Author: Dr Erika Kovacs, Senior Director of Bioassay
Bridging the Gap Between in vitro Models and Clinical Success
Pre‑clinical drug discovery is at an inflection point. The recent announcement from the U.S. Food and Drug Administration (FDA) – its Roadmap to Reducing Animal Testing in Pre‑clinical Safety Studies issued in April 2025 – marks a decisive shift toward New Approach Methodologies (NAMs) that prioritize human‑relevant data over traditional animal models. Central to this roadmap is the adoption of complex in vitro cellular systems such as three‑dimensional spheroids, organ‑on‑a‑chip devices and patient‑derived organoids that faithfully reproduce tumor heterogeneity, extracellular‑matrix barriers and immune‑cell interactions.
By modelling these complex cellular environments, scientists can gain deeper insights into drug behavior that traditional 2D cultures cannot provide. These 3D models – by recapitulating the tumor microenvironment – help refine candidate selection while reducing reliance on animal studies, all while providing human-relevant insights to better predict clinical outcomes.
The discussion that follows illustrates how Abzena is acting on the FDA’s vision by leveraging advanced in vitro platforms to model tumor complexity and generate decision‑quality data to move Antibody-drug conjugates (ADCs) and other complex biologics like Antibody-oligonucleotide conjugates (AOCs) and bi-multi-specifics toward the clinic with greater confidence.
Why Add Cellular Complexity?
Traditional 2D cell cultures can’t capture the full complexity of tumors. These simplified models can be useful tools in early development, where throughput is key, or later development for lot release assays, where precision and accuracy is of utmost importance. However, to truly understand the mechanism-of-action (MoA), and how a potential drug might perform in patients, three-dimensional models, co-cultures, and organoids offer a richer, more realistic setting to evaluate both biologics and bioconjugates. Here’s why:
- Heterogeneity tolerance: in real tumors, not all cells express the same antigen levels. Some cells have low or no antigen expression, posing a challenge for bioconjugates. A cleavable payload can help target these cells indirectly through bystander effects – essentially hitting cells that aren’t the primary target but are in close proximity to those that are.
- Matrix penetration: solid tumors often have dense extracellular matrices that impede drug diffusion. Assessing how well a bioconjugate can penetrate these barriers is crucial for its efficacy.
- Immune cooperation: the immune system can significantly enhance efficacy. Evaluating how well a bioconjugate recruits immune cells, like NK cells and macrophages, can provide a critical measure of its ability to harness immune-mediated killing. This is especially relevant for ADCs designed to leverage Fc-mediated functions, where immune cell recruitment can significantly impact overall efficacy.
Understanding these cellular complexities provides essential context for evaluating how distinct bioconjugate design parameters – such as payload type and linker stability – influence efficacy within tumor microenvironments.
Putting Kadcyla® and Enhertu® to the Test
An ADC is composed of a cytotoxic payload linked to an antibody. Their mechanism of action involves the ADC binding to a surface receptor on a cancer cell, internalization, and payload release to kill the cancer cells. However, this process is not as linear as it may seem. Factors resulting from the three-dimensional cancer cell environment such as bystander effect and the tumor microenvironment interactions add layers of complexity to ADC design and testing.
To illustrate how ADC design parameters influence therapeutic performance in complex tumor settings, Abzena conducted comparative studies on two HER2-targeting ADCs: Kadcyla® and Enhertu®.
- Kadcyla®: contains a non-cleavable linker and DM1 cytotoxic payload targeting tubulin polymerization to prevent cancer cells from dividing by disrupting their internal structure.
- Enhertu®: features a cleavable linker and a topoisomerase inhibitor (DXd) payload, allowing a bystander killing effect.
Abzena’s comparative studies on Kadcyla® and Enhertu® demonstrate how ADC design influences performance in complex tumor settings. While Kadcyla® showed cell killing due to its potent DM1 payload, Enhertu® leveraged its cleavable linker to induce bystander killing in mixed cell populations, extending its reach beyond HER2-positive targets (Figure 1). These findings correlate with what is seen in the clinic in heterogenous tumor indications and demonstrate the importance of considering both direct and bystander effects in ADC lead selection.
Integrating Immune Components in ADC Evaluation
One of the key advancements in complex cellular modelling is the integration of immune components. Adding primary Peripheral Blood Mononuclear Cells (PBMCs) into in vitro assays offers a deeper look at how ADCs leverage immune-mediated and payload-mediated killing.
To better predict clinical outcomes, researchers at Abzena have developed the following in vitro assays that integrate immune components to simulate complex cellular environments:
- Antibody-dependent cellular cytotoxicity (ADCC) assays via live cell imaging: these assays assess both immune-mediated and payload-mediated cell killing in real time. The use of the Incucyte® S3 Live-Cell Analysis System enables monitoring of cell death kinetics over time.
- Spheroid ADCC assays: 3D tumor spheroids (Figure 2) are used to more accurately model solid tumor microenvironments. These assays assess the impact of biologics and ADCs in dense cellular structures, revealing the extent of drug penetration and cytotoxicity.
- Bystander ADCC assays in co-cultures: co-culturing HER2-positive and HER2-negative cells in the presence and absence of PBMCs, to evaluate the bystander effect of ADCs in ‘hot’ and ‘cold’ immune environments.
Figure 2: Spheroid ADCC assay via live cell imaging. Visualization of 3D tumor cell killing demonstrates the impact of varying antibody/ADC concentrations and effector-to-target (E:T) ratios on efficacy. Enhertu®, Kadcyla® and Trastuzumab show distinct response profiles.
By building this extra level of cellular complexity by integrating immune components into in vitro assays, Abzena provides deeper insights into ADC mechanisms of action, guiding lead selection with data that more accurately predicts clinical success.
Extension to Organoids and Emerging Targets
As Abzena continues to expand its biologic and bioconjugate development capabilities, the focus is shifting to incorporate more complex and clinically relevant tumor models. The use of organoids and organoids mimicking tumors (“tumoroids”) offer a unique opportunity to assess ADC efficacy in a broader array of target profiles. Implementing a tumoroid platform allows for real-time imaging of payload diffusion into a model that is as close to a clinical mimic of a tumor as possible in vitro, with characteristics such as a necrotic core and cellular layers.
In addition, by looking at tumor cell viability in tumoroid models down to the single cell level, a better understanding of anti-cancer treatment can be achieved on these mixed population of cells. For example, monitoring viability of cancer stem cells, responsible for metastasis and relapse, provides invaluable insight into the potential performance of the biologic in patients.
By adopting these advanced methodologies, Abzena is not only enhancing its lead selection strategies but also identifying potential off-target effects and refining payload strategies early in drug design and development. As our customers extend the next phase of their research to evaluate ADCs against a broader array of targets such as TROP2 and CDH17, we are keen to support them with the best in vitro models during their development journey.
By leveraging the in vitro technologies and platforms discussed here, Abzena provides comprehensive insights into biologic and bioconjugate performance, addressing challenges like tumor heterogeneity, drug penetration, and immune interactions. Our integrated approach not only aligns with regulatory shifts towards reducing animal testing but also accelerates the journey from discovery to clinical application.
To learn more about how Abzena’s integrated in vitro platforms can accelerate your biologic or bioconjugate program and align with regulatory expectations, reach out to our team.
Read Part 1 of 4: Timing is Everything: Choosing the Right Bioassay at the Right Time for Successful Drug Development
