Why Potency Assays Matter in NK and CAR-NK Development

Potency is one of the hardest questions in cell therapy development.

For conventional biologics, potency may often be connected to a defined molecular interaction, such as receptor binding, enzyme activity, neutralization, or ligand blockade. For living cell therapies, the challenge is different. The product is a population of cells, and the biological activity may depend on phenotype, viability, activation state, receptor expression, metabolic condition, cytotoxic function, cytokine production, migration, persistence, and sensitivity to handling.

For NK and CAR-NK therapies, the NK cell potency assay matters because it connects the product to its intended biological function.

A batch of cells can meet identity, purity, viability, and sterility expectations and still fail to perform the function that matters. Potency assays are designed to reduce that risk.

What potency means in cell therapy

Potency is the specific ability or capacity of a product to achieve a defined biological effect. In cell therapy, potency testing should be linked to the intended mechanism of action.

For NK cell products, relevant biological functions may include:

• Tumor-cell killing
• Degranulation
• Cytokine secretion
• Antibody-dependent cellular cytotoxicity
• Missing-self recognition
• Activation through NK-cell receptors
• Persistence or proliferative capacity, depending on the product hypothesis
• Migration or infiltration in more advanced characterization assays

For CAR-NK products, the potency challenge is broader. A CAR-NK product may act through both native NK-cell mechanisms and CAR-mediated antigen recognition. That means the CAR-NK potency assay strategy may need to evaluate:

• Baseline NK cytotoxicity
• CAR-specific killing
• Antigen-dependent activation
• CAR expression
• Functional activity against antigen-positive target cells
• Specificity versus antigen-negative controls
• Cytokine release profile
• Post-thaw function
• Repeat-dose suitability
• Persistence-related attributes, where relevant

No single assay automatically captures all of this. A credible potency strategy usually requires a staged analytical package.

Why NK potency is biologically complex

NK cells are not passive delivery vehicles. They are active innate immune cells.

They can kill target cells through natural cytotoxicity receptors, NKG2D-mediated recognition, missing-self biology, CD16-mediated antibody-dependent cellular cytotoxicity, perforin/granzyme release, death receptor pathways, and cytokine-mediated immune modulation.

This biology is valuable, but it complicates potency testing. If an NK product kills K562 cells in vitro, that may indicate cytotoxic potential, but it does not necessarily prove disease-specific activity in every clinical indication. If a CAR-NK product kills a target cell line, developers still need to understand whether the activity is CAR-dependent, native NK-mediated, or both.

This distinction matters for release testing, comparability, mechanism-of-action claims, and clinical interpretation.

Why CAR-NK potency is not just “NK potency plus CAR expression”

CAR expression is important, but expression alone is not potency.

A CAR-NK product may express a chimeric receptor and still have limited functional activity if the cells are exhausted, poorly viable, damaged by cryopreservation, inadequately activated, poorly expanded, or functionally impaired after thawing.

Conversely, a CAR-NK product may show cytotoxicity through native NK mechanisms even when CAR-dependent function is weak. That can create an analytical problem: the assay may look positive while failing to measure the engineered mechanism that justifies the CAR.

For this reason, CAR-NK potency strategies should generally include both:

• NK-cell functional assessment
• CAR-dependent functional assessment

Where possible, assays should compare antigen-positive and antigen-negative targets, include appropriate controls, and support a mechanistic interpretation. See our companion piece on CAR-NK and CAR-T biology for related context.

Release assay, characterization assay, or exploratory assay?

Not every useful assay is suitable for batch release.

A release potency assay must be practical, reproducible, timely, controlled, and tied to predefined acceptance criteria. It must support batch disposition within the operational constraints of the product, including fresh or cryopreserved release timelines.

A characterization assay can be more detailed. It can help explain the product’s biological profile, mechanism, stability, and comparability.

An exploratory assay can support research and product understanding, but it may not yet be qualified or validated enough for regulated decision-making.

Potency and cryopreservation

Cryopreservation can change the functional state of NK cells.

Post-thaw viability alone is not sufficient. A product may look viable but show reduced cytotoxicity, altered receptor expression, impaired migration, reduced cytokine production, or delayed functional recovery. For off-the-shelf NK and CAR-NK products, this is a central CMC issue.

Potency assays should therefore consider the product as it will be administered. If the clinical product is cryopreserved, developers must understand post-thaw potency, post-thaw hold time, recovery, and the effect of thawing on the intended mechanism of action.

This is especially important for Brazil and LATAM logistics, where chain of custody, cold-chain validation, post-thaw handling, and clinical-site readiness can materially affect the final administered product.

Potency and comparability

Comparability is one of the main reasons potency assays matter.

Cell therapy development often involves process changes:

• New raw materials
• New cytokine source
• Culture-system changes
• Scale-up or scale-out changes
• Engineering-method changes
• Vector or construct changes
• Site transfer
• Cryopreservation changes
• Analytical-method changes
• Formulation or container-closure changes

Each change raises a question: does the post-change product retain the biological activity that matters?

Potency assays in cell therapy help answer that question. Without a credible potency assay, comparability becomes weaker, and clinical data become harder to interpret.

Potency and clinical interpretation

A clinical signal is only meaningful if the product is understood.

If one batch differs materially from another in cytotoxicity, CAR expression, post-thaw recovery, activation state, or cytokine profile, clinical outcomes may reflect product variability rather than dose, patient biology, or target selection.

Potency assays help connect manufacturing to clinical development. They allow teams to ask:

• Was the administered product functionally active?
• Did activity change after process modification?
• Did post-thaw handling affect function?
• Are clinical outcomes interpretable against product attributes?
• Does the potency readout align with the intended mechanism of action?

Potency assays do not guarantee clinical benefit. But without them, development becomes scientifically weaker and regulatorily riskier.

Why this matters for BioNK

For BioNK, potency assays are central to responsible NK and CAR-NK development.

A non-modified NK product and a CAR-NK product require separate development tracks. Progress in an unmodified NK platform does not automatically establish readiness of a CAR-NK product. CAR-NK introduces additional requirements related to engineering, antigen-specific function, comparability, safety, persistence, and long-term follow-up.

BioNK’s potency strategy should therefore be product-specific and phase-appropriate:

• Unmodified NK products require assays that reflect NK-cell identity and functional cytotoxicity
• CAR-NK products require assays that also assess CAR-dependent activity and specificity
• Cryopreserved products require post-thaw potency evaluation
• Process changes require comparability-relevant functional testing
• Clinical trials require potency data that support interpretation of safety and activity signals

Bottom line

Potency assays matter because NK and CAR-NK therapies are living products whose function cannot be inferred from identity or viability alone.

For NK products, potency connects cell phenotype to cytotoxic function. For CAR-NK products, potency must also address antigen-specific engineered activity. For cryopreserved products, potency must be understood after thawing and handling. For process changes, potency is central to comparability. For clinical trials, potency helps make safety and activity signals interpretable.

In NK and CAR-NK development, potency is not an isolated QC test. It is a core part of CMC strategy, regulatory readiness, and scientific credibility. Learn more about our broader work on the BioNK platform and explore related clinical development context.