JSL is the Director of the MSK Radiochemistry and Molecular Imaging Probe Core that provides contract, development and manufacturing services for ImaginAb. N.P. is/has served as a consultant/advisory board and has received honoraria for Actinium Pharma, Progenics, Medimmune/ Astrazeneca, Illumina, Imaginab, and conducts research supported by Ymabs, Imaginab, BMS, Bayer, Clarity pharma, Janssen and Regeneron.

Antibody-based PET (immunoPET) with radiotracers that recognize specific cells of the immune system provides an opportunity to monitor immune cell trafficking at the organismal scale. We previously reported the visualization of human CD8+ T cells, including CD8+ tumor-infiltrating lymphocytes (TIL), in mice using a humanized CD8-targeted minibody. Given the important role of CD4+ T cells in adaptive immune responses of health and disease including infections, tumors, and autoimmunity, we explored immunoPET using an anti-human-CD4 minibody. We assessed the ability of [64Cu]Cu-NOTA-IAB41 to bind to various CD4+ T-cell subsets in vitro. We also determined the effect of the CD4-targeted minibody on CD4+ T-cell abundance, proliferation, and activation state in vitro. We subsequently evaluated the ability of the radiotracer to visualize CD4+ T cells in T-cell rich organs and orthotopic brain tumors in vivo. For the latter, we injected the [64Cu]Cu-NOTA-IAB41 radiotracer into humanized mice that harbored intracranial patient-derived glioblastoma (GBM) xenografts and performed in vivo PET, ex vivo autoradiography, and anti-CD4 IHC on serial brain sections. [64Cu]Cu-NOTA-IAB41 specifically detects human CD4+ T cells without impacting their abundance, proliferation, and activation. In humanized mice, [64Cu]Cu-NOTA-IAB41 can visualize various peripheral tissues in addition to orthotopically implanted GBM tumors. [64Cu]Cu-NOTA-IAB41 is able to visualize human CD4+ T cells in humanized mice and can provide noninvasive quantification of CD4+ T-cell distribution on the organismal scale.

Liquid biopsy has emerged as a novel noninvasive tool in cancer diagnostics. While significant strides have been made in other malignancies using liquid biopsy for diagnosis, disease monitoring, and treatment selection, development of these assays has been more challenging for brain tumors. Recently, research in primary and metastatic brain tumors has begun to harness the potential utility of liquid biopsy—particularly using circulating tumor DNA (ctDNA). Initial studies to identify ctDNA in plasma of brain tumor patients have shown feasibility, but the yield of ctDNA is far below that for other malignancies. Attention has therefore turned to the cerebrospinal fluid (CSF) as a more robust source of ctDNA. This review discusses the unique considerations in liquid biopsy for glioma and places them in the context of the work to date. We address the utility of CSF liquid biopsy for diagnosis, longitudinal monitoring, tracking tumor evolution, clinical trial eligibility, and prognostication. We discuss the differences in assay requirements for each clinical application to best optimize factors such as efficacy, cost, and speed. Ultimately, CSF liquid biopsy has the potential to transform how we manage primary brain tumor patients.

Supplementary Data from Noninvasive Imaging of CD4⁺ T Cells in Humanized Mice

<div>AbstractPurpose:<p>Glioblastoma (GBM) is the most common malignant brain tumor in adults. Various immunotherapeutic approaches to improve patient survival are being developed, but the molecular mechanisms of immunotherapy resistance are currently unknown. Here, we explored the ability of a humanized radiolabeled CD8-targeted minibody to noninvasively quantify tumor-infiltrating CD8-positive (CD8⁺) T cells using PET.</p>Experimental Design:<p>We generated a peripheral blood mononuclear cell (PBMC) humanized immune system (HIS) mouse model and quantified the absolute number of CD8⁺ T cells by flow cytometry relative to the [⁶⁴Cu]Cu-NOTA-anti-CD8 PET signal. To evaluate a patient-derived orthotopic GBM HIS model, we intracranially injected cells into NOG mice, humanized cohorts with multiple HLA-matched PBMC donors, and quantified CD8⁺ tumor-infiltrating lymphocytes by IHC. To determine whether [⁶⁴Cu]Cu-NOTA-anti-CD8 images brain parenchymal T-cell infiltrate in GBM tumors, we performed PET and autoradiography and subsequently stained serial sections of brain tumor tissue by IHC for CD8⁺ T cells.</p>Results:<p>Nontumor-bearing NOG mice injected with human PBMCs showed prominent [⁶⁴Cu]Cu-NOTA-anti-CD8 uptake in the spleen and minimal radiotracer localization to the normal brain. NOG mice harboring intracranial human GBMs yielded high-resolution PET images of tumor-infiltrating CD8⁺ T cells. Radiotracer retention correlated with CD8⁺ T-cell numbers in spleen and tumor tissue. Our study demonstrates the ability of [⁶⁴Cu]Cu-NOTA-anti-CD8 PET to quantify peripheral and tumor-infiltrating CD8⁺ T cells in brain tumors.</p>Conclusions:<p>Human CD8⁺ T cells infiltrate an orthotopic GBM in a donor-dependent manner. Furthermore, [⁶⁴Cu]Cu-NOTA-anti-CD8 quantitatively images both peripheral and brain parenchymal human CD8⁺ T cells.</p></div>