CAR-based therapies, originally developed for oncology, are now advancing into autoimmune diseases with promising early clinical results.¹ In this setting, the biological context differs, and so does the way CAR kinetics are evaluated and understood.

To explore what this means in practice, we sat down with Laïla-Aïcha Hanafi, Director Global Assay Development here at CellCarta. With a background in immuno-oncology and translational biomarkers for cell therapies, Laïla works closely with sponsors to design and execute CAR kinetic strategies across programs.

In this Q&A, she shares how immunologists approach CAR kinetic monitoring in autoimmune disease and what sponsors should consider when designing their strategy.

How does CAR kinetic monitoring in autoimmune diseases differ from oncology?

It may sound obvious, but the differences in monitoring strategies really come down to the differing goals of CAR therapies in oncology versus autoimmune diseases. In oncology, the aim is to achieve strong CAR expansion and maintain those cells over time to sustain tumor control. Whereas in autoimmune disease, the focus is on eliminating diseased B cells, allowing the immune system to reset, and then letting CAR levels decline. From a monitoring perspective, that difference translates into two key considerations.

First, the timeline. In oncology, monitoring can extend for six months to a year or more because persistence is part of the intended outcome. In autoimmune programs, the most informative window is often much earlier, typically around the first one to two weeks post-infusion, when CAR expansion peaks. After that, the focus shifts to confirming contraction and monitoring immune cell recovery rather than tracking long-term maintenance.

Second, assay sensitivity. In autoimmune disease, starting doses are lower; as a result, CAR expansion may be harder to detect than in oncology. Detecting and quantifying those lower levels reliably requires highly sensitive, well-optimized assays.

What key factors should be considered when choosing an assay for CAR kinetic monitoring in autoimmune diseases?

Assay selection in autoimmune CAR programs largely comes down to sensitivity, specificity, and what type of information you need to generate.

In practice, two main assay families are used to monitor CAR kinetics: flow cytometry and PCR-based approaches. Digital PCR can provide highly sensitive, quantitative detection of the CAR construct and can be run in batches. It gives a clear numerical readout of CAR signal in the sample, but it does not indicate which cells are expressing the CAR.

Flow cytometry, on the other hand, allows direct detection of CAR-expressing cells and makes it possible to identify which cell types have been transfected. This is important in certain contexts, such as in vivo CAR approaches, where the CAR construct may not be restricted to a single predefined cell population. Flow cytometry also enables phenotyping alongside enumeration, providing additional biological insight.

In autoimmune programs, that biological context is particularly relevant, as sponsors need to understand how CAR levels relate to downstream immune effects, especially B-cell depletion and recovery.

In most cases, it’s best to use both approaches. PCR offers sensitivity and quantitative measurement of the construct, while flow cytometry provides cell-level resolution and biological context.

How should immunologists use CAR enumeration and absolute counts to interpret CAR kinetics in autoimmune disease?

Enumeration is the starting point for understanding CAR kinetics—it shows whether CAR cells are present and how their levels change over time. But interpreting those numbers meaningfully requires looking at absolute counts rather than percentages alone.

Absolute counts allow teams to construct the full kinetic curve: how quickly the CAR cells expand, how high they peak, and how long they remain detectable. That information helps in understanding dose, exposure, and how the CAR levels relate to B-cell reduction.

The early expansion phase, typically around day 7–14 post-infusion, is where absolute counts really add value, allowing the assessment of peak levels and overall exposure. Later in the timeline, when CAR levels are much lower, small numerical differences become less meaningful. At that stage, interpretation focuses more on whether CAR cells are still detectable rather than on detailed quantitative comparisons.

What are the biggest interpretation challenges in autoimmune CAR monitoring?

As mentioned, one of the main challenges is sensitivity. Because CAR expansion may be lower in autoimmune programs, detecting small populations reliably can be difficult. Increasing assay input or optimizing the assay design may be necessary to capture low-level signals.

Specificity is just as important, particularly when using flow cytometry. Background signal or non-specific binding can obscure low-level CAR detection. Addressing this may require increasing assay input to improve sensitivity, refining gating strategies, or adding additional markers, such as negative selection or “dump” channels, to reduce background.

Finally, as CAR levels decline, deeper phenotyping becomes more difficult. To ensure detailed biological insight can be extracted, it is better to conduct subpopulation analysis during the expansion peak, when there are sufficient events to analyze.

Overall, what would you say are the key things sponsors should keep in mind when designing CAR kinetic monitoring strategies for autoimmune programs?

In autoimmune programs, timing and sensitivity are key. Plan monitoring around the most informative window (days 7–14 post-infusion), make sure assays are sensitive enough for lower signals, and think beyond simple detection.

At the same time, it’s important not to look at CAR levels in isolation. In autoimmune disease, what ultimately matters is how those kinetics translate into biological effect. Monitoring B-cell depletion and recovery, including changes in specific subsets, helps connect CAR exposure to immune reset.

In the end, good CAR kinetic monitoring in autoimmune programs is about seeing the full picture, not just whether CAR cells are present, but how the kinetic profile aligns with downstream immune changes and the therapeutic goal.

Supporting CAR Kinetic Monitoring in Autoimmune Programs

To support robust CAR kinetic monitoring from early expansion through immune reconstitution, CellCarta provides integrated assay solutions tailored to autoimmune programs.

• Digital PCR: custom and off-the-shelf assays for sensitive, quantitative detection of CAR constructs.
• Flow cytometry: custom CAR detection panels for enumeration and phenotyping, and spectral flow cytometry for deeper immune profiling.
• Advanced immune profiling: CyTOF for high-dimensional phenotyping and single-cell genomics for deeper biological characterization.
• Ready-to-deploy assays for pharmacodynamic monitoring, including: B-cell aplasia and recovery, memory B-cell phenotyping, TBNK CD20 panels, and absolute B-cell enumeration.

Explore how CellCarta’s immunology platforms can help you generate high-quality CAR kinetic and immune monitoring data for your autoimmune programs

Reference

1. https://pmc.ncbi.nlm.nih.gov/articles/PMC12488630/pdf/fimmu-16-1613622.pdf

CAR-based therapies, originally developed for oncology, are now advancing into autoimmune diseases with promising early clinical results.¹ In this setting, the biological context differs, and so does the way CAR kinetics are evaluated and understood.

To explore what this means in practice, we sat down with Laïla-Aïcha Hanafi, Director Global Assay Development here at CellCarta. With a background in immuno-oncology and translational biomarkers for cell therapies, Laïla works closely with sponsors to design and execute CAR kinetic strategies across programs.

In this Q&A, she shares how immunologists approach CAR kinetic monitoring in autoimmune disease and what sponsors should consider when designing their strategy.

How does CAR kinetic monitoring in autoimmune diseases differ from oncology?

It may sound obvious, but the differences in monitoring strategies really come down to the differing goals of CAR therapies in oncology versus autoimmune diseases. In oncology, the aim is to achieve strong CAR expansion and maintain those cells over time to sustain tumor control. Whereas in autoimmune disease, the focus is on eliminating diseased B cells, allowing the immune system to reset, and then letting CAR levels decline. From a monitoring perspective, that difference translates into two key considerations.

First, the timeline. In oncology, monitoring can extend for six months to a year or more because persistence is part of the intended outcome. In autoimmune programs, the most informative window is often much earlier, typically around the first one to two weeks post-infusion, when CAR expansion peaks. After that, the focus shifts to confirming contraction and monitoring immune cell recovery rather than tracking long-term maintenance.

Second, assay sensitivity. In autoimmune disease, starting doses are lower; as a result, CAR expansion may be harder to detect than in oncology. Detecting and quantifying those lower levels reliably requires highly sensitive, well-optimized assays.

What key factors should be considered when choosing an assay for CAR kinetic monitoring in autoimmune diseases?

Assay selection in autoimmune CAR programs largely comes down to sensitivity, specificity, and what type of information you need to generate.

In practice, two main assay families are used to monitor CAR kinetics: flow cytometry and PCR-based approaches. Digital PCR can provide highly sensitive, quantitative detection of the CAR construct and can be run in batches. It gives a clear numerical readout of CAR signal in the sample, but it does not indicate which cells are expressing the CAR.

Flow cytometry, on the other hand, allows direct detection of CAR-expressing cells and makes it possible to identify which cell types have been transfected. This is important in certain contexts, such as in vivo CAR approaches, where the CAR construct may not be restricted to a single predefined cell population. Flow cytometry also enables phenotyping alongside enumeration, providing additional biological insight.

In autoimmune programs, that biological context is particularly relevant, as sponsors need to understand how CAR levels relate to downstream immune effects, especially B-cell depletion and recovery.

In most cases, it’s best to use both approaches. PCR offers sensitivity and quantitative measurement of the construct, while flow cytometry provides cell-level resolution and biological context.

How should immunologists use CAR enumeration and absolute counts to interpret CAR kinetics in autoimmune disease?

Enumeration is the starting point for understanding CAR kinetics—it shows whether CAR cells are present and how their levels change over time. But interpreting those numbers meaningfully requires looking at absolute counts rather than percentages alone.

Absolute counts allow teams to construct the full kinetic curve: how quickly the CAR cells expand, how high they peak, and how long they remain detectable. That information helps in understanding dose, exposure, and how the CAR levels relate to B-cell reduction.

The early expansion phase, typically around day 7–14 post-infusion, is where absolute counts really add value, allowing the assessment of peak levels and overall exposure. Later in the timeline, when CAR levels are much lower, small numerical differences become less meaningful. At that stage, interpretation focuses more on whether CAR cells are still detectable rather than on detailed quantitative comparisons.

What are the biggest interpretation challenges in autoimmune CAR monitoring?

As mentioned, one of the main challenges is sensitivity. Because CAR expansion may be lower in autoimmune programs, detecting small populations reliably can be difficult. Increasing assay input or optimizing the assay design may be necessary to capture low-level signals.

Specificity is just as important, particularly when using flow cytometry. Background signal or non-specific binding can obscure low-level CAR detection. Addressing this may require increasing assay input to improve sensitivity, refining gating strategies, or adding additional markers, such as negative selection or “dump” channels, to reduce background.

Finally, as CAR levels decline, deeper phenotyping becomes more difficult. To ensure detailed biological insight can be extracted, it is better to conduct subpopulation analysis during the expansion peak, when there are sufficient events to analyze.

Overall, what would you say are the key things sponsors should keep in mind when designing CAR kinetic monitoring strategies for autoimmune programs?

In autoimmune programs, timing and sensitivity are key. Plan monitoring around the most informative window (days 7–14 post-infusion), make sure assays are sensitive enough for lower signals, and think beyond simple detection.

At the same time, it’s important not to look at CAR levels in isolation. In autoimmune disease, what ultimately matters is how those kinetics translate into biological effect. Monitoring B-cell depletion and recovery, including changes in specific subsets, helps connect CAR exposure to immune reset.

In the end, good CAR kinetic monitoring in autoimmune programs is about seeing the full picture, not just whether CAR cells are present, but how the kinetic profile aligns with downstream immune changes and the therapeutic goal.

Supporting CAR Kinetic Monitoring in Autoimmune Programs

To support robust CAR kinetic monitoring from early expansion through immune reconstitution, CellCarta provides integrated assay solutions tailored to autoimmune programs.

• Digital PCR: custom and off-the-shelf assays for sensitive, quantitative detection of CAR constructs.
• Flow cytometry: custom CAR detection panels for enumeration and phenotyping, and spectral flow cytometry for deeper immune profiling.
• Advanced immune profiling: CyTOF for high-dimensional phenotyping and single-cell genomics for deeper biological characterization.
• Ready-to-deploy assays for pharmacodynamic monitoring, including: B-cell aplasia and recovery, memory B-cell phenotyping, TBNK CD20 panels, and absolute B-cell enumeration.

Explore how CellCarta’s immunology platforms can help you generate high-quality CAR kinetic and immune monitoring data for your autoimmune programs

Reference

1. https://pmc.ncbi.nlm.nih.gov/articles/PMC12488630/pdf/fimmu-16-1613622.pdf