Cell and Gene Therapy Answers: The critical role of flow cytometry in adoptive cell therapies

Your source for answers to the complex challenges of cell and gene therapy development.

Adoptive cell therapy (ACT) is a promising type of immunotherapy that involves engineering a patient's immune cells to recognize and attack their cancer. As this field continues to grow, researchers are focused on refining the process to make ACT more effective in supporting the patient immune response. One critical tool for developing better ACT treatments is flow cytometry. To learn more about how flow cytometry enables advances in ACTs, we spoke with Leanne Flye, associate director of scientific operations, general laboratory, at Labcorp.

Can you talk about current applications of flow cytometry in the development of adoptive cell therapies or advanced therapies, including CAR T-cells?
 

1. Target identification

Currently in flow cytometry, we're able to use either targeted cell isolation—or we can look at circulating tumor cells technology also based on those flow cytometric approaches and we can investigate those isolated tumor cells and figure out what's unique to them using flow or even post-flow sequencing, combining our methodologies. From there, that provides us some information on what those targets could be for the adoptive cell therapies. We can also explore the neoantigens, produced by those cells using other non-flow proteomic approaches that can enhance the on-target directing of those cell therapeutics. So, flow, not just as a standalone but in combination with other modalities of detection, becomes a powerful tool.

2. Patient immune response

Once you have that target, the next step is to create your pharmaceutical product, which is going to be the targeted cell therapy. We’ll either reinfuse the patients with those cell therapeutics or, prior to that, we'll monitor those patients and see if they have been properly lympho-depleted. Specifically, we’ll monitor how a patient’s immune system profile compares to their basic immune health before cell therapeutics were infused. Once determined, we can assess:

• Immune system exhaustion, and whether their NK cells are functioning or if there are issues with those
• Anything that could impact the patient's innate ability to form an effective immune response

These factors may have an impact on the success of the adoptive cell therapy overall.

3. Cell therapy efficacy

Once we've infused those, we can monitor changes in both the native cells that were already there, as well as track the persistence of those adoptive cells that were infused. We can:

• Assess both the native cells and the adoptive cells to see if they're proliferating
• Do deep phenotyping, looking at the memory, naive and effector subsets
• Watch the NK cells reinvigorate as a first line of response to tumors and measure it by flow
• Monitor the myeloid-derived suppressor cells and watch those clear as patients respond to treatment

4. Persistence

More importantly, we're going to be able to determine what that adoptive cell therapy is doing within the patient. Are there subpopulations that are persisting better than others? Are those indicative of responders versus non-responders? And can we, with that information, provide finer tuning of those cell products for future product generation—to know what lineage of those cells are going to persist better and result in a response versus a non-response?

It's important that we understand all the aspects of those cells, and flow allows us to do that. This is unlike a PCR-type approach where you know a transgene is present, but don’t necessarily know what cell population it’s in.

Now, researchers are investigating how you can incorporate tumor neoantigens into the CAR T-cell therapies, specifically for solid tumors. So you can design a CAR that recognizes a patient's specific neoantigen and create a very precise treatment for that patient's very precise disease. It gets a little more complex, but it's better than having a treatment with a bunch of off-target effects. And that's one of the really cool things that flow can do. We can fine-tune and hone in on exactly what those populations are doing and which ones should be better targets.

Compared to other therapeutic modalities, what are the challenges and unique attributes to flow cytometry with adoptive cell therapies?
 

Unique and time-consuming

From a therapeutic standpoint, every one of these is a new unique product, which means a new unique assay to detect it. And from flow, we're often looking at clone-specific antibody therapeutics, such as the monospecific, bi-specific, tri-specific therapeutics, which are all very clone dependent and require often custom reagents to detect them. It's not necessarily an insurmountable challenge, but it does mean that sometimes we have a longer runway from a development and validation standpoint when compared to basic phenotyping that doesn't require any type of custom detection reagent.

Challenges from a reagent standpoint

The other challenge is that, once infused in a patient, the adoptive cell therapies are treated almost like a rare cell event, which is similar to what our minimal residual disease, or measurable residual disease, assays would be. So you're really looking for that needle in a haystack rather than a population that's going to be prevalent within the periphery. That does create some unique challenges from a reagent standpoint because we have to make sure that those detection reagents are very, very clean. We have many methods we put into place to avoid introducing background noise and to make sure what we are actually measuring is what we say we are measuring.

Toxicity profile

One of the other unique things to cell therapies is the toxicity profile and how that relates to the mechanism of action for these things. This may mean that you need to search for the cells in a compartment of the body that you would not typically see the presence itself, like the cerebrospinal fluid for patients that have ICANS. ICANS stands for immune effector cell-associated neurotoxicity syndrome; basically, what that is saying is that the blood-brain barrier is compromised, meaning these cells and native cells can make their way into the cerebrospinal fluid.

This is a safety issue that we don't see with other therapeutics necessarily.

Deeper neurotoxicity insights

From a flow cytometry standpoint, we can detect if CAR cells are present within the cerebrospinal fluid, which is great. More importantly, flow cytometry not only allows you to detect the presence of CAR cells, but it also enables you to determine if there's a specific subset or phenotype profile of those cells present versus something you might see in the periphery that didn't make it across that blood-brain barrier. If those cells are present in the cerebrospinal fluid in the first two weeks—ICANS typically presents the first two weeks of treatment — how does that population that was present in the cerebrospinal fluid correlate to disease progression versus responders who actually wind up in remission? So if those cells making it into the cerebrospinal fluid are not the cells getting my patient into remission, then can I filter them out using some cleaning techniques, which we're also able to do by flow via some sorting technique, before infusing those cells back into the patient and maybe decrease the incidence of neurotoxicity? It’s exciting that we have an assay that can track the native cell as well as the adoptive cell within the same assay and not need additional sampling for that.

What are the utilizations of these therapies beyond oncology?

It's not just cancer treatment anymore. We're now moving into autoimmune disorders and seeing some really good responses with those. With autoimmune disorders, the adoptive cell therapies have two modes of approach here. It's either eliminate the autoreactive cells or dampen that autoreactivity. And if you take Type 1 diabetes, for example, then by flow, we can measure the number and the function of T-cells that are specific to the beta cells in those patients before they're treated with their therapy, whatever it may be. And we can also track those cells over time to see if they are diminishing as the adoptive cell therapy begins to persist and proliferate. Flow is uniquely positioned to be able to tell you the phenotype, the persistent phenotype, which cells are causing my regression of disease, which cells are those that may be causing potential issues and safety concerns within my patient and provide that information back to the sponsors so they can fine-tune their therapies for later treatments. The end goal is to find a way to get to a treatment that has the least amount of a safety profile, and flow allows us to do that very nicely.

What are some potential benefits of connecting early-stage preclinical flow cytometry insights with clinical assay development?

This is an area that Labcorp really excels at because of the widths and breadths of our subject matter experts in all of the different divisions of a clinical trial.

One of the benefits of having (early preclinical work) done early is that you know the profile of those adoptive cell therapies before they ever make it into the clinical setting. The approach we've taken with that is we’ve already developed backbone panels where we can then take the assay-specific or drug-specific detection reagents and just plug them into our backbone assays. So we've got assays that will tell us the memory, naive, and effector subsets; assays that will tell us the Treg compartments; and basic T-cell, B-cell, NK-cell assays. These all have open channels, and all we have to do is take that detection reagent that has already been fully vetted through our early development and preclinical folks and then plug it into our assay.

What that ultimately means is that the time from assay development and validation to patient enrollment is significantly shorter than if we had to start from ground zero every single time. We know what that cell population should look like. Even if it's created in a different animal model, we still know what that human cell should look like. It just helps us get there a lot more quickly.

In your view, what are some exciting developments on the horizon for flow cytometry with adoptive cell therapy development and beyond?

1. Broader-spectrum platforms

The instruments of old are not the instruments we have now. Previously, we had between five and eight color flow cytometry, so there were a limited number of markers you could put into a panel with a given volume of blood.

And we have to keep in mind that these patients are very frequently lympho-depleted before they're in treatment, so taking a lot of their blood is not a kind thing to do. With the newer platforms, we have the BD FACSLyric™ 12-color instrument, and then we have the Bio-Rad ZE5, which is a 5-laser, or a comfortable 22-color panel, although we're working on a 27-color panel now. What that means is that we can do much deeper phenotyping. We can dive down into not just the native cell populations within a patient, but also the adoptive cell therapies and look at exhaustion markers simultaneously with the phenotypic markers. We can look at proliferative markers. We can look at all of those different markers simultaneously on a very small volume of blood compared to what we would have needed previously for that same type of readout. 

2. Extracellular vesicles

We're also developing techniques to detect extracellular vesicles that may express some of the things that are going to hone in on disease. They're talking now about using extracellular vesicles as delivery methods for some of the gene therapies. So that's very exciting that we will be able to not only detect the extracellular vesicles but potentially even watch them deliver a payload into a target cell.

3. Circulating tumor cells

The ability to use flow cytometry to look at circulating tumor antigen autoantibodies will help us fine-tune and target the next generation of therapeutics to the different tumor types, or even in a patient-specific tumor-type setting, which is potentially a bit more problematic from a therapy development standpoint.

mRNA detection

One of the other things that we're going to be looking at is a product called PrimeFlow™, which is used to detect messenger RNA in flow cytometry. What we're hoping is that we'll be able to very cleanly detect cells that are expressing the transgene of interest that says, “Okay, this is my cell therapy.” But unlike with PCR, we'll be able to tell which specific cell types those are in. Not only can we tell which cells are transgene positive, but also which cells are proliferating in the long-term follow-up studies, which of those are correlated to responders versus non-responders, and if that can help us get to a better treatment design in the future.

Learn how Labcorp can support your flow cytometry needs to advance your cell and gene therapies at each of our central laboratory locations.

You can also read more blogs in our CGT Answers blog series on additional topics: ddPCR, CMC, Companion Diagnostics, Gene Therapy, Preclinical Oncology and Adoptive Cell Therapy.