New CD4-specific minibodies to predict cancer treatment outcomes

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‍Pezzana et al. In-depth cross-validation of human and mouse CD4-specific minibodies for noninvasive PET imaging of CD4+ cells and response prediction to cancer immunotherapy, Theranostics, 2024

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New CD4-targeting minibodies were developed for live positron emission tomography (PET) imaging of the dynamics of CD4⁺ cells. This tool helps in predicting the outcome of cancer treatments and understanding the CD4⁺ T-cell antitumoral responses triggered by immunotherapy. The specificity of these minibodies to human CD4⁺ cells was tested by employing the humanized CD4 mouse model developed by genOway. 

While several cancer therapies are now available, such as T-cell therapies, T-cell engagers, and immune checkpoint inhibitors (ICIs), they exhibit varying response rates and a lack of reliable predictive biomarkers^1,2. Furthermore, it is now known that beyond the role of cytotoxic CD8⁺ T cells in the anti-tumoral response, CD4⁺ T cells also play a critical role in developing and sustaining effective anti-tumor immunity^3,4. As such, Pezzana and colleagues aimed to use CD4⁺ T cells as a biomarker to assess the efficacy of cancer therapy and understand the contribution of these cells to the antitumoral response⁵.

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The CD4 minibodies are specific and biologically inert

The authors started by validating the specific binding of their ⁸⁹Zr-radiolabelled minibodies in vitro. No cross-reactivity was observed between the human minibody and mouse CD4⁺ T cells and vice-versa, confirming their specificity. Importantly, the human CD4 minibody did not induce the activation and proliferation of PBMCs, showing that the construct is biologically inert and does not affect the CD4⁺ cells, as also previously shown⁶.

To analyze the specific binding of the human CD4 minibody in vivo, Pezzana and colleagues employed immunodeficient mice implanted with human tumors that were positive (HPB-ALL leukemia) or negative (HDL B-cell lymphoma) for CD4 expression. A much higher tracer uptake was observed in the tumors of mice implanted with the CD4⁺ HBP-ALL tumor, confirming the specificity of the human CD4 minibody tracer. 

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Monitoring the dynamics of CD4⁺ T cells by PET imaging

Given the importance of CD4⁺ T cells in antitumoral immunity, the whole-body dynamics of human CD4⁺ cells upon tumor implantation and tumor treatment were analyzed. The authors employed an immunocompetent mouse model with humanized CD4 (hCD4-KI) developed by genOway. Wild-type (WT) and hCD4-KI mice were implanted with mammary PyMT tumors and then subjected to combined αPD-1 and α4-1BB immunotherapy^7,8 to increase the T-cell density in tumors. Analysis of tracer distribution and uptake revealed a higher signal accumulation in the lymphatic organs, in a species-specific manner. Importantly, increased signal was also detected in the tumor microenvironment (TME) of hCD4-KI mice compared to WT mice (see figure below), demonstrating that these tracers are a useful tool in understanding the spatial dynamics of CD4⁺ cell populations.

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Figure 4 in the paper – Non-invasive visualization of tumor-infiltrating endogenous CD4⁺ cells in hCD4-KI and WT PyMT mammary tumor-bearing mice. Representative in vivo PET/MR images of PyMT mammary tumors acquired 48h post-⁸⁹Zr-CD4-Mbs i.v. injection (day 4 post-αPD-1/α4-1BB mAb injection). Tumors are highlighted by white circles. Figure adapted from Pezzana et al., 2024⁵.

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hCD4 minibodies for predicting the outcome of immunotherapies

The sensitivity of the mouse minibody for detecting changes in the density of CD4⁺ T cells in the TME and for predicting the outcome of immunotherapies was then evaluated. WT mice bearing murine colorectal carcinomas were treated with combined αPD-L1/αLag-3 monoclonal antibodies. Flow cytometry analysis revealed that some mice responded to treatment (responders) while others did not (non-responders). Treatment resulted in an increase in signal in the TME of responders compared to non-responders, which was also higher than in untreated mice, demonstrating that this tracer can detect changes in CD4⁺ T-cell density. Immunofluorescence analysis confirmed enhanced CD4⁺ T-cell infiltration in responding tumors, highlighting the tracer's potential for monitoring therapy effectiveness.

Altogether, the data shows that the combination of minibodies with non-invasive PET imaging can be a powerful tool to closely follow the response and progression of treatment in cancer patients. Importantly, it was shown that using this tool could help identify patients who do not respond to treatment, aiding to avoid potentially fatal outcomes. Additionally, this technology helps to understand the roles of CD4⁺ T cells in the context of the tumor microenvironment. As shown in this study, the hCD4 mouse model developed by genOway facilitates understanding the dynamics of CD4⁺ T-cell recruitment, while it is also a useful model to evaluate the efficacy and safety of hCD4 targeting compounds.

A hCD4 mouse model is available off the shelf at genOway, designer and provider of multiple preclinical models in several research areas, including immuno-oncology, metabolism, cardiovascular diseases and neuroscience.

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References

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4. Tay, R. E., Richardson, E. K. & Toh, H. C. Revisiting the role of CD4+ T cells in cancer immunotherapy—new insights into old paradigms. Cancer Gene Ther. 28, 5–17 (2021).
5. Pezzana, S. et al. In-depth cross-validation of human and mouse CD4-specific minibodies for noninvasive PET imaging of CD4+ cells and response prediction to cancer immunotherapy. Theranostics 14, 4582–4597 (2024).
6. Nagle, V. L. et al. Noninvasive Imaging of CD4+ T Cells in Humanized Mice. Mol. Cancer Ther. 21, 658–666 (2022).
7. Wang, Y. et al. Targeting 4-1BB and PD-L1 induces potent and durable antitumor immunity in B-cell lymphoma. Front. Immunol. 13, 1004475 (2022).
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