Agilent has steadily been developing innovative cell-based solutions designed to enable researchers and developers to overcome the challenges and capture the opportunities faced in the rapidly growing field of immunotherapy. Recently David Ferrick, an executive in the Cell Analysis Division at Agilent, caught up with Carl June, a professor at the University of Pennsylvania and Abramson Cancer Center, to discuss their perspectives on these unique challenges and opportunities. Dr. June has not served as a consultant for Agilent and has not received payments from Agilent.
David Ferrick: Carl, as you know, our relationship with you over the past few years has really helped us to advance several innovative solutions comprised of purpose-built tools in immunotherapy. Your guidance on the needs of this rapidly evolving field are invaluable. I’d like to focus on three areas: the engineering of human cells, how cell analysis tools are enabling cell therapy to reach its potential, and next steps in cell manufacturing QA/QC for cell-based therapies. As a pioneer in the engineering of human cells, how do you see the field evolving, and what do you think are the greatest challenges today?
Carl June: We’re at an early stage where the proof of concept has been shown, and now accepted, that you can make synthetically engineered T cells that have enhanced performance characteristics in a variety of ways compared to the natural immune system. For a long time that was a question – could that be done? Can you improve upon a Darwinian, if you will, naturally evolved T cell, and if so can you do that safely?
I think we’ve had more than 1,000 patients treated with genetically engineered T cells, primarily with cancer, and there have not been any instances where the cells have transformed or had evidence of genotoxicity. This is a major turning point, where we have this orthogonal approach of combining the knowledge of the entire human genome and epigenome. We’re using that to look at vulnerabilities in the tumor microenvironment and to make T cells that are designed and “purpose-built” to overcome the barriers that are in toxic tumor microenvironments.
David Ferrick: How do you see the potential for engineering other immune cells, such as natural killer cells (NKs), gamma deltas, or macrophages?
Carl June: That’s one of the reasons it’s so exciting to be in this field now. We realize that the immune system is not just one instrument, to analogize, it’s an orchestra. They have non-overlapping roles in the entire immune system. NK cells, for instance, kill and recognize targets in a way that is different from T cells. Similarly, there are subsets of T cells, gamma delta cells that are more like an innate immune cell, but they can also kill tumors. They have a different metabolism and can survive in different environments in a better way than alpha beta T cells. Because gamma delta cells don’t have alpha beta T cell receptors, they won’t cause graft versus host disease.
And recently macrophages have come to the forefront, as they kill and eliminate cells by phagocytosis rather than using a cytolytic mechanism like NK cells and T cells. In addition, we’ll see engineered stem cells and their progeny, that after engraftment into patients can produce engineered cells of all the types we’ve just discussed.
David Ferrick: You’ve hit on an important point, that the immune system is like an orchestra, with homeostatic principles, and there are many cell types that cooperate in both time and space to achieve that. One of the things we’ve focused on is the advancement of cell analysis tools that can generate information that is of a quality [time-resolved] that can help to understand this behavior along the timelines that occur in vivo. Could you comment on the value of real-time kinetic assays—let’s call them the ‘newer types’ of cell-based solutions?
Carl June: That’s a critical issue. The emerging data in basic immunology is showing that cells have major metabolic reprogramming steps. Acute effector T cells have, in general, dominant glycolysis for metabolism, whereas memory cells proliferate slower but live longer, and mostly use metabolism that’s based on fatty acid oxidation, Krebs Cycle, and mitochondrial biogenesis. From what we’ve seen in mouse models of chronic infection and tumors, you want to have populations of both cells. Some cells that would be potent effector cells but are going to be short-lived and another set of cells that would be able to establish long-term cellular memory and function, for long-term immunosurveillance.
Metabolic assays such as Seahorse, are well poised to identify those cells, with those properties. I see in the future that there may be cell-based release assays for potency, and also predictive assays, as biomarkers of response in cell products.
David Ferrick: It’s amazing what’s happened so quickly. To think there would be, as you point out, this qualitative dichotomy between mitochondrial respiration and glycolysis that fits immune functionality so well—but hey, here we are. One question we hear regarding this ‘newer type’ of cell-based assay is about functional potency testing in terms of what the new product can do, and how long it will be able to perform. What do you see?
Carl June: One potential use of these new kinds of assays for cell analysis [Agilent ACEA xCELLigence, Agilent Seahorse XF] may be the ability to know which individuals could possibly make a curative product with current technologies and in others it would be futile. If you determine that an individual is not a good candidate, then that means you would go down the line of using third-party cells for instance, so that’s going to be a major change in the future. In addition, even in candidates where you determine you can manufacture a successful product, another application of this new “tool kit” may be finding, if you will, the heavy lifters. Some assays have shown, that when you do adoptive transfer, the T cells that carry out most of the tumor elimination are the progeny of just a few cells. If we can identify those T cells, up front, then potentially we can manufacture fewer cells, meaning the cost of manufacturing would go down, leading to a number of benefits in treatment.
David Ferrick: To follow up on that, how do you see some of the newer technologies like CRISPR being used to increase the fidelity and minimize the footprint of cell engineering so that we can get that protective immunity with a more natural approach which may be the key to durability and minimal side effects?
Carl June: There are a few genome-wide discovery approaches that identify ‘targets of opportunity’ in T cells that, in preclinical models, enhance their performance. It’s a great time because of these technological advances in genome editing using CRISPR, meganucleases, and so on, that make this possible.
The issues are somewhat different between solid tumors and hematologic malignancies. In hematologic malignancies, T cells, after infusion, generally traffic right to the bone marrow, that’s a natural aspect to them. But in solid tumors, it may be rate limiting in many instances for T cells to enter the solid tumor. So, strategies that edit T cells to enhance their homing, penetration, and persistence of the solid tumor microenvironment are of great interest.
Another opportunity is the tumor and T cell “tug-of-war” between metabolites, and where CRISPR engineering can help increase access to nutrients in the tumor microenvironment. CRISPR approaches can help develop cells that are resistant to that tug-of-war situation, so they can survive longer, and therefore proliferate better in a solid tumor microenvironment.
David Ferrick: That leads me to cell manufacturing and the QA/QC component of cells as a therapeutic modality. Is there a role for cell-based assays of the kind that we’re talking about, the ‘newer type’ that are being developed?
Carl June: Very much so. With autologous cell therapies, at least one issue has been that it’s always going to be more expensive than having third-party cells that can be made in large batches. If you’re going to make something expensive, you want to make sure it works. So, any [cell-based] assay that improves the probability that you’re going to have an effective cell product will be something that everyone wants— patients, physicians, and third-party payors, etc.
Understanding the basis by which cells from candidates can manufacture an effective cell product is an important first step. To find these answers flow cytometry approaches, kinetic measures of live-cell metabolism, and quantifying the ability of T cells to kill targets over time are all aspects that can be investigated now with tools that Agilent provides.
David Ferrick: To wrap up, what would you tell Agilent and others, who are working on building tools and trying to enable people in this field, about how they can contribute?
Carl June: Functional assays. For a long while, we only had flow cytometry. I believe that we can learn a lot from a weakness in the pharmaceutical industry, where many trials were done without much emphasis on studying the reason why some trials failed.
But when using engineered cells, you can retrieve them back from the patient and study them and investigate more thoroughly the immunophenotyping and metabolic health of the cells and find out was this T cell exhaustion, had the cells senesced, or perhaps it never engrafted? I’m optimistic that we may find solutions through analysis of where it has failed. With that knowledge, I believe we’re going to be able to make better “next generation” T cells that will overcome those vulnerabilities.
David Ferrick: Carl, it’s a great note to end on. Thank you so much for helping us with this, it’s been great.
Carl June: Well thanks David, it’s been great to work with Agilent as well.
This interview has been edited for length and clarity.