From left: Helene Negre, Heather Garrity, Karl Stasko, and Heather Daley lead the production of CAR T cells.
Turning Immune Cells into CAR T Cells
From Paths of Progress 2018
By Robert Levy
The news about CAR T-cell therapies for cancer keeps getting better. After many years of studies and clinical trials, these therapies, made from genetically modified immune cells, have been
generating early remissions in more than 80 percent of patients with acute lymphoblastic leukemia (ALL) and nearly half of patients with B-cell non-Hodgkin lymphoma (NHL).
Last year, the U.S. Food and Drug Administration (FDA) approved one CAR T-cell therapy as standard treatment for children and young adults with relapsed ALL and a second for adults with advanced B-cell lymphomas. Approvals for other blood cancers are expected in the next two years, and trials of genetically
modified T cells for patients with some solid tumors are beginning to open.
Amid such progress, it can be easy to overlook the enormous technical complexity of making CAR T-cell therapies. They are, as is often noted, "living drugs," manufactured by collecting millions of disease-fighting T cells from a patient's bloodstream,
equipping them with new genes that improve their ability to track down and destroy cancer cells, and infusing the upgraded cells back into the same patient. As such, they represent a combination of gene therapy, immunotherapy, and personalized therapy
unlike virtually anything else in medicine.
Only a small number of labs and biotechnology companies have the expertise and capacity to produce high-quality CAR T cells in quantities necessary for a clinical trial or standard patient treatment. Many manufacturers of CAR T-cell therapies are commercial
biotechnology or pharmaceutical firms, but Dana-Farber's Connell and O'Reilly Families Cell Manipulation Core Facility (CMCF), a hub of production
for cell-based therapies, has made CAR T-cell therapies for several clinical trials. One such trial was for patients with acute myeloid leukemia (AML) or multiple myeloma. It is one of the highest-volume academic centers for cell therapy production
in the world. The facility and its 50-member staff will be moving to larger, specially designed quarters on Dana-Farber's Longwood campus later this year.
"We are fortunate to have access to world-class nurses and physicians, a robust program in clinical trials, and a state-of-the-art manufacturing facility which cooperate to bring the latest cell therapy advances safely and efficiently to patients," said
Sarah Nikiforow, MD, PhD, assistant medical director of the CMCF and technical director of Dana-Farber's Immune Effector Cell Program.
At the CMCF, every step in the process of making CAR T cells is meticulously supervised and documented. There are daily worksheets with checklists for each phase of production. Each technician's work is overseen by a verifier. Quality assurance specialists
are on hand at key stages of the process with the authority to bring production to a halt if problems are detected. For each CAR T-cell therapy produced, there are 100 pages of documentation, with signoffs required every step of the way.
Here is a look at how CAR T cells were produced at the CMCF for the trial for patients with AML and multiple myeloma.
Making CAR T Cells
1. Collecting T Cells
The first stop for CAR T-cell production is the Kraft Family Blood Donor Center at Dana-Farber and Brigham and Women's Hospital, where a patient's immune system T cells are collected
through a process known as leukapheresis. Blood from the patient's arm is channeled to an apheresis machine, which separates out T cells and other cells and returns the remaining blood components to the other arm. When the process is complete, the
collection bag holds 10-20 billion cells, about half of which are T cells.
In the Kraft Family Blood Donor Center, a patient’s blood is channeled through an apheresis machine to separate out white blood cells.
2. Purifying and Growing
A courier hand-delivers the sample to the CMCF, where it is placed in a biosafety cabinet — an enclosed, ventilated workspace inside a closely monitored clean room. Flow cytometry equipment counts and categorizes the cells in the sample. Technicians test
the product for sterility, and cull out excess red blood cells and platelets if necessary. The T cells are placed in a flask, bag, or device containing nutrients to keep the cells alive and antibodies to stimulate their growth.
A technologist purifies T cells using a cliniMACS® device.
3. Going Viral
After a few days in this nutrient-rich environment, the T cells are ready to receive their new genetic instructions. A CMCF technician retrieves a vector — a solution of commercially produced viruses — from a lab freezer and allows it to thaw. The viruses,
engineered to be harmless, contain three key genes that carry cellular instructions. The technician uses a syringe or pipette to add the vector to the cell culture.
Cell culture media and vector viruses are added to the T cells, which are then grown in an incubator.
4. Becoming CAR Ts
The viruses make their way inside the T cells and download their genetic material, including the three specially installed genes, into the cells' DNA. The trio of genes order the cells to produce a three-part protein structure — known as a chimeric antigen
receptor, or CAR — on the cells' surface. The CARs will help the T cells recognize the patient's cancer cells and latch onto and destroy them.
5. Multiplying by Dividing
The cells remain in their nutrient bath for seven to 10 days, until they become fully fledged CAR T cells and reach a sufficient quantity. During this time, technicians periodically remove used-up growth media — the mixture cells feed on — and replenish
it with new media.
The culture media is changed every two days.
6. Quality First and Last
A technician "harvests" the CAR T cells by drawing them into a syringe and placing them in a bag or tubes. A centrifuge spins them at high speed to remove the culture media. The cells then undergo a variety of quality control checks — ensuring there are
enough cells, that they're the right type and function properly, that the product is sterile, that the CAR cells have acquired the proper number of genes, and, lastly, that the vector viruses have been fully removed. While it takes just 10-14 days
to manufacture CAR T cells, quality-control testing may consume one to two additional weeks. If any of these quality checks fails, the cells will not be used.
Containers of CAR T cells are placed in a centrifuge, which spins them at high speed to wash the cells before infusion.
7. Homeward Bound
With a final signoff from the medical director of the CMCF, CAR T cells are ready to be re-infused. They can be infused fresh, but they're usually frozen so they'll be available when the patient is ready. Close coordination between the facility's staff
and infusion nurses ensures they're thawed/released at precisely the right time. At this point, the cells make their final trip to the bedside, where they're infused into the patient from whom their forebearers were taken weeks earlier. Back in their
"home" body, they immediately join the battle against the patient's cancer cells.
Says Nikiforow: "Despite the dramatic clinical benefits that current, ‘second generation' CAR T cells have delivered to some patients, there are many opportunities to fine-tune our genetic and cell-manufacturing approaches to achieve better safety and
effectiveness and target additional cancer types in the years to come. We are looking forward to the challenge."
A bag of CAR T cells is prepared for infusion into a patient.