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Yes, we have over 30 years of expertise in developing and validating cell based assays for FDA IND, lot release or stability study approval. Our team can also liaise directly with the FDA on your behalf, ensuring a smooth and compliant process. Please contact us to learn more about how we can assist you with your assay development and FDA approval needs.

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MarinBio scientists are experienced and creative in designing, developing, validating assays. Some of the assays we perform are listed below, but there are many more that we created, developed and validated. Give us call so that we may help you.

Potency Assays and Release Testing of CAR T-Cell Therapies

CAR T-cell therapy represents a significant shift in the treatment landscape for cancer, particularly in hematologic malignancies. Unlike traditional immunotherapies, Chimeric Antigen Receptor (CAR) T-cells are genetically engineered T lymphocytes that can recognize specific tumor antigens independently of the major histocompatibility complex (MHC). After being redirected to identify cancer cells through an extracellular antigen-binding domain, CAR T-cells can initiate a range of effector functions, including cytotoxic activity, cytokine secretion, and proliferation. To ensure the clinical efficacy and safety of CAR T-cell therapies, it is essential to assess their biological functionality through standardized in vitro assays. These potency assays evaluate the extent to which CAR T-cells can engage and destroy tumor cells, proliferate upon antigen encounter, release cytokines, and resist regulatory checkpoints. This document provides an overview of these crucial assay methodologies and highlights their significance for the development and quality control of CAR T-cell therapies.

Molecular Mechanism of CAR T-Cell Tumor Killing

CAR T-cell therapy operates through a clearly defined sequence of molecular events that begins with antigen recognition and culminates in tumor cell lysis. The engineered CAR construct typically includes a single-chain variable fragment (scFv) that binds a tumor-associated antigen. Upon recognition of the target antigen on tumor cells, the intracellular signaling domains within the CAR, typically CD3ζ and co-stimulatory motifs like CD28 or 4-1BB, initiate robust T-cell activation. This activation triggers several downstream functions, including the release of cytotoxic granules containing perforin and granzymes that perforate the tumor cell membrane and induce apoptosis. Simultaneously, CAR T-cells upregulate activation markers and begin to secrete inflammatory cytokines such as IFN-γ and TNF-α, amplifying both autocrine and paracrine immune responses. These events not only result in direct killing of tumor cells but also stimulate CAR T-cell proliferation and persistence, factors necessary for sustained therapeutic response. Understanding these mechanisms underpins the design of effective assays aimed at measuring the functional activity of CAR T-cells in preclinical and clinical development.

Design of an In Vitro Tumor Killing Assay Using a Co-Culture of CAR T-cells with Tumor Cell Lines

In vitro tumor killing assays are among the most widely used approaches to assess the potency of CAR T-cell therapies. These assays are designed to emulate the cellular interactions between CAR T-cells and tumor targets under controlled laboratory conditions. A typical setup involves co-culturing CAR T-cells with tumor cell lines that express the relevant antigen. Prior to co-culture, tumor cells can be fluorescently labeled with dyes such as CFSE or genetically modified to express a luminescent reporter such as luciferase, allowing the extent of tumor cell death to be measured through fluorescence intensity or luminescence. CAR T-cells are added at varying effector-to-target (E:T) ratios, and measurements are taken at predetermined time points to quantify cytotoxicity. Common readouts include flow cytometry, ATP content assays, and LDH release, which together provide insight into cell viability, lysis kinetics, and killing efficiency. Data from co-culture assays help validate CAR construct optimization, predict functional potency, and confirm antigen specificity, making them a cornerstone of CAR T-cell assay development pipelines.

Selecting the Right Tumor Cell Line for Co-Culture Assays

The choice of tumor cell line is arguably the most consequential decision in co-culture assay design. The biggest driver of assay reliability is matching antigen density and biology to the clinical target, not simply using a convenient cell line.

Antigen expression level. The target cell line must express the relevant antigen at physiologically relevant levels. Overexpression systems can artificially inflate potency readings, producing results that do not reflect clinical performance. Where possible, use native tumor lines; when engineered lines are necessary, control expression levels carefully.

Antigen uniformity and stability. Heterogeneous antigen expression leads to variable killing readouts across replicates and passages. Choose lines with stable, uniform expression and low antigen drift over time in culture.

Sensitivity to killing mechanisms. Some tumor lines are intrinsically resistant to apoptosis, while others are overly fragile. Lines that are too sensitive produce false high-potency readings; lines that are too resistant underestimate potency. Characterize the killing sensitivity of candidate lines before committing to an assay platform.

Growth characteristics and assay compatibility. Whether a line grows in suspension or as an adherent monolayer affects assay format, readout selection, and throughput. Doubling time also impacts readout timing, as fast-growing targets may outpace killing at lower E:T ratios.

Background signal and assay interference. Autofluorescence, endogenous ATP levels, or LDH leakage from target cells can skew quantitative readouts. Validate that the chosen line produces acceptable signal-to-noise ratios in your specific detection platform.

Basics of CAR T-cell Potency Assays

CAR T-cell potency assays are critical tools used to evaluate and quantify the functional capacity of genetically engineered T cells in preclinical research and during clinical development. These assays are designed to assess key functions that determine therapeutic efficacy, including cytotoxic activity, activation, cytokine release, immune checkpoint inhibition, and proliferative capability. By accurately measuring these parameters, researchers can validate CAR constructs, define potency thresholds, monitor batch-to-batch consistency during manufacturing, and ensure compliance with regulatory requirements. Each assay type isolates a specific aspect of CAR T-cell function, offering a comprehensive view of how the therapeutic cells behave in response to antigen challenge. Collectively, potency assays play a vital role in refining CAR T-cell therapies and improving patient outcomes.

Common Pitfalls in CAR T-cell Potency Assay Design

Potency assay development for CAR T-cell therapies presents several recurring challenges. Recognizing these pitfalls early in assay design can prevent costly redesigns and regulatory delays.

Over-reliance on a single functional readout. Many assays focus exclusively on cytotoxicity (for example, an LDH or luciferase readout), ignoring activation, cytokine secretion, or proliferation. This gives an incomplete picture of potency. An orthogonal assay panel, such as cytotoxicity combined with an IFN-γ ELISA and CD69/CD25 expression, provides the biologically relevant, multi-attribute assessment that regulators increasingly expect.

Poor control design. A lack of appropriate controls leads to false positives or inflated potency readings. Every co-culture assay should include antigen-negative tumor cells, mock or untransduced T cells, and a maximum killing (lysis) control.

Inconsistent effector-to-target ratios and timing. Cytotoxicity results are highly sensitive to E:T ratios and assay duration. Standardize multiple E:T ratios (for example, 1:1, 5:1, and 10:1) and optimize timepoints based on the mechanism of action, as 4-hour, 24-hour, and 48-hour reads may each reveal different aspects of CAR T-cell function.

Signal saturation or poor dynamic range. Luminescence and fluorescence assays can saturate at high killing levels, masking differences between manufacturing lots. Validate the linear range of the detection system and adjust target cell numbers or reporter expression levels so the assay discriminates across the potency range that matters.

Donor variability. For autologous products, intrinsic variability in T-cell fitness between donors skews assay reproducibility. Normalize results to internal reference standards and consider relative potency models rather than absolute values to account for donor-to-donor variation.

Types of CAR T-cell Potency Assays

Cytotoxicity Assays are used to determine the direct tumor-killing ability of CAR T-cells. These assays require incubating CAR T-cells with antigen-expressing tumor cell lines, enabling researchers to measure cell death by several methods. Techniques include measuring membrane integrity disruption using lactate dehydrogenase (LDH) release, fluorescent dye-based live/dead cell assays via flow cytometry, and ATP-based luminescent measurements of target cell viability.

Table 1: CAR T-cell cytotoxicity (tumor killing) assays

Assay Name	Mechanism	Example Tumor Antigen
LDH Release Assay	Detects lactate dehydrogenase (LDH) enzyme released into the media upon tumor cell lysis, which correlates with cytotoxic activity of CAR T-cells.	HER2 (Solid tumors)
Flow Cytometry–Based Killing	Uses dyes or markers (e.g., propidium iodide, Annexin V) to differentiate dead from live tumor cells after co-culture; allows assessment of cell viability and subsets.	BCMA (Multiple myeloma)
Bioluminescence Imaging	Tumor cells engineered to express luciferase. Tumor cell death decreases luminescent signal, as measured with a luminometer, reflecting CAR T-dependent killing.	EGFRvIII (Glioblastoma)
ATP Release / ATP Level Assay	Killing of tumor cells causes release or changes in ATP levels measurable by luminescence or fluorescence-based assays, indicating cytotoxicity.	CD22 (B cell malignancies)

T Cell Activation Assays assess how effectively CAR T-cells become activated upon tumor engagement. Upon binding their target antigen, CAR T-cells initiate signal transduction pathways that upregulate activation markers such as CD25 and CD69. These markers can be detected using flow cytometry. In parallel, the secretion of effector cytokines like IFN-γ and IL-2 provides a readout of functional activation, measurable via ELISA or ELISpot platforms. Additional activation metrics include calcium flux and proliferation rates post-stimulation.

Table 2: CAR T-cell activation assays

Assay Name	Mechanism	Tumor Antigen Example
Granzyme B or Perforin Detection Assay	Measures intracellular or secreted cytotoxic granules, indicating effector function of CAR T cells	Mesothelin
Flow Cytometry-based Activation Marker Upregulation	Detects surface or intracellular activation markers (e.g. CD69, CD25, 4-1BB) upregulated on CAR T-cells upon antigen-specific stimulation using tumor cells or artificial targets.	Multiple
Transcription Factor Reporter Assays	CAR T cells engineered with a reporter gene under control of a transcription factor; activation triggers reporter expression like luciferase or GFP	CD19 (B-cell malignancies)
Calcium Flux Assay	Measures intracellular calcium release in CAR T cells upon antigen recognition, indicative of early activation signaling	Multiple

Immune Checkpoint Assays evaluate the role of inhibitory receptors in regulating CAR T-cell activity. These assays determine how checkpoint molecules are expressed following activation and whether blocking these pathways restores full effector function. While early checkpoint assays focused primarily on PD-1 and CTLA-4, the field has moved toward a broader, more functional view of immune suppression.

Expanded checkpoint landscape. Current approaches use multiplex checkpoint profiling that simultaneously measures TIM-3, LAG-3, and TIGIT alongside PD-1 via multi-parameter flow cytometry, providing a more complete picture of the inhibitory receptor landscape on CAR T-cells after antigen encounter.

Functional rescue assays. Expression-level measurement alone is insufficient. CAR T-cells are co-cultured with target cells in the presence or absence of checkpoint-blocking antibodies, and the resulting changes in cytokine recovery, proliferation rescue, and cytotoxicity restoration are quantified. This reveals whether checkpoint engagement is actively suppressing CAR T-cell function, not merely whether the receptors are present.

Exhaustion and chronic stimulation models. Repeated antigen exposure better mimics the tumor microenvironment, where CAR T-cells face sustained antigenic challenge. These chronic stimulation assays reveal exhaustion phenotypes that single-exposure co-cultures miss, providing insight into the durability of CAR T-cell function.

Combination checkpoint testing. Synergistic blockade effects, such as combined PD-1 and LAG-3 inhibition, are increasingly evaluated to identify optimal strategies for overcoming immune suppression in the tumor microenvironment. Together, these approaches mark a shift from static expression assays to dynamic functional suppression and rescue models.

Table 3: CAR T-cell immune checkpoint assays

Assay Approach	What It Measures	Key Readouts
Multiplex Checkpoint Profiling	Simultaneous expression of PD-1, TIM-3, LAG-3, and TIGIT on CAR T-cells post-activation	Multi-parameter flow cytometry
Functional Rescue (Single Blockade)	Effector function recovery upon blocking a single checkpoint pathway	IFN-γ recovery, proliferation rescue, cytotoxicity restoration
Combination Blockade	Synergistic effects of blocking multiple checkpoints (e.g., PD-1 + LAG-3)	Comparative cytokine and killing recovery vs. single blockade
Chronic Stimulation / Exhaustion Model	CAR T-cell function under repeated antigen exposure over days to weeks	Progressive checkpoint upregulation, killing decay, proliferation loss

T Cell Proliferation Assays monitor CAR T-cell division upon target recognition. These assays commonly use fluorescent dyes like CFSE or Cell Trace Violet (CTV), which dilute progressively with each round of mitosis. Alternative methods include incorporation of BrdU, a thymidine analog, into replicating DNA; or analysis of Ki-67, a nuclear protein expressed during active cell cycling. These approaches quantify antigen-dependent CAR T-cell expansion, a marker of sustained bioactivity.

Table 4: CAR T-cell proliferation assays

Assay	Mechanism	Example Tumor Antigen Targeted
Flow Cytometry (CFSE/CTV)	T cells labeled with a fluorescent dye (CFSE or CellTrace Violet). Dye dilution by flow cytometry after antigen stimulation reflects cell division and proliferation.	CD19 (B-cell leukemia/lymphoma)
BrdU Incorporation Assay	BrdU, a thymidine analog, incorporates into newly synthesized DNA of dividing CAR T cells during the S phase. Incorporated BrdU is detected by anti-BrdU antibodies after DNA denaturation, quantifying proliferation.	EGFRvIII (glioblastoma)
Ki-67 Staining Assay	Ki-67 protein is expressed in all active phases of the cell cycle (except G0). Fixed CAR T cells are stained with anti-Ki-67 antibodies and analyzed by flow cytometry or microscopy to quantify proliferating cells.	Mesothelin (pancreatic cancer)

Cytokine Assays are used to profile the immunomodulatory effects and potency of CAR T-cells by measuring expressed cytokines such as IFN-γ, IL-2, or TNF-α. Methods used include traditional ELISA assays for individual cytokines, ELISpot to quantify cytokine-producing cells at the single-cell level, intracellular cytokine staining (ICS), and multiplex bead-based assays for simultaneous multi-analyte detection. These data provide insights into the type and magnitude of immune activation elicited by CAR T products.

Table 5: Cytokine expression assays

Assay	Mechanism	Example Tumor Antigen Targeted
Enzyme-Linked Immunosorbent Assay (ELISA)	Measures specific cytokines present in culture supernatants by antibody capture and enzymatic detection.	CD19 (hematological malignancies)
Flow Cytometry (Intracellular Cytokine Staining)	Detects cytokine production at the single-cell level by fixing and permeabilizing cells, staining with cytokine-specific antibodies, and analyzing fluorescence.	HER2 (solid tumors)
Multiplex Cytokine Assays (Luminex)	Simultaneously quantifies multiple cytokines from supernatants using bead-based or electrochemiluminescence immunoassays.	CD19 (B-cell tumors)
Enzyme-Linked Immunospot Assay (ELISPOT)	Detects cytokines at the single-cell level by capturing secreted cytokines onto antibody-coated PVDF membrane wells, followed by enzyme-linked detection and visualization of spots representing individual cytokine-producing cells.	Mesothelin (pancreatic and ovarian cancers)

FDA Expectations for CAR T-cell Release Testing

Release testing is a critical component of CAR T-cell manufacturing, and the FDA has clear expectations for the potency assays that support it. Understanding these expectations early in development prevents costly assay redesigns and regulatory delays during IND or BLA submissions.

What the FDA Expects

A biologically relevant potency assay. The assay must reflect the product’s mechanism of action. For CAR T-cells, this typically means demonstrating cytotoxicity against antigen-expressing targets, T-cell activation (such as cytokine secretion or CD69 upregulation), or both. A surrogate marker alone, such as CAR surface expression by flow cytometry, is generally insufficient unless thoroughly justified.

Quantitative and stability-indicating. The assay should detect meaningful changes in potency over time and differentiate between strong and weak manufacturing lots. It must be sensitive enough to serve as a release criterion that protects patients from subpotent product.

Validated or qualified. The FDA expects assay validation addressing accuracy, precision, linearity, specificity, and robustness. Early-phase products may use qualified assays, but a clear path to full validation should be in place.

Defined acceptance criteria. Potency specifications should be based on clinical data where available, manufacturing history, and a demonstrated link between the assay readout and patient outcomes.

Where Sponsors Most Often Fall Short

Using non-functional assays as potency measures. Relying solely on CAR expression (percent CAR+) or viability without a functional readout is a common deficiency in regulatory submissions.

Poor assay validation or high variability. Assays with unacceptable coefficients of variation or limited characterization create significant regulatory risk.

No demonstrated link between assay results and clinical outcomes. Regulators expect sponsors to show, or at least plan to show, that the potency assay predicts clinical response.

No bridging strategy when assays evolve. As products move from Phase I through commercialization, assays inevitably change. Failing to bridge old and new assays creates data continuity gaps that delay approvals.

Overly complex assays not scalable for QC release. An assay that works in R&D but cannot be reliably executed in a GMP quality control environment will not serve as a release test.

Conclusion

As CAR T-cell therapies continue to evolve and expand into new oncologic indications, the importance of functional assays that assess potency grows in parallel. These assays are critical for preclinical evaluation and serve as key release criteria for clinical manufacturing, ultimately guiding safety and efficacy at the patient level.

However, one of the most common misconceptions in the field is equating in vitro killing with clinical efficacy. In vitro co-culture systems lack the tumor microenvironment, immune suppression, and the trafficking and persistence factors that determine real-world outcomes. A CAR T-cell product can kill effectively in vitro and still fail clinically due to exhaustion, poor expansion, or inhibition by the tumor microenvironment.

Potency is fundamentally multidimensional. Durable clinical responses depend not only on cytotoxic activity but also on T-cell activation, cytokine production, proliferative capacity, and resistance to inhibition. Proliferation assays, such as CFSE dilution and Ki-67 staining, are just as important as killing assays, because durable responses require robust expansion after infusion and long-term persistence in vivo.

By comprehensively monitoring these parameters through orthogonal assay panels, researchers and manufacturers can move beyond convenience-driven assay design toward mechanism-driven assessment. Aligning assay strategy early with regulatory expectations, choosing biologically relevant tumor models, and designing for the multidimensional nature of potency will support the next generation of CAR T-cell platforms, including those targeting solid tumors, allogeneic products, and armored CAR constructs with enhanced functionalities.

Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

1. Development of VNX-101, an Adeno-Associated Virus with Less Immunogenicity and Efficient Long-Term Expression of a CD19 T-Cell Engager. Molecular Therapy Methods & Clinical Development, published online July 24, 2025.

2. Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1.

4. American Society of Hematology (ASH) Annual Meeting 2024.
Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

Comprehensive Assay Solutions for In Vitro and Cell Based Potency Assays and Pharmacokinetics Studies- Our Expertise

With 30 years of expertise in cell culture, cell-based assays, and preclinical/clinical PK/PD analysis, we specialize in offering assay services essential for a wide variety of therapeutic drug development programs, preclinical studies, IND/BLA applications, and commercialization. Our comprehensive services include both preclinical non-GLP and GLP assays, as well as non-GMP and GMP assays, providing critical support throughout the entire development pipeline.

Watch the following video and explore our latest presentation on the development and validation of potency and pharmacokinetic (PK) assays for AAV vectors, highlighting innovative methodologies and industry-leading expertise.