Function-based antibody screening evaluates whether a candidate antibody actually triggers the biological response a therapeutic program needs, rather than simply measuring whether it sticks to a target antigen. For programs involving CAR-T constructs, T cell engagers, and other multispecific formats, binding data alone frequently fails to predict how a candidate will perform once built into the final molecule.
The questions below explain how function-based screening works, why it matters for complex modalities, and how it compares to conventional binding-first discovery.
What is function-based antibody screening?
Function-based antibody screening selects antibody candidates according to the biological activity they trigger, such as T cell activation, cytotoxicity, or cytokine release, rather than selecting them purely on affinity for a target antigen. This approach enables identification of HCAbs based on actual CAR function rather than binding characteristics alone.
The distinction is important because a molecule can bind its target tightly in solution yet fail to generate the desired signaling response once expressed in a membrane-anchored format. Research has shown that not all binders that perform well in conventional screening translate effectively into CAR formats, as the membrane-anchored configuration imposes structural and spatial constraints that can alter both binding behavior and downstream signaling. Function-based screening addresses this challenge by evaluating signaling output directly while candidates are still being selected, helping identify molecules with the greatest potential for functional performance in a CAR setting.
Why doesn’t strong binding affinity guarantee good CAR-T or T cell engager function?
Strong binding affinity does not reliably predict CAR-T or T cell engager performance because cellular activation depends on factors beyond how tightly an antibody fragment binds its target. In functional screening studies, some of the highest-performing HCAb-derived single domains exhibited only moderate binding affinity, demonstrating that superior functional activity does not necessarily correlate with the strongest target binding.
Other characteristics can also influence performance. For example, surface expression levels may be similar among constructs that display markedly different biological activity, indicating that cell-surface density alone does not explain functional differences. Researchers have proposed several additional factors that may contribute, including recognition of distinct epitopes and increased avidity. Avidity, in particular, is widely recognized within the cell therapy field as an important determinant of CAR binder performance. Because multiple mechanisms can influence activation and therapeutic function, activity is best measured directly rather than inferred from affinity data alone.
How does Nona’s Direct CAR-Based Functional Library Screening Platform work?
NonaCarFx™ (Nona’s CAR-based functional screening platform) screens candidate HCAb VH domains by building them directly into CAR constructs and measuring real signaling output in reporter cells. The workflow begins by immunizing HCAb Harbour Mice® (transgenic mice engineered to produce fully human heavy-chain-only antibodies), recovering the VH sequences, and cloning them into proprietary CAR lentiviral vectors to build a CAR lentiviral vector library.
Researchers transduce a reporter T cell line, Jurkat cells containing an NFAT-GFP reporter, so that when a CAR actually interacts with its target and becomes activated, the reporter cell turns on GFP. The pool is then stimulated with target antigen and both GFP-positive and RFP-positive cells are sorted out, representing the VH sequences that drive real CAR activation, which are enriched for the next round. Because RFP marks transduction and expression while GFP marks true functional activation, the platform separates candidates that merely express well from candidates that actually signal.
What happens to clones that show non-specific or “tonic” signaling?
Clones exhibiting tonic or non-specific signaling can be identified and removed before they ever reach later-stage development. Even without stimulating the cells, some GFP-positive cells appear in the absence of target, representing HCAb VH domains showing non-specific or tonic signaling, though this percentage is described as very low, which was seen as a testament to the suitability of the HCAb VH domains for CAR T cell therapy. The platform sorts GFP-negative cells in the absence of target, isolating RFP-positive, GFP-negative cells that are CAR-expressing but unactivated, and this sort routinely achieves over 90% post-sort purity. Those clean, unactivated cells are then stimulated with the actual target antigen, and only the ones that turn GFP-positive under real antigen exposure move forward. Multiple rounds of this stimulation and sorting cycle can be layered to further eliminate background activation and enrich for antigen-specific function.
How does this compare to Beacon®, phage display or standard HCAb direct expression screening?
Beacon® single B cell screening, phage display and standard HCAb direct expression screening are both binding-based methods, which limits how predictive they are for CAR-T or T cell engager development. Beacon offers high-throughput, rapid identification of binders, but like the other methods it does not provide a direct CAR function readout — and because it requires plasma cells specifically, it isn’t able to draw on the full B cell population the way broader library-based screens can.
Phage display has the advantage of being able to include all of the B cells in a large library-based screening, but it is fundamentally binding-based screening, and it is not clear how well those binders will convert to CARs and show good CAR function. Standard HCAb direct expression systems carry similar limitations to other binding-based methods, are not a direct CAR function readout, and tend to have a somewhat lower positivity rate. Function-based screening was built specifically to close this gap.
|
Screening Method |
Selection Basis |
CAR Function Readout |
Library Coverage |
|---|---|---|---|
|
Beacon single B cell screening |
Binding only |
No |
Narrow — plasma cells only |
|
Phage display |
Binding only |
No |
Broad, includes all B cells |
|
Standard HCAb direct expression |
Binding only |
No |
Moderate, lower positivity rate |
|
NonaCarFx™ functional screening |
Direct CAR activation (NFAT-GFP) |
Yes |
Broad, covers memory and plasma B cells |
Is this platform limited to CAR-T cell therapy, or can it support other modalities?
NonaCarFx™ extends beyond CAR-T into other therapeutic modalities, including antibody therapeutics broadly and T cell engagers specifically. The majority of HCAbs identified from this platform show good developability in general, even as soluble HCAbs, which is a testament to the suitability of the platform for antibody therapeutics in general. The platform supports multi-round stimulation and sorting that can be tailored to any project need, whether targeting a unique epitope, eliminating non-specific binding, or focusing on specific tumor cells. Enriched VH regions can also be cloned directly into a T cell engager construct, enabling function-based T cell engager screening rather than screening for binding alone. This flexibility reflects a broader industry recognition that clustering behavior around CD3 and the T cell receptor shares mechanistic similarities across T cell engagers and CAR-T constructs alike.
How does functional screening apply to T cell engager development specifically?
T cell engager development benefits from functional screening because both arms of a bispecific molecule must be tuned to balance potency against safety, not just optimized for raw affinity. VH domains with specificity for the T cell receptor 4-1BB were identified using a functional discovery workflow, and in T cell engagers, both arms of the bispecific molecule must be carefully optimized to balance potency with safety. Selecting VH domains with fine-tuned 4-1BB affinity is especially critical to avoid excessive T cell activation and cytokine release, while ensuring sufficient engagement for effective tumor cell killing. Nona’s approach evaluated candidate leads by testing their capacity to activate T cells in the presence and absence of target-expressing cells, benchmarking results against established reference antibodies.
What clinical or preclinical evidence supports function-based screening as a predictor of real-world performance?
A BCMA CAR-T case study demonstrates that function-based selection translates into measurable in vivo efficacy, not just favorable reporter assay signals. Co-culturing the resulting CAR-T cells with BCMA-positive target cells produced strong cytotoxicity, robust interferon-gamma and other cytokine secretion, along with clear activation and healthy proliferation. A dual VH configuration produced even stronger and more consistent signaling response in the reporter assay compared to a single VH BCMA CAR, showing that the fully human HCAb-derived VH not only binds BCMA well but also translates into potent CAR signaling. In a xenograft model conducted in collaboration with the Dana-Farber Cancer Institute, the HCAb CAR constructs showed rapid tumor clearance and strong suppression of tumor burden over time, with survival curves and total BLI data confirming potent and sustained anti-tumor efficacy. These outcomes connect functional reporter assay data directly to in vivo tumor control, reinforcing that activity-based selection is predictive rather than purely theoretical.
Why do fully human HCAb VH domains from Harbour Mice® perform well in this functional context?
Fully human HCAb VH domains generated by HCAb Harbour Mice® combine a fully human sequence with favorable biophysical properties that support both binding and downstream function. Because the HCAb has a fully human sequence, it does not need humanization and carries a very low risk of immunogenicity, while affinity can reach up to picomolar ranges similar to camelid-derived binders, and the HCAb format also carries global IP protection. This is the core distinction between fully human and humanized antibodies: fully human sequences arise from natural in vivo immune selection in Harbour Mice® and carry no residual non-human residues, while humanized antibodies are engineered from non-human starting sequences and retain some immunogenicity risk even after optimization. Because HCAb VH domains are single-domain building blocks rather than paired heavy and light chains, they avoid the chain mispairing complications that plague conventional bispecific formats, simplifying incorporation into CAR constructs, T cell engagers, and other multispecific designs. Nona Biosciences’ Hu-mAtrIx™ AI-platform integrated in discovery extends this further by guiding the incorporation of developability-optimized sequences, complementing the functional screening data generated through NonaCarFx™.
What is the typical timeline for a function-based screening program?
A function-based HCAb discovery program typically takes two to three months from immunization to identification of functional binders. The majority of this time is spent in the immunization stage, and if the antigen is ready, immunization can begin immediately, with binder identification typically taking two to three months depending on titer. Programs requiring additional purification or in vitro validation steps will extend somewhat beyond this window. Sponsors with well-characterized antigens and clear functional endpoints, such as a defined reporter assay or in vivo model, tend to move through this timeline most efficiently.
Choosing a discovery partner for CAR-T, T cell engager, or other multispecific programs comes down to whether they generate functional data before you commit to lead sequences, not after. NonaCarFx™ pairs fully human HCAbs from Harbour Mice® with direct CAR functional screening, connecting antigen binding to real signaling output from the earliest stages of discovery.
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