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The Long Evolution Of CAR-T Assays

July 22, 2019 | The evolution of CAR-T cell study has been a long process, says Preet Chaudhary. Current assays lack the sensitivity and high throughput for robust research. As Chief of Hematology and the Director of Blood and Marrow Transplant at the Keck School of Medicine at USC, Chaudhary works to overcome these limitations, and for the past seven years, has been developing assays that cut through current limitations. Dr. Chaudhary is also the Founder of Angeles Therapeutics, Inc., a next generation cell therapy company.

On behalf of Diagnostics World News, Mary Ann Brown spoke with Chaudhary about his work developing assays, his background that led him to the Keck School of Medicine, and what technology needs to be developed for the second generation of adoptive cell therapies.

Editor's note: Brown, Executive Directors of Conferences at Cambridge Healthtech Institute, is planning a track dedicated to Adoptive Cell Therapy at the upcoming Immuno-Oncology Summit in Boston, August 5-9. Chaudhary will be speaking on the program. Their conversation has been edited for length and clarity.

Diagnostics World News: Could you share a bit about your background, and how it led you to the Keck School of Medicine?

Preet Chaudhary: I went to medical school in India, and after I finished medical school, I was looking for the next opportunity, and I realized that I wanted to get into the area of gene therapy.

The way I came across that was in the standard textbook of medicine, which is Harrison's. I read a chapter on prospects for human gene therapy, and I was fascinated by that. I decided to pursue PhD studies, and came to the University of Illinois at Chicago to do my PhD work focused on the area of gene therapy.

My PhD topic was to put the human multidrug resistance 1 (MDR1) gene – which codes for P-glycoprotein, which in turn happens to be a multidrug efflux pump – into the bone marrow stem cells. The idea was that if the stem cells express this drug efflux pump, they will become resistant to chemotherapy, so you could give multiple rounds of chemotherapy to the patient with cancer without having the consequences of a drop in blood count.

As often happens in science, I discovered that actually the bone marrow stem cells naturally express the P-glycoprotein protein at very high levels, which was a surprising discovery at that time because people had noticed that stem cells don't stain with a dye called Rhodamine 123, and also another dye called Hoechst, but they had attributed the lack of staining with these dyes to the lack of mitochondria or inactive mitochondrion in these stem cells because Rhodamine 123 is a mitochondrial dye or to very condensed chromatin because Hoechst dye binds with DNA. What our work showed was that the dye dull phenotype of the cells has nothing to do with mitochondrial potential or the chromatin-condensed nature of these cells, but due to P-glycoprotein-mediated efflux of the dyes.

We used this discovery to actually develop a method for isolation of hematopoietic stem cells because P-glycoprotein is a cell surface protein for which very high-quality antibodies are available. We subsequently discovered that the same protein is also expressed in human prolymphocytes and all different subset of lymphocytes. Other investigators followed up on our work of the presence of drug efflux pumps in stem cells, which led to the commonly used method to identify stem cells in other organs and tissues based on staining with Hoechst dyes called the “side-population”.

After I finished my PhD, I did my medical residency training at Northwestern and then fellowship at Fred Hutchinson Cancer Center/ University of Washington where I did research work in the lab of Dr. Leroy Hood, using large-scale automated sequencing and bioinformatics tools looking for novel genes expressed in stem cells and happened to clone several members of the TNF-receptor family.

I got my first faculty position for the UT Southwestern in Dallas and then subsequently worked at University of Pittsburgh Cancer Institute in this area of cell signaling. In approximately 2008, the Division of Hematology at USC received a very large gift of approximately 60 million dollars. After a national search, I was selected to be the chief of the division to develop the research enterprise in the division using the gift.

The first thing we did was to start the allogeneic stem cell program at USC in 2011. Our program has been consistently one of the top performing adult allogeneic program in the country in terms of one-year survival as published by CIBMTR for the past several years. Last year we were the top center in one-year survival in the state of California and one of the top in the country. After the allogeneic stem cell transplant program was established, we decided, "Okay, what is the next challenge in the field of hematology and cell therapies". Impressive results with CAR-T therapies were getting published, and so we decided to get into this area.

Over the last six to seven years, we have been working on the CAR-T cell field. The first insight we gained was that despite their success, the CAR-T cell constructs in current clinical use are actually based on a nearly 25 years old design with the main modification being the incorporation of a co-stimulatory domain, which is frequently derived from TNF family receptors. Based on my previous work in TNF signaling field, I came to the conclusion that a number of limitations of the current generation CAR constructs, including toxicity and lack of efficacy in solid tumors, can be traced to their defective design, including the non-physiological signaling via the co-stimulatory domains. Therefore, we decided to develop a next generation CAR platform that provides more physiological signaling. However, in developing the next generation CAR-T platform, we ran into other obstacles. In particular, we realized that some of the assays being used to study the functionality of CARs and to study their expressions are not very robust and high throughput. The assays were time consuming, expensive, and not very sensitive or specific. As we needed to test a large number of constructs to develop our next generation CAR-T platform, we did a lot of work to develop non-radioactive luciferase-based assays to overcome the above limitations.

Can you describe the first assays you developed?

The first assay we developed was called the Matador Assay. By the way, most of our assays are named after our local beautiful beaches in southern California, so the Matador Assay is named after the El Matador State Beach. it's a non-radioactive cytotoxicity assay based on marine luciferases and is extremely sensitive, with sensitivity down to a single cell level, and very specific. The assay can be performed in a 96 or a 384 well plate without the need of any expensive equipment like a flow cytometry. It's our workhorse assay that we have used to test thousands of CAR-T constructs. The assay is very simple in design and involves expression of marine luciferase inside the target cell in a stable fashion. When the target cells are killed by the immune effector cells, the substrate can rapidly interact with the luciferase that has leaked outside the cells and that which is still trapped inside the cells to produce a light signal that can be easily measured using a simple luminometer. My laboratory has already developed close to 80 cells lines that can be used in the Matador Assay.

Your recent paper, "A Novel Luciferase-Based Assay for the Detection of Chimeric Antigen Receptors," addresses one of the challenges of CAR-T therapies by developing an assay for the detection of CARs on the surface of immune effector cells. Would you please explain further?

Another challenge in the CAR-T cell field is there's no easy assay to measure the expression of CARs on the surface of, engineered T-cells. People have used a variety of assays for this purpose, but no single assay satisfies all the requirements that scientists need. For example, one can use flow cytometry-based assays using immunoadhesions, but they require multiple labeling steps and there's a high background. The other method that has been used is based on Protein-L staining but, again, Protein-L has a lot of background, shows variable binding depending on the type of scFv fragment or the vL region which is incorporated in the CAR, and requires multi-step staining steps.

The second assay that we developed to detect expression of CARs is called Topanga Assay and is named after the Topanga Beach in Malibu. The Topanga Assay is a very straight-forward assay in which we fuse the extra-cellular domain of a receptor that is targeted by a CAR to a marine luciferase. For example, if the CAR is targeting CD19, we will fuse the extra-cellular domain of CD19 to a marine luciferase. We express this fusion construct in mammalian cells by transient transfection. Using the crude supernatant containing the secreted fusion protein, we can easily detect the expression of CARs on T cells. The assay is very simple, can be done in a 96 well plate and finished in 30 to 45 minutes.

We can detect close to one cell in the background of 1 million mononuclear cells. The signal-to-noise ratio is excellent so there's very clear demarcation between the cells which are expressing the CAR versus those which do not. The assay can also be used to measure what number of CAR-T cells or the expression of CAR on the surface of T-cells. Topanga assay can also be used as a measure of the functional binding affinity or the avidity of the CARs to the target antigens. It has multiple potential applications, not only in the detection of CARs but also to monitor their expansion and persistence in patients following infusion. For example, it can be potentially used to monitor the expansion of CAR-T in patients after administration to make sure the CARs are not expanding too quickly which can lead to toxicity.

Are you developing any other assays?

Yes, so we have also just submitted another paper on a third assay which is called the Malibu Assay, again after the Malibu Beach. This assay again uses marine luciferases and is a non-radioactive assay. Malibu Assay is the opposite side of the Topanga Assay. Instead of fusing the target antigen to a marine luciferase, we fused the antibody or the antigen-binding domain to the marine luciferase. We use the assay for scFv engineering to find the best scFv for subsequent incorporation of the CAR-T cells. Similar to Topanga assay, the Malibu assay can be done relatively quickly and it's extremely economical and sensitive.

In your view, what technology needs to be developed for the second generation of adoptive cell therapies?

As I stated previously, the design of the current generation of CARs is almost 25-30 years old. This design was first described in 1989 by investigators from Israel. While some minor modifications, such as inclusion of a co-stimulatory domain, have been done to the design over the last two or three decades, the design is pretty much the same. The design does work and obviously the fact that CAR-T cells are in clinical use attests to that. However, I believe that the design has several limitations which manifest in the various toxicities of current generation of CAR-T cells, such as cytokine release syndrome (CRS) and neurotoxicity. Obviously, I do not agree with the prevailing thesis that CRS is a marker of efficacy and therefore cannot be controlled without losing efficacy. The lack of long-term persistence of current generation CARs is another major problem, which results in disease relapse in blood cancers and lack of efficacy in solid tumors. While it is generally believed that hostile microenvironment is a major factor in the lack of efficacy of CAR-T cells in solid tumors, my personal view is that this also has more to do with the poor design of the current generation CARs with the hostile microenvironment playing a contributory role.

We believe that one of the big challenges in the field is to come up with next generation CAR design, which can provide more physiological signaling and can overcome some of these limitations of toxicity and lack of persistence and efficacy. My lab at USC has been developing such a platform using the assays that I described.

Then, beyond a more physiological CAR platform, there are other problems such as identification of suitable target antigens, especially for myeloid malignancies and solid tumors. We need to come up with better tools to identify the antigens that can be safely targeted in myeloid malignancies and solid tumors. You obviously need a better way to measure, for example in the case of TCR T-cell therapy, better way to find peptide antigens or neoantigens, which are highly or selectively expressed in tumor versus normal tissues, and then to target them.

A number of groups are developing allogeneic off-the-shelf CAR-T cells. However, the problem of limited persistence of allogeneic CAR-T cells needs to be addressed. In my view, there are two major contributors to the lack of persistence of allogeneic CAR-T cells; immune rejection and exhaustion. While a number of groups are working on different strategies to improve the persistence of allogeneic CAR-T cells by overcoming immune rejection, I believe that the poor design of the current generation CARs, which results in their exhaustion and lack of functional persistence, remains a major impediment. Therefore, a next generation CAR platform that overcomes the problem of lack of persistence is also needed to fully capitalize on the potential of the allogeneic CAR-T cell approach. Finally, a practical and commercially viable method for isolation of stem like T cells for genetic modification by CARs (and TCRs) will help improve the persistence of these cells.

The cell therapy field is just in the second inning and there is going to be need for innovations in multiple different areas, including on the manufacturing side. The above areas, however, are some of the critical areas that we need to pay attention to in the immediate future.