Quantitative Analysis of Membrane Components using Super-Resolution Microscopy

B cell maturation antigen (BCMA) is the primary target for chimeric antigen receptor (CAR)-T cell therapies and other immunotherapies in multiple myeloma (MM). Frequent relapses and the need for sequential treatments underscore the importance of alternative targets. We previously developed a dual-antigen-specific CAR recognizing both BCMA and transmembrane activator and CAML interactor (TACI). Here, we efficiently expressed this CAR in patient-derived marrow-infiltrating lymphocytes (MILs) to investigate MM cell killing via both endogenous T cell receptor (TCR) specificities and the CAR. While the patients' MIL TCRs did not recognize or kill autologous malignant plasma cells (PCs), the CAR consistently mediated efficient PC killing even at low or undetectable antigen levels by flow cytometry. Quantification of BCMA and TACI molecules on target cells by direct stochastic optical reconstruction microscopy (dSTORM) revealed that both BCMA and TACI were significantly underestimated by flow cytometry. Analysis by dSTORM allowed us to delineate the dual-antigen sensitivity of the CAR, indicating that CAR-MILs were polyfunctional and killed targets at very low antigen densities. Our findings support the further exploration of TACI as target in MM and underline the value of super-resolution microscopy in assessing antigen density thresholds for CAR-T cells.

Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.

Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T-cell therapies.

Development of a Regulated, Optimized Mesothelin Chimeric Antigen Receptor (CAR) for the Treatment of Mesothelin Positive Cancers

Chimeric antigen receptor (CAR) T-cell therapy holds great promise as cancer immunotherapy. While impressive responses have been achieved in patients with hematological malignancies there are challenges for CAR T therapy such as toxicity complications due to cytokine release syndrome (CRS) and limitations due to CAR T cell exhaustion. As increasing number of novel CAR T products are in the discovery and preclinical phase of development, preclinical models to assess efficacy as well as potential toxicity of CAR T in vivo are needed. Here, we describe a new NSG™ mice model to examine CAR T cells for efficacy and potential CRS. Immunodeficient NSG™ mice are commonly used to evaluate CAR T functions. However, human T cells can cause acute xeno-GVHD in NSG™ limiting the use of the model system. A system to study human T cell function in the absence of xeno-GVHD is required. A new NSG™ derivative NSG-MHC Class I/II knockout (KO) strain has MHC class I and MHC class II deficiency in addition to B, T and NK cell deficiency. NSG-MHC Class I/II KO mice are known to have a delayed onset of GVHD and can provide a useful background to study human immunity in the absence of GVHD [M. A. Brehm et al., FASEB J. 33, 3137–3151 (2019)]. Human B-cell lymphoma Raji tumor cells express high levels of CD19 and CD22 which are targets for CAR T therapy. Using NSG-MHC Class I/II KO mice and luciferase-tagged Raji-Luc tumor model we found that compared to controls CD22 CAR T and CD19 CAR T showed significant tumor growth inhibition by Xenogen imaging analysis. No body weight loss was observed with the CAR T treatments. As high tumor burdens are usually associated with the potential of causing CRS we examined CD19 CAR T cells on low and high tumor burdens and the data will be presented in context of efficacy. We have established a new mouse model using NSG-MHCI/II KO mice to enable evaluation of CAR T efficacy and propensity to induce cytokines simultaneously in vivo. Furthermore PBMC-humanized NSG-MHCI/II KO mice can also be established and used to examine CAR T cells prepared from the same PBMC donor to mimic autologous CAR T cell therapy. Citation Format: Jing Jiao, Chunting Ye, Danying Cai, Mingshan Cheng, James Keck. Mouse model for in vivo evaluation of efficacy and potential cytokine release syndrome of chimeric antigen receptor (CAR) T cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4512.

Cellular Dynamics Following CAR T Cell Therapy Are Associated with Response, Resistance and Cytokine Release Syndrome in Relapsed/Refractory Multiple Myeloma

A Novel Method for Molecular Enumeration of Circulating Tisa-Cel and Axi-Cel in Lymphoma Patients

Super-resolution fluorescence microscopy, also known as nanoscopy, has provided us with a glimpse of future impacts on cell biology. Far-field optical nanoscopy allows, for the first time, the study of sub-cellular nanoscale biological structures in living cells, which in the past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy has particular utility in the study of “fenestrations” – phospholipid transmembrane nanopores of 50–150 nm in diameter through liver sinusoidal endothelial cells (LSECs) that facilitate the passage of plasma, but (usually) not blood cells, to and from the surrounding hepatocytes. Previously, these fenestrations were only discernible with EM, but now they can be visualized in fixed and living cells using structured illumination microscopy (SIM) and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy. Importantly, both methods use wet samples, avoiding dehydration artifacts. The use of nanoscopy can be extended to the in vitro study of fenestration dynamics, to address questions such as the following: are they actually dynamic structures, and how do they respond to endogenous and exogenous agents? A logical further extension of these methodologies to liver research (including the liver endothelium) will be their application to liver tissue sections from animal models with different pathological manifestations and ultimately to patient biopsies. This review will cover the current state of the art of the use of nanoscopy in the study of liver endothelium and the liver in general. Potential future applications in cell biology and the clinical implications will be discussed.

Simple SummaryA major barrier to success for chimeric antigen receptor (CAR) T-cell therapies in solid tumors is selecting optimal target antigens. Target antigens should be uniformly expressed by cancer cells and minimally so by healthy tissues in the body. Analyzing single-cell RNA-sequencing data of patient tumors and of healthy tissue reference atlases, we first charted the landscape of existing solid tumor CAR targets, quantifying their tumor selectivity and safety to identify the leading clinical targets. We then compared these benchmark targets to the cell surfaceome, with the aim of systematically identifying new candidate genes that are safer and/or more selective than the best CAR targets in clinical trials, as determined by our measures. Our analysis uncovered twenty new cell surface targets, five of which have both superior selectivity and safety scores, which may serve as promising candidates for CAR treatments in HNSC.Chimeric antigen receptor (CAR) T cell therapies have yielded transformative clinical successes for patients with blood tumors, but their full potential remains to be unleashed against solid tumors. One challenge is finding selective targets, which we define intuitively to be cell surface proteins that are expressed widely by cancer cells but minimally by healthy cells in the tumor microenvironment and other normal tissues. Analyzing patient tumor single-cell transcriptomics data, we first defined and quantified selectivity and safety scores of existing CAR targets for indications in which they are in clinical trials or approved. We then sought new candidate cell surface CAR targets that have better selectivity and safety scores than those currently being tested. Remarkably, in almost all cancer types, we could not find such better targets, testifying to the near optimality of the current target space. However, in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSC), for which there is currently a dearth of existing CAR targets, we identified a total of twenty candidate novel CAR targets, five of which have both superior selectivity and safety scores. These newly identified cell surface targets lay a basis for future investigations that may lead to better CAR treatments in HNSC.

152 Background: Glioblastoma (GBM) is a deadly cancer, with a 5-year survival rate of less than 10%. T cells can be redirected to kill cancer cells using chimeric antigen receptors (CAR), a promising method to treat solid tumors. IL13Rα2 is expressed in many solid tumors but not normal tissues, and provides a target for CAR T cells as a powerful immunotherapy treatment. Methods: Human and canine tumors were screened for IL13Rα2 expression by flow cytometry and RT-PCR. Different IL13Rα2 single-chain variable fragments (scFv) were tested in CARs in vitro against human and canine recombinant IL13Rα2 protein to identify a cross-reactive clone. IL13Rα2 CARs expressed in human and canine T cells and showed antigen-specific stimulation by cytokine secretion and target cell lysis. Solid tumors were used to establish in vivo subcutaneous and orthotopic xenograft models in NSG mice. Mice received a single treatment of 2-5 million CAR T cells IV with or without anti PD-1, CTLA-4 or TIM3 mAb checkpoint blockade. Results: IL13Rα2 was detected on three human GBM (D270, U251 and U87), four canine osteosarcomas (BW-, CS-, MC- and SK-KOSA) and two canine lung cancer cell lines (Cacal3, Cacal5). Two different IL13Rα2 CAR were generated that recognized human IL13Rα2 and not IL13Rα1, one also recognized canine IL13Rα2. Five million IL13Rα2 CAR T-cells delivered IV were able to eliminate established GBM tumors. Use of fewer CAR T cells initially controlled tumor growth, followed by tumor outgrowth correlated with expression of T cell exhaustion markers. Combined treatment with CARs plus checkpoint blockade mAb delivered IP had synergistic effects, restoring anergic T-cell function and improving tumor treatment. Conclusions: IL13Rα2 is highly expressed on human and canine tumors, but not normal tissues. IL13Rα2 specific CAR T cells successfully respond to human and canine tumors and treat GBM in a xenograft mouse model, although this effect was transient at lower treatment doses. Addition of checkpoint blockade mAbs to CAR therapy was beneficial for the treatment of solid tumors. We plan to utilize this treatment in a canine spontaneous cancer preclinical model and translate into clinical trials for patients with GBM.

Mesothelin Targeting IL12-Engineered CAR Memory-like NK Cells Demonstrate Promising Efficacy in Acute Myeloid Leukemia

The „Resolution Revolution in fluorescence microscopy over the last decade has given rise to a variety of techniques that allow imaging beyond the diffraction limit with a resolution power down into the nanometer range. With this, the field of so-called super-resolution microscopy was born. It allows to visualize cellular architecture at a molecular level and thereby achieve a resolution level that had been previously only accessible by electron microscopy approaches. One of these promising techniques is single molecule localization microscopy (SMLM) in its most varied forms such as direct stochastic optical reconstruction microscopy (dSTORM) which are based on the temporal separation of the emission of individual fluorophores. Localization analysis of the subsequently taken images of single emitters eventually allows to reconstruct an image containing super-resolution information down to typically 20 nm in a cellular setting. The key point here is the localization precision, which mainly depends on the image contrast generated the by the individual fluorophore’s emission. Thus, measures to enhance the signal intensity or reduce the signal background allow to increase the image resolution achieved by dSTORM. In my thesis, this is achieved by simply adding a reflective metal-dielectric nano-coating to the microscopy coverslip that serves as a tunable nano-mirror. I have demonstrated that such metal-dielectric coatings provide higher photon yield at lower background and thus substantially improve SMLM performance by a significantly increased localization precision, and thus ultimately higher image resolution. The strength of this approach is that ─ except for the coated cover glass ─ no specialized setup is required. The biocompatible metal-dielectric nano-coatings are fabricated directly on microscopy coverslips and have a simple three-ply design permitting straightforward implementation into a conventional fluorescence microscope. The introduced improved lateral resolution with such mirror-enhanced STORM (meSTORM) not only allows to exceed Widefield and Total Internal Reflection Fluorescence (TIRF) dSTORM performance, but also offers the possibility to measure in a simplified setup as it does not require a special TIRF objective lens. The resolution improvement achieved with meSTORM is both spectrally and spatially tunable and thus allows for dual-color approaches on the one hand, and selectively highlighting region above the cover glass on the other hand, as demonstrated here. Beyond lateral resolution enhancement, the clear-cut profile of the highlighted region provides additional access to the axial dimension. As shown in my thesis, this allows for example to assess the three-dimensional architecture of the intracellular microtubule network by translating the local localization uncertainty to a relative axial position. Even beyond meSTORM, a wide range of membrane or surface imaging applications may benefit from the selective highlighting and fluorescence enhancing provided by the metal-dielectric nano-coatings. This includes for example, among others, live-cell Fluorescence Correlation Spectroscopy and Fluorescence Resonance Energy Transfer studies as recently demonstrated.

H3K27M-mutant diffuse midline gliomas (DMG) are aggressive pediatric brain tumors with dismal prognoses and no effective treatments. GD2, a disialoganglioside overexpressed on DMG cells, is a promising target for chimeric antigen receptor (CAR) T cell therapy. In Arm A of the Phase I clinical trial (NCT04196413), 12 patients (11 on study) received GD2-CAR T cells through an initial intravenous (IV) infusion, and if patients had clinical or radiographic improvement, they subsequently received intracerebroventricular (ICV) administrations. This regimen achieved clinical benefit in ten patients, with >50% tumor volumetric reductions in 5 patients and a durable ongoing complete response in one patient (Majzner, Ramakrishna et al. Nature 2022; Monje et al. Nature 2024). However, disease progression ultimately occurred in 11 patients underscoring challenges in therapeutic optimization. To elucidate mechanisms underlying treatment responses and resistance, we performed a comprehensive multi-omic analysis, integrating single-cell RNA and TCR sequencing, mass cytometry, and clinical and molecular datasets. Sequencing 1, 062, 398 cells from patient apheresis, CAR T products, and cerebrospinal fluid (CSF) samples collected longitudinally at pre-defined trigger events, we reconstructed the dynamic landscape of immune and tumor cell populations in 12 DMG patients. Our analysis revealed profound CAR T cell infiltration into the CSF after treatment, accompanied by increases in endogenous T cell, mast cell, and monocyte populations and a relative decrease in microglia evident in CSF. This works sought to deeply characterize the lymphocyte profiles in these samples. Inflammatory and proliferation pathways were elevated in CAR T cells at early post-ICV time points, transitioning into an NK-like state and TGF-β receptor signaling at later stages. TCR clonal tracking identified persistent CAR T clones, alongside endogenous T cell activation and expansion. Moreover, non-responders to the therapy showed elevated Treg signatures in the CSF. Using CellChat-based cell-cell communication analysis, we uncovered differential cytokine-receptor interactions in responders versus non-responders, offering novel insights into immune-tumor interactions in the context of CAR T cell therapy. By contextualizing single-cell data within multi-omic frameworks and clinical outcomes, this study advances our understanding of the GD2-CAR T cell therapy, the tumor microenvironment, and biomarkers of response and resistance. These findings provide critical insights for optimizing CAR T cell therapies in CNS malignancies and inform the development of next-generation immunotherapies. Citation Format: Yiyun Chen, Moksha H. Desai, Zachary J. Ehlinger, Kun Wei Song, Skyler Rietberg, Hossein Daghagh, Mark P. Hamilton, Snehit Prabhu, Sonia Partap, Jasia Mahdi, Elena Sotillo, Bita Sahaf, Sneha Ramakrishna, Zinaida Good, Michelle Monje, Crystal Mackall. Unravelling temporal dynamics of GD2-CAR T cell therapy in H3K27M-mutant diffuse midline glioma through comprehensive single-cell and multi-omic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7123.

Immunotherapy with Chimeric Antigen Receptor Targeting Intracellular WT1 Gene Product Complexed with HLA-a*24:02 Molecule

Improving Anti-BCMA CAR-T Functionality with Novel Celmods in Multiple Myeloma