bound GFP was estimatedby analysis of cell lysates by SDS-PAGE and immunoblotting againstGFP and comparing the resultant signal to a GFP standard (right lanes).(c) Erythrocyte-depleted splenocytes were incubated with or without500 μM enhancer-LPETG and 20 μM sortase A. After 60 min,500 μM biotin-LPETG was added to reactions where indicated fora further 15 min. Dot plots show the binding of APC-conjugated streptavidinand GFP by sortagged cells after washing. (d) In vitro activated CD8 T cells from OTI RAG–/– mice were incubatedfor 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETGor VHH7-LPETG and 20 μM of sortase A. Sortagged cells were incubatedwith splenocytes from WT mice for 20 h. Histograms show the percentageof propidium iodide-negative CD4 and CD19 cells, compared to cellsincubated with control activated OTI CD8 T cells. Error bars: standarddeviation (n = 3). (e) CAR T cells are geneticallyengineered to express a synthetic receptor composed of an extracellularsingle-chain variable fragment and one or several cytoplasmic activatingmotifs that mediate signal transduction and T cell activation uponantigen binding. (f) Sortase-mediated conjugation of VHHs on activatedT cells affords redirection of cytotoxicity toward cells expressingthe targeted antigen.
Mentions: Expression of tumor-specificchimeric antigen receptors at the surface of CD8 T cells is emergingas practical approach to eradicate leukemic cells.13 To investigate whether LPETG-tagged single domain antibodies(VHH) could be attached to the surface of activated CD8 T cells bymeans of sortagging, we incubated cytotoxic OTI CD8 T cells with increasingconcentrations of a GFP-specific (“enhancer”)33 or a mouse class II MHC-specific (“VHH7”)34 single domain antibody in the presence or absenceof sortase A. To monitor installation of VHHs as well as assess theirspecificity in a cell-bound format, we measured the ability of cellsto bind GFP by flow cytometry. Only cells incubated with enhancer-LPETGand sortase A bound recombinant GFP in a dose-dependent fashion (Figure 2a). The concentration of VHHs used for the reactionis proportional to the number of VHHs installed. We estimated thenumber of VHHs installed per cell by measuring the number of boundGFP molecules by SDS-PAGE and immunoblotting, using a solution ofGFP of known concentration as standard (Figure 2b). Sortagging of T cells in the presence of 500 μM of VHHsand sortase A resulted in the conjugation of ∼1 million VHHsper cell. To investigate whether two different probes could be installedon lymphocytes, we incubated erythrocytes-cell depleted splenocyteswith or without enhancer-LPETG for 60 min, followed by the additionof biotin-LPETG to the reaction for 15 min (Figure 2c). Cells incubated with enhancer-LPETG prior to biotin-LPETGhad similar amounts of surface-conjugated biotin compared to cellsincubated with biotin-LPTEG alone (Figure 2c). These data suggest that sortagging of VHHs to cells only minimallyaffect subsequent conjugation of biotin-LPETG. The smaller LPETG-taggedprobes may have more ready access to surface-displayed nucleophilesleft unoccupied by larger LPETG-tagged proteins.

Bottom Line: Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen.Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies.This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.

ABSTRACT
Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.

Show MeSH
Related in: MedlinePlus