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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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Establish non-toxic MTX and MMF dose regimens for in vivo selection.6–8 week old NSG mice (n = 6) were (a), administered MTX by i.p. injection at 25 mg/kg/day twice a week the first week (days 1 and 4) and only once the second week (day 8), and/or (b), fed 0.563% MMF medicated feed for 2 weeks. Serum MTX and MPA levels in each case were analyzed by HPLC. Drug toxicity was then monitored by measuring (c), white blood cell counts, (d), body weight, (e), Hemoglobin, (f), ALT, and (g), creatinine levels. (c–g), Mean levels of each measurement (± S.D) after the last treatment (i.e., at day 14) are depicted. There was no significant difference between control and treatment mice (p≥0.05). Data were evaluated between control and treatment mice using an unpaired, two-tailed Student’s t - test.
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pone-0065519-g005: Establish non-toxic MTX and MMF dose regimens for in vivo selection.6–8 week old NSG mice (n = 6) were (a), administered MTX by i.p. injection at 25 mg/kg/day twice a week the first week (days 1 and 4) and only once the second week (day 8), and/or (b), fed 0.563% MMF medicated feed for 2 weeks. Serum MTX and MPA levels in each case were analyzed by HPLC. Drug toxicity was then monitored by measuring (c), white blood cell counts, (d), body weight, (e), Hemoglobin, (f), ALT, and (g), creatinine levels. (c–g), Mean levels of each measurement (± S.D) after the last treatment (i.e., at day 14) are depicted. There was no significant difference between control and treatment mice (p≥0.05). Data were evaluated between control and treatment mice using an unpaired, two-tailed Student’s t - test.

Mentions: To assess the potential of DHFRFS and IMPDH2IY to allow for in vivo selection of gene-modified T cells, we first optimized the dosage regimens of MTX and MMF (i.e., the prodrug of MPA) that could be tolerated by NSG mice without deleterious effects. Intraperitoneal administration of MTX 3 times over a 2-week period at 25 mg/kg body weight was found to result in average MTX serum concentrations of 17.3±6.0 µM, 27.4±5.5 µM, and 26.6±1.3 µM when blood was harvested 30 minutes post i.p. injection on days 1, 4, and 8 respectively (Fig. 5a). For MMF, mice received medicated feed (0.563% MMF) for two consecutive weeks (Fig. 5b) in order to maintain more continuous active serum MPA levels, since MPA is known to convert over time to the inactive metabolite glucuronide via uridine 5′ diphosphoglucuronyltransferase [26]. Serum concentrations of MPA achieved with this delivery regimen were 15.5±6.2 µg/ml at day 7, 15.0±0.35 µg/ml at day 14, and then undetectable 7 days after the medicated feed was withdrawn (day 21). Pilot experiments established that these MTX and MMF dosing regimens were sufficient for controlling the proliferation of a murine lymphoblast cell line (CTLL2) in NSG mice (data not shown). Importantly, we found that these drug regimens were well tolerated by mice. There was no significant decrease or increase in white blood cell counts (Fig. 5c), mouse bodyweight (Fig. 5d), hemoglobin (Fig. 5e), alanine aminotransferase (ALT) as a measure of liver function (Fig. 5f), or creatinine levels as a measure of kidney function (Fig. 5g) (p≥0.05) compared to control mice. Collectively, these findings suggested that these drug dose concentrations are of minimal of toxicity and enabled the assessment of our DHFRFS/IMPDH2IY platform for in vivo selection of transduced primary human T cells in NSG mice.


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)

Establish non-toxic MTX and MMF dose regimens for in vivo selection.6–8 week old NSG mice (n = 6) were (a), administered MTX by i.p. injection at 25 mg/kg/day twice a week the first week (days 1 and 4) and only once the second week (day 8), and/or (b), fed 0.563% MMF medicated feed for 2 weeks. Serum MTX and MPA levels in each case were analyzed by HPLC. Drug toxicity was then monitored by measuring (c), white blood cell counts, (d), body weight, (e), Hemoglobin, (f), ALT, and (g), creatinine levels. (c–g), Mean levels of each measurement (± S.D) after the last treatment (i.e., at day 14) are depicted. There was no significant difference between control and treatment mice (p≥0.05). Data were evaluated between control and treatment mice using an unpaired, two-tailed Student’s t - test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675038&req=5

pone-0065519-g005: Establish non-toxic MTX and MMF dose regimens for in vivo selection.6–8 week old NSG mice (n = 6) were (a), administered MTX by i.p. injection at 25 mg/kg/day twice a week the first week (days 1 and 4) and only once the second week (day 8), and/or (b), fed 0.563% MMF medicated feed for 2 weeks. Serum MTX and MPA levels in each case were analyzed by HPLC. Drug toxicity was then monitored by measuring (c), white blood cell counts, (d), body weight, (e), Hemoglobin, (f), ALT, and (g), creatinine levels. (c–g), Mean levels of each measurement (± S.D) after the last treatment (i.e., at day 14) are depicted. There was no significant difference between control and treatment mice (p≥0.05). Data were evaluated between control and treatment mice using an unpaired, two-tailed Student’s t - test.
Mentions: To assess the potential of DHFRFS and IMPDH2IY to allow for in vivo selection of gene-modified T cells, we first optimized the dosage regimens of MTX and MMF (i.e., the prodrug of MPA) that could be tolerated by NSG mice without deleterious effects. Intraperitoneal administration of MTX 3 times over a 2-week period at 25 mg/kg body weight was found to result in average MTX serum concentrations of 17.3±6.0 µM, 27.4±5.5 µM, and 26.6±1.3 µM when blood was harvested 30 minutes post i.p. injection on days 1, 4, and 8 respectively (Fig. 5a). For MMF, mice received medicated feed (0.563% MMF) for two consecutive weeks (Fig. 5b) in order to maintain more continuous active serum MPA levels, since MPA is known to convert over time to the inactive metabolite glucuronide via uridine 5′ diphosphoglucuronyltransferase [26]. Serum concentrations of MPA achieved with this delivery regimen were 15.5±6.2 µg/ml at day 7, 15.0±0.35 µg/ml at day 14, and then undetectable 7 days after the medicated feed was withdrawn (day 21). Pilot experiments established that these MTX and MMF dosing regimens were sufficient for controlling the proliferation of a murine lymphoblast cell line (CTLL2) in NSG mice (data not shown). Importantly, we found that these drug regimens were well tolerated by mice. There was no significant decrease or increase in white blood cell counts (Fig. 5c), mouse bodyweight (Fig. 5d), hemoglobin (Fig. 5e), alanine aminotransferase (ALT) as a measure of liver function (Fig. 5f), or creatinine levels as a measure of kidney function (Fig. 5g) (p≥0.05) compared to control mice. Collectively, these findings suggested that these drug dose concentrations are of minimal of toxicity and enabled the assessment of our DHFRFS/IMPDH2IY platform for in vivo selection of transduced primary human T cells in NSG mice.

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