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Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

Jonnalagadda M, Brown CE, Chang WC, Ostberg JR, Forman SJ, Jensen MC - PLoS ONE (2013)

Bottom Line: We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA).These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells.Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America.

ABSTRACT
Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

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Experimental system for evaluating selection of gene-modified T cells in NSG mice.(a), Plasmid map of construct containing drug resistance genes DHFRFS and IMPDH2IY, huEGFRt and T2A gene sequences (black) that was used to genetically alter primary human T cells. Lentiviral vector backbone (epHIV7) - related sequences are depicted in grey. (b), Schematic for isolation, genetic modification and selection of primary human T cells. (c), CD45RA and CD14 staining of mononuclear cells after sorting from PBMC (top), and CD62L and CD45RO staining of T cells after enriching from PBMC (bottom). The percent positive cells in each quadrant are indicated. (d), Primary human T cells were transduced with above lentiviral vector; transduced T cells and immunomagnetic sorted EGFRt+ T cells were stained for EGFRt expression and analyzed for transduction efficiency by flow cytometry. The percent EGFRt+ T (grey histogram; isotype control-dark line) cells are depicted.
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pone-0065519-g001: Experimental system for evaluating selection of gene-modified T cells in NSG mice.(a), Plasmid map of construct containing drug resistance genes DHFRFS and IMPDH2IY, huEGFRt and T2A gene sequences (black) that was used to genetically alter primary human T cells. Lentiviral vector backbone (epHIV7) - related sequences are depicted in grey. (b), Schematic for isolation, genetic modification and selection of primary human T cells. (c), CD45RA and CD14 staining of mononuclear cells after sorting from PBMC (top), and CD62L and CD45RO staining of T cells after enriching from PBMC (bottom). The percent positive cells in each quadrant are indicated. (d), Primary human T cells were transduced with above lentiviral vector; transduced T cells and immunomagnetic sorted EGFRt+ T cells were stained for EGFRt expression and analyzed for transduction efficiency by flow cytometry. The percent EGFRt+ T (grey histogram; isotype control-dark line) cells are depicted.

Mentions: To compare MTX- and MMF-mediated cell selection strategies, either singly or in combination, we designed a lentiviral vector to direct the co-expression of DHFRFS, IMPDH2IY and a truncated human EGF receptor (huEGFRt) [22]. The transgenes are expressed from a single EF-1α promoter, with each polypeptide sequence separated by the ribosomal skip T2A sequence [23] for translation of three proteins from one transcribed message (Fig. 1a). EGFRt functions as a way to mark gene modified cells and allows for alternative cell selection via Erbitux™ [22]. We chose to evaluate MTX and MMF drug selection in central memory derived T (TCM) cells, a sub-population of CD62L+CD45RO+ T cells, which have been shown to have favorable properties for therapeutic application including the capacity for self renewal, proliferation, long-term persistence, and an ability to differentiate into effector T cells [10], [15], [24]. As previously published, we consistently enrich TCM cells to greater than 70% purity from peripheral blood by first depleting naïve T cells (CD45RA) and monocytes (CD14), then positively selecting for CD62L+ cells (Fig. 1b and 1c) [25]. Here, the enriched TCM (83% CD62L+CD45RO+, Fig. 1c) were then transduced with an EGFRt-T2A-DHFRFS-T2A-IMPDH2IY_epHIV lentiviral vector (Fig. 1a), which typically yields greater than 20% cell transduction as assessed by cell surface expression of huEGFRt (Fig. 1d). These engineered cells could then be further immunomagnetically enriched to greater than 98% purity with cetuximab based on expression of huEGFRt (Fig. 1d).


Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

Jonnalagadda M, Brown CE, Chang WC, Ostberg JR, Forman SJ, Jensen MC - PLoS ONE (2013)

Experimental system for evaluating selection of gene-modified T cells in NSG mice.(a), Plasmid map of construct containing drug resistance genes DHFRFS and IMPDH2IY, huEGFRt and T2A gene sequences (black) that was used to genetically alter primary human T cells. Lentiviral vector backbone (epHIV7) - related sequences are depicted in grey. (b), Schematic for isolation, genetic modification and selection of primary human T cells. (c), CD45RA and CD14 staining of mononuclear cells after sorting from PBMC (top), and CD62L and CD45RO staining of T cells after enriching from PBMC (bottom). The percent positive cells in each quadrant are indicated. (d), Primary human T cells were transduced with above lentiviral vector; transduced T cells and immunomagnetic sorted EGFRt+ T cells were stained for EGFRt expression and analyzed for transduction efficiency by flow cytometry. The percent EGFRt+ T (grey histogram; isotype control-dark line) cells are depicted.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675038&req=5

pone-0065519-g001: Experimental system for evaluating selection of gene-modified T cells in NSG mice.(a), Plasmid map of construct containing drug resistance genes DHFRFS and IMPDH2IY, huEGFRt and T2A gene sequences (black) that was used to genetically alter primary human T cells. Lentiviral vector backbone (epHIV7) - related sequences are depicted in grey. (b), Schematic for isolation, genetic modification and selection of primary human T cells. (c), CD45RA and CD14 staining of mononuclear cells after sorting from PBMC (top), and CD62L and CD45RO staining of T cells after enriching from PBMC (bottom). The percent positive cells in each quadrant are indicated. (d), Primary human T cells were transduced with above lentiviral vector; transduced T cells and immunomagnetic sorted EGFRt+ T cells were stained for EGFRt expression and analyzed for transduction efficiency by flow cytometry. The percent EGFRt+ T (grey histogram; isotype control-dark line) cells are depicted.
Mentions: To compare MTX- and MMF-mediated cell selection strategies, either singly or in combination, we designed a lentiviral vector to direct the co-expression of DHFRFS, IMPDH2IY and a truncated human EGF receptor (huEGFRt) [22]. The transgenes are expressed from a single EF-1α promoter, with each polypeptide sequence separated by the ribosomal skip T2A sequence [23] for translation of three proteins from one transcribed message (Fig. 1a). EGFRt functions as a way to mark gene modified cells and allows for alternative cell selection via Erbitux™ [22]. We chose to evaluate MTX and MMF drug selection in central memory derived T (TCM) cells, a sub-population of CD62L+CD45RO+ T cells, which have been shown to have favorable properties for therapeutic application including the capacity for self renewal, proliferation, long-term persistence, and an ability to differentiate into effector T cells [10], [15], [24]. As previously published, we consistently enrich TCM cells to greater than 70% purity from peripheral blood by first depleting naïve T cells (CD45RA) and monocytes (CD14), then positively selecting for CD62L+ cells (Fig. 1b and 1c) [25]. Here, the enriched TCM (83% CD62L+CD45RO+, Fig. 1c) were then transduced with an EGFRt-T2A-DHFRFS-T2A-IMPDH2IY_epHIV lentiviral vector (Fig. 1a), which typically yields greater than 20% cell transduction as assessed by cell surface expression of huEGFRt (Fig. 1d). These engineered cells could then be further immunomagnetically enriched to greater than 98% purity with cetuximab based on expression of huEGFRt (Fig. 1d).

Bottom Line: We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA).These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells.Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America.

ABSTRACT
Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

Show MeSH
Related in: MedlinePlus