Learn how patient-derived organoid co-cultures with T cells can form a powerful preclinical platform to evaluate the efficacy, specificity, and mechanism of action of bispecific antibodies

THE CHALLENGE

THE CHALLENGE

The client, developing bispecific T cell engager (BiTE) for solid tumor indications, required a preclinical system capable of accurately assessing both efficacy and safety of their lead candidate. Conventional 2D models lacked the complexity needed to evaluate tumor-selective killing and immune engagement within a human-relevant microenvironment. Specifically, they needed a translational in vitro immunotherapy model that could preserve tumor heterogeneity, reflect patient-specific antigen expression, and support functional co-culture with T cells to de-risk downstream in vivo studies and guide candidate optimization.

OUR SOLUTION

OUR SOLUTION

We applied our PDO–T cell co-culture platform to model antigen-specific immune engagement and cytotoxicity. Using matched tumor and normal NSCLC patient-derived organoids, we established co-cultures with primary T cells to assess the bispecific antibody’s ability to induce selective killing and activate T cells. The system enabled head-to-head comparison of on-target efficacy and potential off-tumor toxicity, with quantitative functional readouts – providing the client with translationally predictive data ahead of in vivo studies.

EXPERIMENTAL APPROACH

We designed an experiment with 3 key assays:

  • Target Antigen Profiling: Flow cytometry was used to confirm TAA expression across tumor and matched normal PDOs.
  • Cytotoxicity Assay: Cell death in tumor and normal PDOs was measured following treatment with the bispecific antibody in the presence or absence of T cells.
  • T Cell Activation: IFN-γ secretion was quantified as a readout of T cell activation upon engagement with the bispecific antibody.

Here’s what our clients have to say

We were really impressed with the insights we gained from the PDO–T cell co-culture platform. It clearly demonstrated how well our bispecific antibody was working. This has been a major boost for our team, allowing us to move forward with development much faster and with greater certainty.

drug development organoids

KEY RESULTS

Selection of organoid models

Leveraging our large living biobank, we suggested testing the expression of the tumor-associated antigen (TAA) in NSCLC tumor organoids and matched normal lung organoids. Using immunohistochemistry and flow cytometry data, we supported our client in selecting the most relevant organoid models.

Immunohistochemistry and flow cytometry of NSCLC organoid models

Fig1: A) Representative images of IHC staining for the TAA in human tumor tissue and matched normal epithelium. B) Flow cytometry analysis shows expression levels of TAA in 3 pairs of matched normal and tumor patient-derived organoids

Target Antigen Profiling

Flow cytometry confirmed expression of the TAA in both tumor and normal PDOs, with apparent enrichment in NSCLC tumor organoids. This ensured both specificity and the relevance of the chosen models for subsequent efficacy and toxicity testing.

Flow cytometry showing tumor associated target antigen expression
Fig2: Flow cytometry analysis of TAA in NSCLC PDO and matched normal lung PDO

T Cell–Dependent Cytotoxicity

The bispecific antibody induced significant killing of tumor PDOs when co-cultured with T cells. Normal PDOs were largely spared under identical conditions. No cell death occurred in the absence of T cells or when using a non-targeting BiTE, confirming both antigen specificity and T cell dependence.

Graphs showing T-cell mediated cytotoxicity by BiTE

Fig3: Day 2 quantification of cell death fluorescent signal in A) tumor and B) normal PDOs when exposed to a BiTE directed to a tumor-specific antigen. For organoid killing, staurosporine (STS) is used as positive control and medium only as negative control. T cell Trans activator is included as a control for T-cell activation (TnsA). In absence of T- cells, the tested BiTE (TnsA and nonspecific ab) is not able to induce PDO cell death.

IFN-γ Secretion & Immune Activation

IFN-γ secretion was significantly elevated in co-cultures treated with the targeted BiTE, indicating effective activation of T cells in response to TAA recognition on tumor PDOs.

Graph measuring IFN-γ Secretion in non-small cell lung cancer and normal organoids
Fig4: IFN-γ levels secreted by T cells in co-culture with PDOs and in presence of targeted BiTE and a non-specific BiTE as control. Transactivator (TnsA) was included as positive control to induce IFN-γ secretion.

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Conclusion

This study demonstrated that the bispecific antibody elicited antigen-specific, T cell–dependent killing of NSCLC PDOs, with minimal off-tumor effects on matched normal organoids. Elevated IFN-γ secretion confirmed functional T cell engagement. The PDO–T cell co-culture system provided mechanistic insight into the bispecific’s efficacy, selectivity, and mode of action, supporting its progression toward in vivo validation. For bispecific antibody developers, this platform offers a translationally relevant, human-derived system to de-risk immunotherapeutic candidates early in development by modeling tumor–immune dynamics and off-tumor toxicity in vitro.

References

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The Life Science business of Merck KGaA, Darmstadt, Germany Acquires HUB Organoids Holding B.V., Expands Next-Gen Biology Portfolio

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