Flow cytometry enables CAR-T cell therapy

Our interview for this month features Dr. Kimberly Gilmour, Consultant Clinical Scientist and Clinical Lead for the Immunology Laboratory and Director of Cellular Therapy at the Great Ormond Street Hospital for Children (GOSH), London UK. Dr. Gilmour and her team have been focused on diagnosing paediatric immunodeficiencies using protein-based screening assays followed by genetic confirmation.  She is also involved in cell and gene therapy clinical trials and part of the GOSH team that will be the first UK hospital to deliver recently market approved chimeric antigen receptor (CAR) T-cell therapy to patients. Dr. Gilmour kindly agreed to share with us the details of her work, the future of cell therapy, the need for standardisation of flow cytometry and other topics. Read on to learn more about Dr. Gilmour’s exciting work.

CAR T cells and other gene products are used to treat which conditions?

At Great Ormond Street Hospital for Children (GOSH) we run the national diagnostic service for patients with primary immunodeficiencies. These are children born with genetic defects in their immune system. So classically, these were known as the ‘boys in the bubble.’  We work on diagnosing children with severe combined immunodeficiencies. In addition, these conditions are treated by gene therapy or bone marrow transplant. I am also the director of the cell therapy lab which oversees and manipulates cells for bone marrow transplant and cellular therapies. Great Ormond Street Hospital has a very large cell and gene therapy program, ranging from stem cells through to CAR T cells.

Can you talk about cell therapy at your hospital?

GOSH has been doing bone marrow transplants for forty years. That is the only treatment for leukemia, if the patients fail chemotherapy, and for genetic immune and metabolic conditions. So, the whole cell therapy programme grew out of that. Our first gene therapy patient was treated almost eighteen years ago now. That was for a condition called X-linked severe combined immunodeficiency or X-SCID. He was one of ten patients we treated in that trial. A couple of years later we started trials on another SCID called adenosine deaminase SCID or ADA-SCID. Altogether we have treated about 50 patients for genetic causes of immunodeficiency using gene therapy. Then about four years ago, we started working with CAR T cells. In this approach, the patient’s T cells are modified with an artificial receptor that makes them target and kill cancer cells.  Initially we started with anti-CD19 targets against leukemia and lymphoma. Under John Anderson we expanded it to include 1RG, which is a marker against solid tumors or neuroblastomas. CAR T cells are autologous, in which the patient’s own cells are manipulated and given back to the patient. We also use what we call universal CAR T (UCAR T) cells that are allogeneic, in which you take T cells from a donor and replace the T cell receptors with the CAR receptor, giving you about 100 doses of cells that you can administer to a patient.

How are you using CAR-T cell therapies to treat cancer?

Right now, we have a number of trials using CAR T cells that are in collaboration with UCLH. Some are national trials, and we are also treating patients in Manchester. I think you are speaking about Kymriah™, the new Novartis drug. Kymriah™ is a commercially licensed CAR T cell therapy for B cell leukemias and lymphomas.  The U.K. government has decided that six centres in England and Wales, three paediatric and three adult, will be participating. They will be preparing the samples, shipping them off to Novartis for manufacture, receiving them, administering them to patients and monitoring them. The three paediatric centers are GOSH, Manchester Children’s Hospital and Newcastle. How you control and monitor the patients after therapy is relatively complicated. Patients getting these cells often get something called cytokine release syndrome which can send them to intensive care, where they will need intubation and very specialist treatment. Like all things, the more you do, the more experience you get and the better you get at doing things. At the moment, because these are fairly rare treatments, they have chosen hospitals which have a lot of experience in clinical trials for CAR therapy and can handle commercial products. Since 2012, GOSH has treated over 25 patients from all over the UK with CAR T therapies as part of several different clinical trials. In 2015, the hospital successfully treated the first patient in the world with gene-edited ‘universal’ CAR T cells. Researchers at GOSH are currently exploring the use of ‘next generation’ CAR T cells to further reduce the chance of relapse.

Is cell therapy being expanded at your hospital?

Yes, so we are very fortunate that the Zayed Centre for Rare Diseases is being built as a part of GOSH. It is right across from our current lab. Its top floor will have a dedicated GMP facility with seven clean rooms. These will allow us to make many more products to treat many more patients. We expect that at the end of 2019, early 2020, we will be moving both the research and the cell products teams into that building.

What role does flow cytometry play in CAR-T cell therapy?

Flow cytometry is essential because all these products must be quality-approved. You need to be sure that what you are giving to the patient is the right thing. You need to know how many T cells you have, what percentage of those are transduced or express the CAR or whatever gene product you’ve put in and whether the cells are viable. We do all that using FACS. Presently we are using the BD FACSCanto™ II Flow Cytometry System but we are excited as we are getting the BD FACSLyric™ Flow Cytometry System, which we are looking forward to having. This will give us more colours, hopefully more stability, more consistency and less operator variation.  This is because the assays we perform are not routine flow cytometry assays like measuring B, T, NK or CD34 stem cells. All of these are very important and all of these we have to do, but for cell therapy and other procedures, we need to generate modified cells. For example, we do something called an alpha-beta T cell depletion where we remove all alpha-beta T cells from a product. We need to know exactly how many we have removed and we’re enumerating very small cell numbers. So, if a machine acquires five million events and we see four events of interest, we must be convinced that those four are really representative of what is there in the product.  We need to do it accurately, efficiently and quickly because the product is going to a patient and we cannot wait an hour for the assay to run through.

Could flow cytometry standardisation help you improve workflows?

Yes, I think specifically for CAR T cells, there is a real need to standardise the whole procedure. What I mean by that is right now there are a lot of CAR T cells being used for a lot of trials. The first commercial product is now available. In every trial that is set up in the research lab, they use certain antibodies and acquiring a certain number of events, which they analyse this way.  Currently we are running 30 gene and cell therapy trials in our lab and often, each procedure is slightly different. Sometimes they have blocking buffer, sometimes they don’t and so on. So, there is a real opportunity to standardise the way we measure CAR T cells. An example is like how we measure CD34 T cells. We use the BD® Stem Cell Enumeration Kit which has the antibodies, the tubes, the ammonium chloride lysis buffer, and we do it the same way every time. There should be something like this for CAR T cells so that everyone measures everything the same way and the data are comparable.

What future developments would you like to see that could help you work better?

I think standardisation is important. We are still using lots of research antibodies for all kinds of reasons. BD in general is good at research antibodies and usually I can use them at the same concentration. The fluorochromes are usually compatible with other things but I think more compatibility not with cell therapy but with immunodeficiency is desirable.

How we can move forward is a balance. On one hand you want standardised templates for analysis but on the other, you still need people to interpret and who know about whether a gate needs changing, or which quadrant needs to be tweaked.

The other thing is since we are a specialised hospital with paediatric patients, we get a lot of immunoglobulin and monoclonal antibodies. We are having to wash a lot of our samples before we can stain them. Presently, we do it the classic way by putting blood in a tube, adding cell wash, mixing, spinning it down, taking off the cell wash, resuspending and repeating three times before staining the cells. It would be great there was a way to avoid doing that or to do it in an automated way.

Learn more about the BD FACSLyric™ Flow Cytometry System.