Molecular imaging of CAR-T cell therapy

What are CAR-T cells?

Chimeric antigen receptor (CAR)-T cell therapy is a promising type of immunotherapy for patients with certain types of cancer. The backbone of CAR-T cell therapy is – obviously – the T-cells. These are part of the immune system and can directly kill cells that are infected by pathogens.

In general, you obtain CAR-T cells from the patient and re-engineer them in a lab to produce proteins on their surface. These proteins are called chimeric antigen receptors (CARs). CARs recognize and bind to specific proteins or antigens on the surface of cancer cells. If the CAR-T cells are infused into the patient, they will, with guidance from the engineered receptor, recognize and kill cancer cells that harbor the target antigen on their surfaces.¹

Study CAR-T cell migration and expansion.

Despite the enormous potential of CAR-T cell therapy it also has its limitations. These include, for example, lower rates of durable response related to inadequate CAR-T cell expansion and trafficking to tumor sites or lack of objective responses in solid tumors.²

If you image and track CAR-T cells in time in patients, you can establish in vivo characterization of T-cell expansion and trafficking to tumor sites. Therefore, imaging of CAR-T cells could help your CAR-T cell therapy development by studying its migration and expansion and predicting response measures in individual patients. You can choose from several molecular imaging modalities to track your CAR-T cell therapy in humans.

How can you image CAR-T cells in human?

Recently, there has been a strong interest in imaging both T-cells and T-cell activation states. However, these approaches target all T-cells. This makes it difficult for you to use them exclusively for CAR-T cells.³

Another option is to perform direct labeling techniques with for example ⁸⁹Zr-oxine, ¹¹¹In-oxine or ^99mTc-HMPAO for shorter-term T cell trafficking. Using this technique, you can image the injected CAR-T cells and study their migration. However, when the CAR-T cells divide/expand, they do not transfer the imaging label. Therefore, expansion of CAR-T cells goes unnoticed using direct labeling techniques. So, the measured signal reflects the number of injected cells and does not account for cell division and expansion. For example, a small amount of the injected cells may proliferate and largely cause the antitumor response. In this case direct radiolabeling with a radionuclide would only detect the preliminary initial migration and not the expansion. Moreover, it does not generate information whether the engineered gene construct is active, and thus, is not a functional imaging methodology. This methodology only shows you the biodistribution and pharmacokinetics of CAR-T cells.⁴

Labeling CAR-T cells with nanoparticles for MRI.

Another option is to label CAR-T cells with iron or fluorine-19 (¹⁹F) nanoparticles. These enable noninvasive detection of your labeled T-cells with magnetic resonance imaging (MRI).^{5 6} This gives you data on the trafficking of the engineered CAR-T cells (pharmacokinetics (PK)/biodistribution (BD) data). As this is also a direct labeling method, it does not show cell expansion or functionality. Similar to direct labeling with a radionuclide.

Imaging CAR-T cells with reporter genes.

Instead, you can modify your CAR-T cells with reporter genes. This would account for proliferation after injection, as the reporter gene is transferred to the daughter cells. Several groups have developed and investigated strategies to monitor in vivo CAR-T cell trafficking using radionuclide-based imaging (PET or SPECT) with reporter genes.

Reporter genes offer you a genetically linked, nondisruptive way to facilitate specific targeting of PET probes to CAR-T cells. With this approach you can quantify a single-time-point in-tumor invasion while at the same time conduct longitudinal whole-body tracking of CAR-T cells over their entire lifetime and functionality. In short, it lets you monitor the presence of the fused gene activation over a long period of time.

When you select a reporter gene for imaging, you should select a target that is not expressed in healthy tissues. When the target is also expressed elsewhere in the human body, this compromises the detection of the CAR-T cells. You should also keep in mind that if you add another reporter gene to your CAR-T cells it requires an additional ex vivo transfection. This may potentially cause immunogenicity.

The most used PET reporter gene is herpes simplex virus 1 thymidine kinase. Also referred to as HSV1-tk. This reporter gene is naturally not expressed in humans and therefore does not affect the imaging specificity. Many of the enzyme substrates have been labeled with F-18 or I-124 to image HSV1-tk gene expression.^{7 8} Two major substrates for HSV1-tk enzyme/PET include 9-[4-¹⁸F-fluoro-3-(hydroxymethyl)butyl-guanine ([¹⁸F]-FHBG) and ¹²⁴I- or ¹⁸F-labeled 29-fluoro-29-deoxy-59-iodo-1-b-Darabinofuranosyluracil ([¹²⁴I]-FIAU or [¹⁸F]-FIAU).

In summary, imaging of CAR-T cells has broad applications for evaluating and improving your CAR-T cell therapies in vivo. It provides you with early insights into the PK/BD and infiltration/activation at the tumor site.

References

1. CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers
2. Sakemura, R., Can, I., Siegler, E. L., & Kenderian, S. S. (2021). In vivo cart cell imaging: Paving the way for success in CART cell therapy. Molecular Therapy – Oncolytics, 20, 625–633. https://doi.org/10.1016/j.omto.2021.03.003
3. Larimer, B. M. (2018). Reporter genes for PET imaging of car T cells offers insight into adoptive cell transfer. Journal of Nuclear Medicine, 59(12), 1892–1893. https://doi.org/10.2967/jnumed.118.220897
4. Weist, M. R., Starr, R., Aguilar, B., Chea, J., Miles, J. K., Poku, E., Gerdts, E., Yang, X., Priceman, S. J., Forman, S. J., Colcher, D., Brown, C. E., & Shively, J. E. (2018). Pet of adoptively transferred chimeric antigen receptor T cells with 89zr-oxine. Journal of Nuclear Medicine, 59(10), 1531–1537. https://doi.org/10.2967/jnumed.117.206714
5.  Kiru, L., Zlitni, A., Tousley, A. M., Dalton, G. N., Wu, W., Lafortune, F., Liu, A., Cunanan, K. M., Nejadnik, H., Sulchek, T., Moseley, M. E., Majzner, R. G., & Daldrup-Link, H. E. (2022). In vivo imaging of nanoparticle-labeled Car T cells. Proceedings of the National Academy of Sciences, 119(6). https://doi.org/10.1073/pnas.2102363119 
6. Dubois, V. P., Sehl, O. C., Foster, P. J., & Ronald, J. A. (2021). Visualizing car-T cell immunotherapy using 3 Tesla fluorine-19 MRI. Molecular Imaging and Biology, 24(2), 298–308. https://doi.org/10.1007/s11307-021-01672-3
7. Keu, K. V., Witney, T. H., Yaghoubi, S., Rosenberg, J., Kurien, A., Magnusson, R., Williams, J., Habte, F., Wagner, J. R., Forman, S., Brown, C., Allen-Auerbach, M., Czernin, J., Tang, W., Jensen, M. C., Badie, B., & Gambhir, S. S. (2017). Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma. Science Translational Medicine, 9(373). https://doi.org/10.1126/scitranslmed.aag2196
8. Moroz, M. A., Zhang, H., Lee, J., Moroz, E., Zurita, J., Shenker, L., Serganova, I., Blasberg, R., & Ponomarev, V. (2015). Comparative analysis of T cell imaging with human nuclear reporter genes. Journal of Nuclear Medicine, 56(7), 1055–1060. https://doi.org/10.2967/jnumed.115.159855