Limits...
CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

Han J, Chu J, Keung Chan W, Zhang J, Wang Y, Cohen JB, Victor A, Meisen WH, Kim SH, Grandi P, Wang QE, He X, Nakano I, Chiocca EA, Glorioso JC, Kaur B, Caligiuri MA, Yu J - Sci Rep (2015)

Bottom Line: We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells.EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner.In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA [2] The Ohio State University Comprehensive Cancer Center, Columbus, Ohio 43210, USA.

ABSTRACT
Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

No MeSH data available.


Related in: MedlinePlus

NK-92-EGFR-CAR cells suppress in vivo growth of orthotopic human GSCs, prolong the survival of glioma-bearing mice, and localize in the brain without migrating to other organ and tissues.(A) Brain bioluminescence imaging of mice bearing GB30 tumors. NSG mice were inoculated with luciferase-expressing GB30 cells via stereotaxic injection (day 0). Seven days after inoculation, mice were intracranially infused once with empty vector-transduced NK-92 cells (NK-92-EV), EGFR-CAR- transduced NK-92 cells (NK-92-EGFR-CAR) or Hank’s buffered salt solution (HBSS; negative control). (B) Quantification summary of units of photons per second per mouse from (A). * indicates p < 0.05. (C) GB30-bearing mice treated with NK-92-EGFR-CAR cells showed significantly increased overall survival compared to the mice treated with NK-92-EV cells or HBSS (** p < 0.01), as determined by Kaplan-Meier survival curves (n = 5 for each group). (D) Determination of presence of CD56+CD3- human EGFR-CAR NK-92 cells by flow cytometry in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. (E) Determination of EGFR-CAR expression by RT-PCR in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. NC = negative control (no DNA template was added); PC = positive control, EGFR-CAR NK-92 cells. *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496728&req=5

f6: NK-92-EGFR-CAR cells suppress in vivo growth of orthotopic human GSCs, prolong the survival of glioma-bearing mice, and localize in the brain without migrating to other organ and tissues.(A) Brain bioluminescence imaging of mice bearing GB30 tumors. NSG mice were inoculated with luciferase-expressing GB30 cells via stereotaxic injection (day 0). Seven days after inoculation, mice were intracranially infused once with empty vector-transduced NK-92 cells (NK-92-EV), EGFR-CAR- transduced NK-92 cells (NK-92-EGFR-CAR) or Hank’s buffered salt solution (HBSS; negative control). (B) Quantification summary of units of photons per second per mouse from (A). * indicates p < 0.05. (C) GB30-bearing mice treated with NK-92-EGFR-CAR cells showed significantly increased overall survival compared to the mice treated with NK-92-EV cells or HBSS (** p < 0.01), as determined by Kaplan-Meier survival curves (n = 5 for each group). (D) Determination of presence of CD56+CD3- human EGFR-CAR NK-92 cells by flow cytometry in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. (E) Determination of EGFR-CAR expression by RT-PCR in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. NC = negative control (no DNA template was added); PC = positive control, EGFR-CAR NK-92 cells. *p < 0.05, **p < 0.01.

Mentions: To further address the potential therapeutic application of NK-92-EGFR-CAR cells, we examined their antitumor activity in vivo. We established orthotopic glioma by intracranially implanting EGFRvIII-expressing GB30 GSCs which had been genetically manipulated to express firefly luciferase (GB30-FFL) into the brains of NSG mice. The expression of firefly luciferase enabled us to monitor the tumor growth via in vivo bioluminescence imaging. To minimize potential systemic toxicity, we injected the NK-92-EGFR-CAR intratumorally 7 days post tumor cell implantation. As shown in Fig. 6A,B, mice that received either EGFR-CAR- or mock-transduced NK-92 cells had significantly reduced tumor growth as determined by bioluminescence imaging, compared to those injected with Hank’s buffered salt solution (HBSS). Importantly, however, the reduction in tumor growth was significantly greater in mice treated with NK-92-EGFR-CAR cells than those treated with mock-transduced NK-92 cells. In agreement with these data, mice treated with NK-92-EGFR-CAR cells for a single time survived significantly longer than mice treated with mock-transduced NK-92 cells or HBSS (median survival of 38 vs 23 days between NK-92-EGFR-CAR- and NK-92-EV-treated mice, p < 0.01; median survival of 38 vs 17 days between NK-92-EGFR-CAR- and HBSS-treated mice, p < 0.01) (Fig. 6C). To further address the therapeutic efficacy of NK-92-EGFR-CAR cells against wtEGFR-expressing GB tumor, we established an orthotopic GB model by intracranially implanting wtEGFR-expressing U251-FFL cells into NSG mice. We injected NK-92-EGFR-CAR cells, NK-92-EV cells or HBSS as a vehicle control intratumorally 10 days, 40 days and 70 days after tumor cell implantation. As shown in Fig. 7A,B, mice with a single intratumoral injection of EGFR-CAR cells had significantly decreased tumor burden, compared to those infused with HBSS or NK-92-EV cells. Moreover, mice treated with NK-92-EGFR-CAR cells survived significantly longer than those receiving NK-92-EV cells or HBSS (median survival of 187 vs 150 days between NK-92-EGFR-CAR- and NK-92-EV cell-treated mice, p < 0.05; median survival of 187 vs 138 days between NK-92-EGFR-CAR- and HBSS-treated mice, p < 0.01) (Fig. 7C). Taken together, NK-92-EGFR-CAR cells could efficiently target and kill either wtEGFR- or EGFRvIII-expressing GB in vivo.


CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

Han J, Chu J, Keung Chan W, Zhang J, Wang Y, Cohen JB, Victor A, Meisen WH, Kim SH, Grandi P, Wang QE, He X, Nakano I, Chiocca EA, Glorioso JC, Kaur B, Caligiuri MA, Yu J - Sci Rep (2015)

NK-92-EGFR-CAR cells suppress in vivo growth of orthotopic human GSCs, prolong the survival of glioma-bearing mice, and localize in the brain without migrating to other organ and tissues.(A) Brain bioluminescence imaging of mice bearing GB30 tumors. NSG mice were inoculated with luciferase-expressing GB30 cells via stereotaxic injection (day 0). Seven days after inoculation, mice were intracranially infused once with empty vector-transduced NK-92 cells (NK-92-EV), EGFR-CAR- transduced NK-92 cells (NK-92-EGFR-CAR) or Hank’s buffered salt solution (HBSS; negative control). (B) Quantification summary of units of photons per second per mouse from (A). * indicates p < 0.05. (C) GB30-bearing mice treated with NK-92-EGFR-CAR cells showed significantly increased overall survival compared to the mice treated with NK-92-EV cells or HBSS (** p < 0.01), as determined by Kaplan-Meier survival curves (n = 5 for each group). (D) Determination of presence of CD56+CD3- human EGFR-CAR NK-92 cells by flow cytometry in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. (E) Determination of EGFR-CAR expression by RT-PCR in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. NC = negative control (no DNA template was added); PC = positive control, EGFR-CAR NK-92 cells. *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496728&req=5

f6: NK-92-EGFR-CAR cells suppress in vivo growth of orthotopic human GSCs, prolong the survival of glioma-bearing mice, and localize in the brain without migrating to other organ and tissues.(A) Brain bioluminescence imaging of mice bearing GB30 tumors. NSG mice were inoculated with luciferase-expressing GB30 cells via stereotaxic injection (day 0). Seven days after inoculation, mice were intracranially infused once with empty vector-transduced NK-92 cells (NK-92-EV), EGFR-CAR- transduced NK-92 cells (NK-92-EGFR-CAR) or Hank’s buffered salt solution (HBSS; negative control). (B) Quantification summary of units of photons per second per mouse from (A). * indicates p < 0.05. (C) GB30-bearing mice treated with NK-92-EGFR-CAR cells showed significantly increased overall survival compared to the mice treated with NK-92-EV cells or HBSS (** p < 0.01), as determined by Kaplan-Meier survival curves (n = 5 for each group). (D) Determination of presence of CD56+CD3- human EGFR-CAR NK-92 cells by flow cytometry in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. (E) Determination of EGFR-CAR expression by RT-PCR in liver, lung, blood, spleen, bone marrow (BM), and brain 3 days after intracranial injection of the CAR NK cells into brain of GB30-bearing mice. NC = negative control (no DNA template was added); PC = positive control, EGFR-CAR NK-92 cells. *p < 0.05, **p < 0.01.
Mentions: To further address the potential therapeutic application of NK-92-EGFR-CAR cells, we examined their antitumor activity in vivo. We established orthotopic glioma by intracranially implanting EGFRvIII-expressing GB30 GSCs which had been genetically manipulated to express firefly luciferase (GB30-FFL) into the brains of NSG mice. The expression of firefly luciferase enabled us to monitor the tumor growth via in vivo bioluminescence imaging. To minimize potential systemic toxicity, we injected the NK-92-EGFR-CAR intratumorally 7 days post tumor cell implantation. As shown in Fig. 6A,B, mice that received either EGFR-CAR- or mock-transduced NK-92 cells had significantly reduced tumor growth as determined by bioluminescence imaging, compared to those injected with Hank’s buffered salt solution (HBSS). Importantly, however, the reduction in tumor growth was significantly greater in mice treated with NK-92-EGFR-CAR cells than those treated with mock-transduced NK-92 cells. In agreement with these data, mice treated with NK-92-EGFR-CAR cells for a single time survived significantly longer than mice treated with mock-transduced NK-92 cells or HBSS (median survival of 38 vs 23 days between NK-92-EGFR-CAR- and NK-92-EV-treated mice, p < 0.01; median survival of 38 vs 17 days between NK-92-EGFR-CAR- and HBSS-treated mice, p < 0.01) (Fig. 6C). To further address the therapeutic efficacy of NK-92-EGFR-CAR cells against wtEGFR-expressing GB tumor, we established an orthotopic GB model by intracranially implanting wtEGFR-expressing U251-FFL cells into NSG mice. We injected NK-92-EGFR-CAR cells, NK-92-EV cells or HBSS as a vehicle control intratumorally 10 days, 40 days and 70 days after tumor cell implantation. As shown in Fig. 7A,B, mice with a single intratumoral injection of EGFR-CAR cells had significantly decreased tumor burden, compared to those infused with HBSS or NK-92-EV cells. Moreover, mice treated with NK-92-EGFR-CAR cells survived significantly longer than those receiving NK-92-EV cells or HBSS (median survival of 187 vs 150 days between NK-92-EGFR-CAR- and NK-92-EV cell-treated mice, p < 0.05; median survival of 187 vs 138 days between NK-92-EGFR-CAR- and HBSS-treated mice, p < 0.01) (Fig. 7C). Taken together, NK-92-EGFR-CAR cells could efficiently target and kill either wtEGFR- or EGFRvIII-expressing GB in vivo.

Bottom Line: We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells.EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner.In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA [2] The Ohio State University Comprehensive Cancer Center, Columbus, Ohio 43210, USA.

ABSTRACT
Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

No MeSH data available.


Related in: MedlinePlus