Limits...
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.

Show MeSH

Related in: MedlinePlus

Primary human T cells transduced to DHFRFS/IMPDH2IY transgenes are resistant to MTX and MPA.Non-transduced T cells (non-Txd; grey line/bar) and immunomagnetically-enriched EGFRt+ T cells (99.5% EGFRt+; Txd+Enr; black line/bar) were plated on day 8 at the indicated concentrations of MTX (a), MPA (b), and a combination of MTX+ MPA (c), cells were followed for total viable cell number, percentage of viable cells, and fold expansion for12 days. Equal numbers of cells were plated in triplicate wells of 24-well plates. The data represent the mean ± S.D. There was a significant difference in viability and fold expansion at day 12 between the non-transduced (Non-Txd) and the transduced, EGFRt-enriched T cells (Txd+Enr) at each drug concentration. ***, p≤0.0002; **, p≤0.001; *, p≤0.01. The data are representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675038&req=5

pone-0065519-g002: Primary human T cells transduced to DHFRFS/IMPDH2IY transgenes are resistant to MTX and MPA.Non-transduced T cells (non-Txd; grey line/bar) and immunomagnetically-enriched EGFRt+ T cells (99.5% EGFRt+; Txd+Enr; black line/bar) were plated on day 8 at the indicated concentrations of MTX (a), MPA (b), and a combination of MTX+ MPA (c), cells were followed for total viable cell number, percentage of viable cells, and fold expansion for12 days. Equal numbers of cells were plated in triplicate wells of 24-well plates. The data represent the mean ± S.D. There was a significant difference in viability and fold expansion at day 12 between the non-transduced (Non-Txd) and the transduced, EGFRt-enriched T cells (Txd+Enr) at each drug concentration. ***, p≤0.0002; **, p≤0.001; *, p≤0.01. The data are representative of three separate experiments.

Mentions: To examine the resistance of huEGFRt/DHFRFS/IMPDH2IY-transduced TCM to MTX and MPA, we first examined engineered cells that had been immunomagnetically enriched for huEGFRt+ expression (>98% purity; Fig. 1d). As expected, huEGFRt-enriched cells maintained a T cell phenotype as assessed by surface expression of CD4, CD8, CD28, CD45, TCRαβ and CD127 (Fig. S1), and displayed equally potent functional activity, as compared to non-transduced control TCM (Fig. S1). These uniformly positive huEGFRt (>98%) -expressing populations enabled us to determine the optimal drug selection concentrations for huEGFRt+/DHFRFS/IMPDH2IY-engineered TCM. To this end, non-transduced vs. transduced and EGFRt-enriched (EGFRt+) cells were plated in MTX (0–0.25 µM) (Fig. 2a), MPA (0–2.5 µM) (Fig. 2b), and combinations of both (Fig. 2c). In the absence of MTX and MPA, the non-transduced and EGFRt+ T cells expanded at an equivalent rate (70.0±4.6 and 68.6±3.0 fold). Following incubation with MTX for 12 days, non-transduced T cells did not expand at concentrations ≥0.05 µM MTX and a decrease in viability from 92% to 57.1±11.2% (Fig. 2a). In contrast, huEGFRt+/DHFRFS/IMPDH2IY T cells expanded 20.5±1.8-fold at 0.05 µM and 11.4±2.1-fold at 0.1 µM MTX with 93.6±2.7% viable cells. (Fig. 2a). In the presence of MPA, huEGFRt+/DHFRFS/IMPDH2IY T cells expanded 4.7±0.6-fold at 0.75 µM and 3±0.9-fold at 1 µM MPA over 12 days with 83.4±2.1–92.3±1.4% viable cells, whereas non-transduced cells did not expand and cell viability decreased to 65.6±4.1% and 58.3±2.3% at these concentrations (Fig. 2b). Furthermore, we analyzed the survival and expansion of huEGFRt+/DHFRFS/IMPDH2IY T cells in media containing combinations of 0.025 µM MTX +0.5 µM MPA and 0.05 µM MTX +0.75 µM MPA over 12 days. The transduced, huEGFRt-enriched T cells expanded 9.2±0.7 and 5.4±1.0-fold, respectively, with 92.2±1.6% viability, whereas non-transduced cells did not expand and cell viability decreased to 65.7±6.3% and 47.6±4.3% (Fig. 2c). These data indicate that both DHFRFS and IMPDH2IY can confer drug resistance to primary human T cells, allowing them to expand and maintain high cell viability (≥90%) upon culture in MTX and MPA. The growth mediated by DHFRFS in the presence of MTX was more robust than that mediated by IMPDH2IY in MPA over the short-term two-week culture period.


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)

Primary human T cells transduced to DHFRFS/IMPDH2IY transgenes are resistant to MTX and MPA.Non-transduced T cells (non-Txd; grey line/bar) and immunomagnetically-enriched EGFRt+ T cells (99.5% EGFRt+; Txd+Enr; black line/bar) were plated on day 8 at the indicated concentrations of MTX (a), MPA (b), and a combination of MTX+ MPA (c), cells were followed for total viable cell number, percentage of viable cells, and fold expansion for12 days. Equal numbers of cells were plated in triplicate wells of 24-well plates. The data represent the mean ± S.D. There was a significant difference in viability and fold expansion at day 12 between the non-transduced (Non-Txd) and the transduced, EGFRt-enriched T cells (Txd+Enr) at each drug concentration. ***, p≤0.0002; **, p≤0.001; *, p≤0.01. The data are representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675038&req=5

pone-0065519-g002: Primary human T cells transduced to DHFRFS/IMPDH2IY transgenes are resistant to MTX and MPA.Non-transduced T cells (non-Txd; grey line/bar) and immunomagnetically-enriched EGFRt+ T cells (99.5% EGFRt+; Txd+Enr; black line/bar) were plated on day 8 at the indicated concentrations of MTX (a), MPA (b), and a combination of MTX+ MPA (c), cells were followed for total viable cell number, percentage of viable cells, and fold expansion for12 days. Equal numbers of cells were plated in triplicate wells of 24-well plates. The data represent the mean ± S.D. There was a significant difference in viability and fold expansion at day 12 between the non-transduced (Non-Txd) and the transduced, EGFRt-enriched T cells (Txd+Enr) at each drug concentration. ***, p≤0.0002; **, p≤0.001; *, p≤0.01. The data are representative of three separate experiments.
Mentions: To examine the resistance of huEGFRt/DHFRFS/IMPDH2IY-transduced TCM to MTX and MPA, we first examined engineered cells that had been immunomagnetically enriched for huEGFRt+ expression (>98% purity; Fig. 1d). As expected, huEGFRt-enriched cells maintained a T cell phenotype as assessed by surface expression of CD4, CD8, CD28, CD45, TCRαβ and CD127 (Fig. S1), and displayed equally potent functional activity, as compared to non-transduced control TCM (Fig. S1). These uniformly positive huEGFRt (>98%) -expressing populations enabled us to determine the optimal drug selection concentrations for huEGFRt+/DHFRFS/IMPDH2IY-engineered TCM. To this end, non-transduced vs. transduced and EGFRt-enriched (EGFRt+) cells were plated in MTX (0–0.25 µM) (Fig. 2a), MPA (0–2.5 µM) (Fig. 2b), and combinations of both (Fig. 2c). In the absence of MTX and MPA, the non-transduced and EGFRt+ T cells expanded at an equivalent rate (70.0±4.6 and 68.6±3.0 fold). Following incubation with MTX for 12 days, non-transduced T cells did not expand at concentrations ≥0.05 µM MTX and a decrease in viability from 92% to 57.1±11.2% (Fig. 2a). In contrast, huEGFRt+/DHFRFS/IMPDH2IY T cells expanded 20.5±1.8-fold at 0.05 µM and 11.4±2.1-fold at 0.1 µM MTX with 93.6±2.7% viable cells. (Fig. 2a). In the presence of MPA, huEGFRt+/DHFRFS/IMPDH2IY T cells expanded 4.7±0.6-fold at 0.75 µM and 3±0.9-fold at 1 µM MPA over 12 days with 83.4±2.1–92.3±1.4% viable cells, whereas non-transduced cells did not expand and cell viability decreased to 65.6±4.1% and 58.3±2.3% at these concentrations (Fig. 2b). Furthermore, we analyzed the survival and expansion of huEGFRt+/DHFRFS/IMPDH2IY T cells in media containing combinations of 0.025 µM MTX +0.5 µM MPA and 0.05 µM MTX +0.75 µM MPA over 12 days. The transduced, huEGFRt-enriched T cells expanded 9.2±0.7 and 5.4±1.0-fold, respectively, with 92.2±1.6% viability, whereas non-transduced cells did not expand and cell viability decreased to 65.7±6.3% and 47.6±4.3% (Fig. 2c). These data indicate that both DHFRFS and IMPDH2IY can confer drug resistance to primary human T cells, allowing them to expand and maintain high cell viability (≥90%) upon culture in MTX and MPA. The growth mediated by DHFRFS in the presence of MTX was more robust than that mediated by IMPDH2IY in MPA over the short-term two-week culture period.

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