‘Biodegradable’ CAR may aid transplant in AML

Saad S. Kenderian

Photo courtesy of

BMT Tandem Meetings

SAN DIEGO—Researchers have developed a “biodegradable” chimeric antigen receptor (CAR) T-cell therapy that could potentially serve as a preparative regimen for acute myeloid leukemia (AML) patients undergoing allogeneic transplant.

The team created CAR T cells that target CD33 (CART33) and modified them with RNA so the cells would stop expressing CARs over time.

In mouse models of AML, the RNA-CART33 cells had an antileukemic effect and induced myeloablation.

The cells also stopped expressing CARs by the 2-week mark, which would allow for engraftment after allogeneic transplant, according to the researchers.

Saad S. Kenderian, MD, of the University of Pennsylvania in Philadelphia, presented this research at the 2015 BMT Tandem Meetings as one of the meeting’s “Best Abstracts” (abstract 1). The research was funded by Novartis.

“Allogeneic transplantation is the only potentially curative option in relapsed/refractory AML,” Dr Kenderian noted. “Outcomes are poor if patients are transplanted in residual disease  . . . , and these patients are often considered transplant-ineligible. Therefore, novel therapies are desperately needed.”

With this in mind, Dr Kenderian and his colleagues set out to develop a CAR T-cell therapy targeting CD33, which is expressed on AML blasts.

The researchers created a CAR from the anti-CD33 single-chain fragment variable of gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. They transduced T cells with this construct and expanded them in culture using anti-CD3/CD28 magnetic beads.

The team then tested these CART33 cells in NSGS mice engrafted with primary AML blasts. The mice received CART33 cells, another CAR T-cell therapy known as CART123, or control T cells.

At 4 weeks, mice that had received CART33 or CART123 cells were entirely leukemia-free, but the disease continued to progress in mice that received control T cells.

Likewise, when the experiment ended at 200 days, survival was 100% among mice that received CART33 or CART123, but all of the control mice had died. And at 200 days, CAR T cells were still circulating in the CART33- and CART123-treated mice.

Next, the researchers administered CART33 cells to HIS-NSG mice engrafted with human bone marrow and found the treatment resulted in myeloablation. There was a significant reduction of CD34-positive cells in mice that received CART33 compared to mice that received control T cells or no treatment.

“So based on our preclinical data, when we treat refractory AML with lentivirally transduced CART33, that will result in myeloablation, eradication of AML, and persistence of these CARs,” Dr Kenderian said.

“If allogeneic transplantation is performed at this aplastic stage, it will likely lead to rejection of the graft by persisting CAR therapy, which also means that elimination of CARs is necessary prior to stem cell infusion.”

So the researchers decided to create a transiently expressed, mRNA-modified CAR based on CART33. They electroporated T cells with this construct, and the cells expressed CARs for up to 6 days.

In experiments with the MOLM14 cell line, RNA-modified CART33 cells exhibited transient but comparable killing ability as LV-transduced CART33.

The researchers then tested RNA-CART33 in combination with chemotherapy in vivo. They transplanted NSG mice with MOLM14 and treated them with cyclophosphamide plus RNA-CART33 or cyclophosphamide plus control T cells.

Combination RNA-CART33 and chemotherapy prompted stronger, more durable antileukemic activity than cyclophosphamide and control T cells. Furthermore, there was a significant improvement in survival among RNA-CART33-treated mice (P=0.01).

Finally, Dr Kenderian and his colleagues tested the effect of RNA-CART33 on hematopoiesis. The team treated NSGS mice with busulfan and transplanted them with T-cell-depleted bone marrow. Following engraftment, mice received RNA-CART33 cells, LV-CART33 cells, or control T cells.

The researchers followed the mice for 2 weeks and found that both RNA-CART33 and LV-CART33 induced myeloablation. And at 14 days, LV-CART33-treated mice were still expressing CARs, but RNA-CART33-treated mice were not.

“Based on our preclinical data, if we treat refractory AML with RNA-modified CART33, that results in myeloablation, anti-AML activity, and biodegradable, non-persisting CARs,” Dr Kenderian summarized.

“If allogeneic transplantation follows at this stage, it will likely lead to engraftment. Therefore, we conclude from this study that RNA-CART33 could be incorporated in novel conditioning regimens and will be tested in pilot phase 1 studies.”

Saad S. Kenderian

Photo courtesy of

BMT Tandem Meetings

SAN DIEGO—Researchers have developed a “biodegradable” chimeric antigen receptor (CAR) T-cell therapy that could potentially serve as a preparative regimen for acute myeloid leukemia (AML) patients undergoing allogeneic transplant.

The team created CAR T cells that target CD33 (CART33) and modified them with RNA so the cells would stop expressing CARs over time.

In mouse models of AML, the RNA-CART33 cells had an antileukemic effect and induced myeloablation.

The cells also stopped expressing CARs by the 2-week mark, which would allow for engraftment after allogeneic transplant, according to the researchers.

Saad S. Kenderian, MD, of the University of Pennsylvania in Philadelphia, presented this research at the 2015 BMT Tandem Meetings as one of the meeting’s “Best Abstracts” (abstract 1). The research was funded by Novartis.

“Allogeneic transplantation is the only potentially curative option in relapsed/refractory AML,” Dr Kenderian noted. “Outcomes are poor if patients are transplanted in residual disease  . . . , and these patients are often considered transplant-ineligible. Therefore, novel therapies are desperately needed.”

With this in mind, Dr Kenderian and his colleagues set out to develop a CAR T-cell therapy targeting CD33, which is expressed on AML blasts.

The researchers created a CAR from the anti-CD33 single-chain fragment variable of gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. They transduced T cells with this construct and expanded them in culture using anti-CD3/CD28 magnetic beads.

The team then tested these CART33 cells in NSGS mice engrafted with primary AML blasts. The mice received CART33 cells, another CAR T-cell therapy known as CART123, or control T cells.

At 4 weeks, mice that had received CART33 or CART123 cells were entirely leukemia-free, but the disease continued to progress in mice that received control T cells.

Likewise, when the experiment ended at 200 days, survival was 100% among mice that received CART33 or CART123, but all of the control mice had died. And at 200 days, CAR T cells were still circulating in the CART33- and CART123-treated mice.

Next, the researchers administered CART33 cells to HIS-NSG mice engrafted with human bone marrow and found the treatment resulted in myeloablation. There was a significant reduction of CD34-positive cells in mice that received CART33 compared to mice that received control T cells or no treatment.

“So based on our preclinical data, when we treat refractory AML with lentivirally transduced CART33, that will result in myeloablation, eradication of AML, and persistence of these CARs,” Dr Kenderian said.

“If allogeneic transplantation is performed at this aplastic stage, it will likely lead to rejection of the graft by persisting CAR therapy, which also means that elimination of CARs is necessary prior to stem cell infusion.”

So the researchers decided to create a transiently expressed, mRNA-modified CAR based on CART33. They electroporated T cells with this construct, and the cells expressed CARs for up to 6 days.

In experiments with the MOLM14 cell line, RNA-modified CART33 cells exhibited transient but comparable killing ability as LV-transduced CART33.

The researchers then tested RNA-CART33 in combination with chemotherapy in vivo. They transplanted NSG mice with MOLM14 and treated them with cyclophosphamide plus RNA-CART33 or cyclophosphamide plus control T cells.

Combination RNA-CART33 and chemotherapy prompted stronger, more durable antileukemic activity than cyclophosphamide and control T cells. Furthermore, there was a significant improvement in survival among RNA-CART33-treated mice (P=0.01).

Finally, Dr Kenderian and his colleagues tested the effect of RNA-CART33 on hematopoiesis. The team treated NSGS mice with busulfan and transplanted them with T-cell-depleted bone marrow. Following engraftment, mice received RNA-CART33 cells, LV-CART33 cells, or control T cells.

The researchers followed the mice for 2 weeks and found that both RNA-CART33 and LV-CART33 induced myeloablation. And at 14 days, LV-CART33-treated mice were still expressing CARs, but RNA-CART33-treated mice were not.

“Based on our preclinical data, if we treat refractory AML with RNA-modified CART33, that results in myeloablation, anti-AML activity, and biodegradable, non-persisting CARs,” Dr Kenderian summarized.

“If allogeneic transplantation follows at this stage, it will likely lead to engraftment. Therefore, we conclude from this study that RNA-CART33 could be incorporated in novel conditioning regimens and will be tested in pilot phase 1 studies.”

Saad S. Kenderian

Photo courtesy of

BMT Tandem Meetings

SAN DIEGO—Researchers have developed a “biodegradable” chimeric antigen receptor (CAR) T-cell therapy that could potentially serve as a preparative regimen for acute myeloid leukemia (AML) patients undergoing allogeneic transplant.

The team created CAR T cells that target CD33 (CART33) and modified them with RNA so the cells would stop expressing CARs over time.

In mouse models of AML, the RNA-CART33 cells had an antileukemic effect and induced myeloablation.

The cells also stopped expressing CARs by the 2-week mark, which would allow for engraftment after allogeneic transplant, according to the researchers.

Saad S. Kenderian, MD, of the University of Pennsylvania in Philadelphia, presented this research at the 2015 BMT Tandem Meetings as one of the meeting’s “Best Abstracts” (abstract 1). The research was funded by Novartis.

“Allogeneic transplantation is the only potentially curative option in relapsed/refractory AML,” Dr Kenderian noted. “Outcomes are poor if patients are transplanted in residual disease  . . . , and these patients are often considered transplant-ineligible. Therefore, novel therapies are desperately needed.”

With this in mind, Dr Kenderian and his colleagues set out to develop a CAR T-cell therapy targeting CD33, which is expressed on AML blasts.

The researchers created a CAR from the anti-CD33 single-chain fragment variable of gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. They transduced T cells with this construct and expanded them in culture using anti-CD3/CD28 magnetic beads.

The team then tested these CART33 cells in NSGS mice engrafted with primary AML blasts. The mice received CART33 cells, another CAR T-cell therapy known as CART123, or control T cells.

At 4 weeks, mice that had received CART33 or CART123 cells were entirely leukemia-free, but the disease continued to progress in mice that received control T cells.

Likewise, when the experiment ended at 200 days, survival was 100% among mice that received CART33 or CART123, but all of the control mice had died. And at 200 days, CAR T cells were still circulating in the CART33- and CART123-treated mice.

Next, the researchers administered CART33 cells to HIS-NSG mice engrafted with human bone marrow and found the treatment resulted in myeloablation. There was a significant reduction of CD34-positive cells in mice that received CART33 compared to mice that received control T cells or no treatment.

“So based on our preclinical data, when we treat refractory AML with lentivirally transduced CART33, that will result in myeloablation, eradication of AML, and persistence of these CARs,” Dr Kenderian said.

“If allogeneic transplantation is performed at this aplastic stage, it will likely lead to rejection of the graft by persisting CAR therapy, which also means that elimination of CARs is necessary prior to stem cell infusion.”

So the researchers decided to create a transiently expressed, mRNA-modified CAR based on CART33. They electroporated T cells with this construct, and the cells expressed CARs for up to 6 days.

In experiments with the MOLM14 cell line, RNA-modified CART33 cells exhibited transient but comparable killing ability as LV-transduced CART33.

The researchers then tested RNA-CART33 in combination with chemotherapy in vivo. They transplanted NSG mice with MOLM14 and treated them with cyclophosphamide plus RNA-CART33 or cyclophosphamide plus control T cells.

Combination RNA-CART33 and chemotherapy prompted stronger, more durable antileukemic activity than cyclophosphamide and control T cells. Furthermore, there was a significant improvement in survival among RNA-CART33-treated mice (P=0.01).

Finally, Dr Kenderian and his colleagues tested the effect of RNA-CART33 on hematopoiesis. The team treated NSGS mice with busulfan and transplanted them with T-cell-depleted bone marrow. Following engraftment, mice received RNA-CART33 cells, LV-CART33 cells, or control T cells.

The researchers followed the mice for 2 weeks and found that both RNA-CART33 and LV-CART33 induced myeloablation. And at 14 days, LV-CART33-treated mice were still expressing CARs, but RNA-CART33-treated mice were not.

“Based on our preclinical data, if we treat refractory AML with RNA-modified CART33, that results in myeloablation, anti-AML activity, and biodegradable, non-persisting CARs,” Dr Kenderian summarized.

“If allogeneic transplantation follows at this stage, it will likely lead to engraftment. Therefore, we conclude from this study that RNA-CART33 could be incorporated in novel conditioning regimens and will be tested in pilot phase 1 studies.”