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Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V H H nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1 ) associated with high T-cell polyfunctionality. This study demonstrates the potential of V H H-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes. Glypican-1 (GPC1) expression is elevated in pancreatic cancer and has been exploited as a therapeutic target. Here the authors report the development of V H H nanobody-based CAR-T cells targeting GPC1, showing anti-tumor activity in pancreatic cancer preclinical models.

For families and surrogates of patients suffering from COVID-19, the pandemic has made nearly impossible the ability to see their loved ones in the hospital day-to-day. Families and surrogates may now be more hesitant to voice their preferences and concerns, or mistrust of the medical profession on the part of members of the public that may simmer under the surface under ordinary conditions, may be quicker than usual to boil over. Both extremes may compromise the spirit of shared decisionmaking in medicine. 1 Hence, with families absent, providers may find it necessary to adopt a more clinician-directed approach to decision-making. Once a COVID-19 positive patient enters a hospital, and viral spread mitigation policies separate patients and families, it is critically important that physicians be skilled at having end-of-life conversations with families that do not insist that they make decisions to shift to comfort measures only. Skill in these difficult conversations at the highest level is demonstrated when physicians bring families along gradually.

To describe the development of a virtual reality (VR) treatment for phantom limb pain (PLP) and phantom sensations and provide feasibility data from testing the treatment in a population of veterans. Fourteen participants completed a baseline visit evaluating their amputation, PLP, and phantom sensations. Subsequently, participants completed a VR treatment modeled after mirror therapy for PLP, navigating in a VR environment with a bicycle pedaler and motion sensor to pair their cadence to a VR avatar. The VR avatar enabled visualization of the participant's intact phantom limb in motion, a hypothesized mechanism of mirror therapy. Laboratory. Participants completed pre- and post-treatment measures to evaluate changes in PLP, phantom sensations, and rate helpfulness, realism, immersion, adverse experiences, and treatment satisfaction. Eight of 14 participants (57.1%) reported PLP pre-VR treatment, and 93% (13/14) reported one or more unpleasant phantom sensations. After treatment, 28.6% (4/14) continued to report PLP symptoms (t[13] = 2.7, P = 0.02, d = 0.53) and 28.6% (4/14) reported phantom sensations (t[13] = 4.4, P = 0.001, d = 1.7). Ratings of helpfulness, realism, immersion, and satisfaction were uniformly high to very high. There were no adverse experiences. Four participants completed multiple VR treatments, showing stable improvements in PLP intensity and phantom sensations and high user ratings. This feasibility study of a novel VR intervention for PLP was practical and was associated with significant reductions in PLP intensity and phantom sensations. Our findings support continued research in VR-based treatments in PLP, with a need for direct comparisons between VR and more established PLP treatments.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V H H nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1 ) associated with high T-cell polyfunctionality. This study demonstrates the potential of V H H-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

Abstract Background and Significance Neuroblastoma is a rare pediatric cancer that forms in immature nerve tissue of infants and accounts for 10 to 15 percent of cancer-related deaths in children. The five-year survival for high-risk neuroblastoma is 50% with current treatment practices being a combination of surgery, chemotherapy, and radiation. A more effective therapy is therefore needed to improve overall patient outcomes. Methods The CT3 mouse antibody that targets GPC2 was previously identified in the lab and has shown activity in the chimeric antigen receptor (CAR) T cell format against neuroblastoma. Humanization of the CT3 antibody was also done through CDR grafting in human germline sequences to prevent potential adverse immunogenic effects when treating patients. In the present study, the CT3 antibody and humanized CT3 (hCT3) antibody were engineered into T cells based on the engineered gamma/delta TCR scaffold (called AbTCR). The activities of the CT3 and hCT3 AbTCRs were tested in luciferase-based cell killing assays and xenograft mouse models. Results Humanized CT3 retains a comparable binding affinity for GPC2. The hCT3 CAR T cell showed its ability to regress tumor expression in mice. Furthermore, the mice treated with the CT3 AbTCR showed tumor regression while the mice treated with the hCT3 AbTCR became tumor free three weeks after treatment. Conclusions Overall, the hCT3 AbTCR T cells are very active when combating neuroblastoma tumors in mice. The efficacy at a low treatment dosage indicates that the GPC2 targeted hCT3 AbTCRs are a promising therapeutic for the treatment of neuroblastoma and other GPC2 positive cancers in patients.

Cederquist et al discuss a case involving shared decision making in the presence of COVID-19 and the absence of families. A 47 years old woman was brought by ambulance to her community hospital emergency room, where she was diagnosed with hypoxic respiratory failure secondary to COVID-19. The family was updated regularly by the Intensive Care Unit (ICU) team. The family requested, then insisted, on continued, full life-sustaining treatmen. Over a period of a week or more, multiple goals of care conversations were conducted, including vide-oconferences to demonstrate the patient's devastating neurologic injury. Like so many in the acute hospital setting, clinical ethicists are learning as this pandemic progresses. What is certain is that processes for engaging in shared decision-making will look quite different post-pandemic than they did pre-pandemic. The pandemic will almost certainly change how ICU physicians communicate with patients and/or families at the end of life.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V H nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of V H-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in solid tumors, in part due to variability in autologous T cells derived from heavily pretreated patients with advanced disease. To address these constraints, we developed an off-the-shelf allogeneic CAR-T platform using CRISPR-Cas9-mediated genome editing in T cells from healthy donors to enable targeted CAR insertion at the locus with concurrent disruption of . Using adeno-associated virus (AAV) delivery, we designed CAR-T cells targeting glypican-2 (GPC2) and glypican-3 (GPC3), emerging antigens expressed in pediatric and adult solid tumors. Genome-edited allogeneic CAR-T cells exhibited potent, antigen-specific cytotoxicity across multiple tumor models. GPC2-directed allogeneic CAR-T cells demonstrated enhanced or comparable activity relative to conventional lentiviral CAR-T cells in neuroblastoma models and mediated tumor regression with prolonged survival in preclinical models. Notably, repeated dosing augmented antitumor efficacy without evidence of toxicity, supporting multi-dose regimens for solid tumors. Similarly, GPC3-targeted allogeneic CAR-T cells based on a single-domain antibody showed robust activity against hepatocellular carcinoma cells in vitro and in vivo. These findings establish a scalable, genome-engineered allogeneic CAR-T strategy with strong therapeutic potential and support the clinical development of off-the-shelf cell therapies for pediatric and adult solid tumors.

A poem is presented.