Clinical trials use Sleeping Beauty gene transfer to create CAR T cells

First-in-human application of T cells modified  by “awakened” DNA sequence system reveals no toxicities from treatment after stem cell transplantation for leukemia, lymphoma

MD Anderson News Release 12/08/13

The first clinical trials using the Sleeping Beauty gene transfer system to customize immune system T cells to attack specific types of leukemia and lymphoma indicate the approach to be safe and feasible, investigators reported at the 55th American Society of Hematology (ASH) Annual Meeting and Exposition.

“Five months into these clinical trials, no acute or late toxicities have been noted from treatment with genetically modified chimeric antigen receptor (CAR) T cells in nine patients after they received blood stem cell transplantation,” said Partow Kebriaei, M.D., associate professor of Stem Cell Transplantation and Cellular Therapy at The University of Texas MD Anderson Cancer Center.

T cells collected ahead of treatment from the patient are modified to find the CD19 receptor on the surface of immune system B cells, which make antibodies against infection.  They are infused after blood stem cell transplantation for acute lymphocytic leukemia or B-cell lymphomas.

“Delivering CAR T cells after transplant targets minimal residual disease in hopes of maintaining remission for people with high-risk B cell malignancies,” Kebriaei said.

The team has prepared CAR T cells for 25 patients, nine of whom have been treated after receiving either their own blood stem cells, those from a matched donor or derived from umbilical cords.

Of the first five patients, three treated with the initial, minimal T cell dose had their disease progress while the first treated at the next highest dose remains in remission.  It was too early to evaluate the fifth, who received an umbilical cord blood transplant.

Four non-Hodgkin lymphoma patients treated with the higher T cell dose all remained in remission after three months.

Sleeping Beauty’s advantages

Compared with recombinant viral DNA delivery methods used to create CAR T cells, Sleeping Beauty is faster, simpler and therefore a more nimble process for customizing T cells,  as well as far less expensive, said Laurence Cooper, M.D., Ph.D., professor of Pediatrics at MD Anderson.

Cooper read about Sleeping Beauty in a study published by Perry Hackett, Ph.D., professor of Genetics, Cell Biology and Development  at the University of Minnesota and creator of the process. The system is named Sleeping Beauty because Hackett was able to “awaken” an extinct transposon – DNA that can replicate itself and insert the copy back into the genome – and package it with a gene he wants to transfer into a DNA molecule called a plasmid.

An associated transposase enzyme binds to the plasmid, cuts the transposon and gene out of the plasmid and pastes it into the target DNA sequence.  

Hackett first developed the system for gene transfer in fish and then expanded its use to other vertebrates.

Cooper contacted Hackett to collaborate using the Sleeping beauty transposon and transposase to plug a gene into T cells that creates an artificial, or chimeric, antigen receptor on the T cell that recognizes and binds to CD19, a cell surface molecule on B cells.  

The MD Anderson team does this by combing the Sleeping Beauty/CD19 plasmid with the patient’s T cells and applying an electrical field that loosens the T cell membranes to facilitate the gene transfer.

The modified T cells are then cultured with artificial antigen-presenting cells that stimulate expansion of the T cells with the help of response boosters interleukin-2 and interleukin-21.  The CAR T cells are retrieved over 28 days and frozen for later infusion.

These cells are now made in a Good Manufacturing Process lab at MD Anderson.  Cooper’s ultimate ambition is to refine and develop the process to make CAR T cells available off the shelf, or premade like blood and platelets are now in blood banks.

Idea springs from paucity of pediatric cancer treatments

Cooper treats children with blood-based cancers. Anti-cancer drugs are rarely developed for childhood cancers because they are so rare.

“In pediatrics, we’re in a situation where many companies don’t really pay attention to pediatric needs, simply because there’s no return on investment for them,” Cooper said. “It’s up to those of us who are  investigators to develop and implement therapies for children with cancer.  So you have to ask yourself, ‘what can we manufacture?’ Well, we can make new cells to use as drugs.”

The researchers have opened a new clinical trial to treat people with B cell malignancies with CAR T cells right after they finish chemotherapy. “The goal here would be to actually avoid stem cell transplant,” Cooper said.  Plans for solid tumor trials are in the works.

Continued development of this approach is proceeding under MD Anderson’s Moon Shots Program to accelerate the pace of converting scientific discoveries into clinical advances that reduce cancer deaths.

Co-authors of the study with Kebriaei, Cooper and Hackett are Helen Huls, Harjeet Singh, Ph.D.,  Simon Olivares, Matthew Figliola, Pappanaicken Kumar, Bipulendu Jena, Ph.D., Sonny Ang, Ph.D., and Rineka Jackson of Pediatrics; Doyle Bosque, Ian McNiece, Ph.D., Gabriela Rondon, M.D., Chitra Hosing, M.D., Elizabeth Shpall, M.D., and Richard Champlin, M.D., all of Stem Cell Transplantation and Cellular Therapy.