Photo courtesy of MDACC
NEW YORK—Researchers have used a nonviral approach to create chimeric antigen receptor (CAR) T cells and tested these cells in safety trials.
Patients with advanced lymphoma or leukemia were infused with the nonvirally modified CD19-directed CAR T cells after autologous or allogeneic hematopoietic stem cell transplant (HSCT).
Eighty-six percent of autologous HSCT recipients were alive 24 months after infusion, and 53% of allogeneic HSCT recipients were alive with a median follow-up of 6.5 months.
“Gratifyingly, the patients have not demonstrated any acute or late toxicity to these CAR T-cell infusions,” said Laurence Cooper, MD, PhD, formerly of MD Anderson Cancer Center (MDACC) in Houston, Texas, and now with Ziopharm Oncology.
Dr Cooper presented these results at the inaugural CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference.
Some of the technology he described was conducted at MDACC. Dr Cooper is currently a visiting scientist there and will continue to supervise the development of this technology.
Dr Cooper said the appeal of this nonviral approach, which is a modified Sleeping Beauty approach, “is it essentially avoids the complexity of making a virus, a lentivirus or a retrovirus, it can be done at quite low cost, and really allows for a nimbleness to this system.”
Using a simple blood draw of 200 cc of peripheral blood—the process does not require apheresis—the T cells can be expanded on a feeder cell layer and genetically reprogrammed.
Sleeping Beauty system
The researchers reprogrammed the T cells using a 2-plasmid Sleeping Beauty system, which is a transposon/transposase system.
The transposon DNA plasmid codes for the cargo load, which, in this case, is the CAR. At the same time, the transposase DNA plasmid is electroporated, “which is really the secret sauce of the transposition event,” Dr Cooper explained.
After electroporation, the transposon/transposase are co-cultured with K562-derived artificial antigen-presenting cells (aAPC) and expanded with the integrated transposon of K562-aAPC. In this case, CD19 is on the aAPC.
CD19 is co-expressed with other co-stimulatory molecules, CD86 and 4-1BB ligand.
In addition, the researchers added a molecule of interleukin 15 that’s sewn in frame to the Fc region of an immunoglobulin that then activates the T cell in the context of these co-stimulatory molecules.
The T cells that have stable integrants of the CAR grow out over time. And those that have transient expression of the CAR die by neglect.
“By day 14, most of the T cells have the CAR sewn into the genome and are stably expressed,” Dr Cooper said.
The CAR used for these safety trials at MDACC targets CD19 and uses mouse scFv held in frame with an immunoglobulin 4 Fc (IgG4Fc) stalk.
It’s tunneled through the T-cell membrane and has 2 costimulatory molecules, signal 1 delivered by phosphorylation of the immunoreceptor tyrosine-based activation motif in CD3ζ and signal 2 by the costimulatory domain CD28.
The researchers tested the CD19 CARs in 2 clinical settings—one with T cells that were patient-derived and infused after autologous HSCT, and the second with T cells that were derived from a third party and infused after allogeneic HSCT.
Infusion after autologous HSCT
The researchers first tried the CARs in 7 non-Hodgkin lymphoma patients who had an autologous HSCT. Their median age was 52 (range, 36-61).
Five patients received a starting CAR T-cell dose of 5x108 cells/m2, and 2 received 5x109 cells/m2.
Six patients (86%) remain alive and are in complete remission (CR) at a median follow-up of 24 months.
Infusion after allogeneic HSCT
The researchers expanded the investigation to a wider cohort of 19 patients who had undergone allogeneic HSCT.
Seventeen patients had advanced CD19-positive acute lymphoblastic leukemia, and 2 had non-Hodgkin lymphoma. Their median age was 35 (range, 21-56).
All patients were on graft-versus-host disease (GVHD) prophylaxis with tacrolimus at the time of CAR infusion. A subset of these allogeneic transplant patients had haploidentical donors rather than matched sibling donors.
Five patients received a CAR T-cell dose of 106, 6 patients received 107, 5 received 5x107, and 3 received 5x108 cells/m2 based on recipient body surface area.
Fifty-eight percent of patients (11/19) achieved a CR, and 10 remain alive a median of 6.5 months after CAR T-cell infusion.
Three patients developed GVHD, 1 with steroid-refractory acute liver disease, 1 with grade 2 acute skin disease, and 1 with chronic limited skin disease. The incidence of GVHD was lower than historical controls at MDACC, Dr Cooper said.
“[G]ratifyingly, in this clinical setting of minimal disease, patients did not have any acute or late toxicity from these infusions,” he added.
And the rate of cytomegalovirus reactivation after CAR T-cell infusion was 24%, compared with 41% for patients after transplant at MDACC without CAR T-cell infusion.
Eight patients received haploidentical HSCT followed by CAR T-cell infusion, and 75% (6/8) remain in CR.
Persistence of infused T cells
The researchers used 2 forms of PCR—qPCR and droplet PCR—to map the fate of the CARs.
“Roughly speaking, for these patients, and this is in line with the literature, in terms of those T cells that are activated through CD28 in contrast to 4-1BB, these T cells are, on average, living about 28 or so days post-infusion,” Dr Cooper noted.
He said this is similar to results observed with CARs being tested at the National Cancer Institute and Memorial Sloan-Kettering Cancer Center.
