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Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells 1 , 2 . Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity 3 – 5 ; however, a truly safe and effective logic-gated CAR has remained elusive 6 . Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity 7 and fibrosis 8 . In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering. Logic gating is used to develop a CAR T cell platform that is highly specific and allows the activity of T cells to be restricted to the encounter of two antigens, thus reducing on-target, off-tumour toxicity.

The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI’s performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform. Content-aware frame interpolation (CAFI) improves the temporal resolution in time-lapse imaging by accurately predicting images in between image pairs. By allowing fewer frames to be imaged, CAFI also enables gentler live-cell imaging.

BackgroundFDA-approved monoclonal antibodies (mAbs) that recognize the ganglioside GD2 (glycolipid) are used in the treatment of high-risk neuroblastoma (NBL). Despite successful integration of anti-GD2 mAbs into upfront treatment protocols, 40–50% of patients relapse, often with poor outcomes. We previously identified that downregulation of the sialyltransferase ST8SIA1 reduces GD2 levels and renders resistance to anti-GD2 therapy. Interestingly, we discovered that the loss of GD2 synthesis as a result of ST8SIA1 downregulation, is accompanied by a compensatory increase in GM2, another ganglioside. We have engineered anti-GM2 chimeric antigen receptors (CAR) and mAbs that recognize this antigen on NBL cells. Our overall hypothesis is that GD2 downregulation in NBL can be overcome by co-targeting GD2 and GM2.MethodsNSG mice were injected with GD2-high NBL cells and treated with anti-GD2 mAbs or CAR-T cells 7 days after tumor inoculation. Tumors were harvested at endpoint and analyzed by flow cytometry for ganglioside levels.We developed GM2-specific CAR T-cells and mAbs and tested them in vitro and in vivo.GM2-specific CAR T-cells were generated from primary human T cells by retroviral gene delivery. For in vivo experiments, NSG mice were injected with NBL cell lines and treated with CAR T-cells or mAbs 4–6 days after tumor inoculation. After meeting endpoint criteria, mice were euthanized and tumors were analyzed for ganglioside levels.ResultsTumors from mice treated with anti-GD2 therapy (mAb and CARs) showed reduced GD2 levels and increased GM2 at end point.GM2-specific CAR T-cells showed robust in vitro activity (cytokine production and cytotoxicity) and a durable response in vivo with no signs of toxicity. Similar to our previous findings, regarding changes in ganglioside levels after treatment, tumors from mice treated with GM2-CAR T-cells, showed a reduction in GM2 and an increase in GD2 levels.Anti-GM2 mAbs, alone or in combination with anti-GD2 mAbs, were well tolerated by mice, with no evidence of pain or weight loss. Dual administration of anti-GD2 and anti-GM2 mAbs, showed higher efficacy and better survival than targeting each ganglioside individually with mAbs.ConclusionsGM2 specific CAR T-cells and mAbs showed robust activity in vitro and in vivo, against multiple NBL lines, suggesting targeting GM2 could overcome anti-GD2 resistance in NBL.Ethics ApprovalVERIFICATION OF INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) APPROVALDate: April 18, 2023To: Robbie Majzner, Pediatrics - Hematology/Oncology Peng Xu, Cynthia A Klein, Alyssa RayAssurance Number: A3213–01Approval Period: 04/18/2023 THROUGH 01/23/2026Protocol ID: 33698The IACUC approved this protocol transaction on 04/18/2023. Prior to initiation of animal studies, if this study involves biohazardous or radioactive agents, you must obtain Biosafety Panel or Radiological Safety Panel approval. The expiration date of this approval is 01/23/2026 at Midnight. If this project is to continue past that date, you must submit an updated protocol (renewal) in advance for IACUC re-approval. Proposed changes to approved research must be reviewed and approved prospectively by the IACUC. No changes may be initiated without prior approval by the IACUC, except where deemed necessary by veterinary staff. (Any such exceptions must be reported to the IACUC within 10 working days).

While CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity3–7; however, a truly safe and effective logic-gated CAR has remained elusive8. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3ζ domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumors in vivo while bypassing upstream signaling proteins such as CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineer Logic-gated Intracellular NetworK (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity9 and fibrosis10. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering.