Extracellular domains of CARs reprogramme T cell metabolism without antigen stimulation.

Metabolism is an indispensable part of T cell proliferation, activation and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are composed of extracellular domains-often single-chain variable fragments (scFvs)-that determine ligand specificity and intracellular domains that trigger signalling following antigen binding. Here, we show that CARs differing only in the scFv variously reprogramme T cell metabolism. Even without exposure to antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observed basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harbouring a rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14G2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Modest overflow metabolism of CAR-T cells and metabolic compatibility between cancer cells and CAR-T cells are identified as features of efficacious CAR-T cell therapy.

Perception and memory reinstatement engage overlapping face selective regions within human ventral temporal cortex.

Humans have the remarkable ability to vividly retrieve sensory details of past events. According to the theory of sensory reinstatement, during remembering, brain regions specialized for processing specific sensory stimuli are reactivated to support content specific retrieval. Recently, several studies have emphasized transformations in the spatial organization of these reinstated activity patterns. Specifically, studies of scene stimuli suggest a clear anterior shift in the location of retrieval activations compared with activity observed during perception. However, it is not clear that such transformations occur universally, with inconsistent evidence for other important stimulus categories, particularly faces. One challenge in addressing this question is the careful delineation of face-selective cortices, which are inter-digitated with other selective regions, in configurations that spatially differ across individuals. Therefore, we conducted a multi-session neuroimaging study to first carefully map individual participants (9 males and 7 females) face-selective regions within ventral temporal cortex (VTC), followed by a second session to examine the activity patterns within these regions during face memory encoding and retrieval. While face-selective regions were expectedly engaged during face perception at encoding, memory retrieval engagement exhibited a more selective and constricted reinstatement pattern within these regions, but did not show any consistent direction of spatial transformation (e.g., anteriorization). We also report on unique human intracranial recordings from VTC under the same experimental conditions. These findings highlight the importance of considering the complex configuration of category selective cortex in elucidating principles shaping the neural transformations that occur from perception to memory.Significance statement Sensory reinstatement suggests that brain regions involved in the initial sensory processing of a stimulus are reactivated to support successful memory retrieval. However, recent findings have suggested reinstated cortical activations occur anterior to perceptually driven activities, particularly for scene stimuli. It remains unclear if this anteriorization occurs for other stimuli, such as faces. To address this question, we conducted a multi-session fMRI study to identify face selective regions in ventral temporal cortex, and examined activities within these regions during face-memory reinstatement. Results showed retrieval activity closely aligns with the perceptual neural substrate, confirming individual-specific face-selective regions without consistent spatial shifts. This underscores the importance of considering individual functional organizations when investigating the neural substrates of perception-memory transformations.

Self-regulating CAR-T cells modulate cytokine release syndrome in adoptive T-cell therapy.

Cytokine release syndrome (CRS) is a frequently observed side effect of chimeric antigen receptor (CAR)-T cell therapy. Here, we report self-regulating T cells that reduce CRS severity by secreting inhibitors of cytokines associated with CRS. With a humanized NSG-SGM3 mouse model, we show reduced CRS-related toxicity in mice treated with CAR-T cells secreting tocilizumab-derived single-chain variable fragment (Toci), yielding a safety profile superior to that of single-dose systemic tocilizumab administration. Unexpectedly, Toci-secreting CD19 CAR-T cells exhibit superior in vivo antitumor efficacy compared with conventional CD19 CAR-T cells. scRNA-seq analysis of immune cells recovered from tumor-bearing humanized mice revealed treatment with Toci-secreting CD19 CAR-T cells enriches for cytotoxic T cells while retaining memory T-cell phenotype, suggesting Toci secretion not only reduces toxicity but also significantly alters the overall T-cell composition. This approach of engineering T cells to self-regulate inflammatory cytokine production is a clinically compatible strategy with the potential to simultaneously enhance safety and efficacy of CAR-T cell therapy for cancer.

CAR-T cell manufacturing: Major process parameters and next-generation strategies.

Chimeric antigen receptor (CAR)-T cell therapies have demonstrated strong curative potential and become a critical component in the array of B-cell malignancy treatments. Successful deployment of CAR-T cell therapies to treat hematologic and solid cancers, as well as other indications such as autoimmune diseases, is dependent on effective CAR-T cell manufacturing that impacts not only product safety and efficacy but also overall accessibility to patients in need. In this review, we discuss the major process parameters of autologous CAR-T cell manufacturing, as well as regulatory considerations and ongoing developments that will enable the next generation of CAR-T cell therapies.

Successes and challenges in clinical gene therapy.

Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of diseases. There are active clinical trials approaching a variety of inherited and acquired disorders of different organ systems. Approaches include ex vivo modification of hematologic stem cells (HSC), T lymphocytes and other immune cells, as well as in vivo delivery of genes or gene editing reagents to the relevant target cells by either local or systemic administration. In this article, we highlight success and ongoing challenges in three areas of high activity in gene therapy: inherited blood cell diseases by targeting hematopoietic stem cells, malignant disorders using immune effector cells genetically modified with chimeric antigen receptors, and ophthalmologic, neurologic, and coagulation disorders using in vivo administration of adeno-associated virus (AAV) vectors. In recent years, there have been true cures for many of these diseases, with sustained clinical benefit that exceed those from other medical approaches. Each of these treatments faces ongoing challenges, namely their high one-time costs and the complexity of manufacturing the therapeutic agents, which are biological viruses and cell products, at pharmacologic standards of quality and consistency. New models of reimbursement are needed to make these innovative treatments widely available to patients in need.

Advances in promoting chimeric antigen receptor T cell trafficking and infiltration of solid tumors.

T cells engineered to express chimeric antigen receptors (CARs) have demonstrated robust response rates in treating hematological malignancies. However, solid tumors present multiple challenges that hinder the antitumor efficacy of CAR-T cells, including antigen heterogeneity, off-tumor and systemic toxicities, and the immunosuppressive milieu of the tumor microenvironment (TME). Notably, the TME of solid tumors is characterized by chemokine dysregulation and a dense architecture consisting of tumor stroma, extracellular matrix, and aberrant vasculature that impede migration of CAR-T cells to the tumor site as well as infiltration into the solid-tumor mass. In this review, we highlight recent advances to improve CAR-T-cell trafficking to and infiltration of solid tumors to promote effective antigen recognition by CAR-T cells.

Individual-specific memory reinstatement patterns within human face-selective cortex.

Humans have the remarkable ability to vividly retrieve sensory details of past events. According to the theory of sensory reinstatement, during remembering, brain regions involved in the sensory processing of prior events are reactivated to support this perception of the past. Recently, several studies have emphasized potential transformations in the spatial organization of reinstated activity patterns. In particular, studies of scene stimuli suggest a clear anterior shift in the location of retrieval activations compared with those during perception. However, it is not clear that such transformations occur universally, with evidence lacking for other important stimulus categories, particularly faces. Critical to addressing these questions, and to studies of reinstatement more broadly, is the growing importance of considering meaningful variations in the organization of sensory systems across individuals. Therefore, we conducted a multi-session neuroimaging study to first carefully map individual participants face-selective regions within ventral temporal cortex (VTC), followed by a second session to examine the correspondence of activity patterns during face memory encoding and retrieval. Our results showed distinct configurations of face-selective regions within the VTC across individuals. While a significant degree of overlap was observed between face perception and memory encoding, memory retrieval engagement exhibited a more selective and constricted reinstatement pattern within these regions. Importantly, these activity patterns were consistently tied to individual-specific neural substrates, but did not show any consistent direction of spatial transformation (e.g., anteriorization). To provide further insight to these findings, we also report on unique human intracranial recordings from VTC under the same experimental conditions. Our findings highlight the importance of considering individual variations in functional neuroanatomy in the context of assessing the nature of cortical reinstatement. Consideration of such factors will be important for establishing general principles shaping the neural transformations that occur from perception to memory.

Handedness effects on imagery of dominant- versus non-dominant-hand movements: An electroencephalographic investigation.

Mental representations of our bodies are thought to influence how we interact with our surroundings. We can examine these mental representations through motor imagery, the imagination of movement using scalp EEG recordings. The visual modality of motor imagery emphasises 'seeing' the imagined movement and is associated with increased activity in the alpha rhythm (8-14 Hz) measured over the occipital regions. The kinaesthetic modality emphasises 'feeling' the movement and is associated with decreased activity in the mu rhythm (8-14 Hz) measured over the sensorimotor cortices. These two modalities can be engaged in isolation or together. We recorded EEG activity while 37 participants (17 left-hand dominant) completed an objective hand motor imagery task. Left-handers exhibited significant activity differences between occipital and motor regions only during imagery of right-hand (non-dominant-hand) movements. This difference was primarily driven by less oscillatory activity in the mu rhythm, which may reflect a shift in imagery strategy wherein participants placed more effort into generating the kinaesthetic sensations of non-dominant-hand imagery. Spatial features of 8-14 Hz activity generated from principal component analysis (PCA) provide further support for a strategy shift. Right-handers also exhibited significant differences between alpha and mu activity during imagery of non-dominant movements. However, this difference was not primarily driven by either rhythm, and no differences were observed in the group's PCA results. Together, these findings indicate that individuals imagine movement differently when it involves their dominant versus non-dominant hand, and left-handers may be more flexible in their motor imagery strategies.

Extracellular Domains of CAR Reprogram T-Cell Metabolism Without Antigen Stimulation.

Metabolism is an indispensable part of T-cell proliferation, activation, and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are comprised of extracellular domains that determine cancer specificity, often using single-chain variable fragments (scFvs), and intracellular domains that trigger signaling upon antigen binding. Here we show that CARs differing only in the scFv reprogram T-cell metabolism differently. Even in the absence of antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observe basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harboring rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14g2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Since CAR-dependent metabolic reprogramming alters cellular energetics, nutrient utilization, and proliferation, metabolic profiling should be an integral part of CAR-T cell development.

Rational Protein Design Yields a CD20 CAR with Superior Antitumor Efficacy Compared with CD19 CAR.

Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens. See related Spotlight by Scheller and Hudecek, p. 142.

Synthetic Biology in the Engineering of CAR-T and CAR-NK Cell Therapies: Facts and Hopes.

The advent of modern synthetic-biology tools has enabled the development of cellular treatments with engineered specificity, leading to a new paradigm in anticancer immunotherapy. T cells have been at the forefront of such development, with six chimeric antigen receptor-modified T-cell products approved by the FDA for the treatment of hematologic malignancies in the last 5 years. Natural killer (NK) cells are innate lymphocytes with potent cytotoxic activities, and they have become an increasingly attractive alternative to T-cell therapies due to their potential for allogeneic, "off-the-shelf" applications. However, both T cells and NK cells face numerous challenges, including antigen escape, the immunosuppressive tumor microenvironment, and potential for severe toxicity. Many synthetic-biology strategies have been developed to address these obstacles, most commonly in the T-cell context. In this review, we discuss the array of strategies developed to date, their application in the NK-cell context, as well as opportunities and challenges for clinical translation.

CD19/CD20 Bispecific Chimeric Antigen Receptor (CAR) in Naive/Memory T Cells for the Treatment of Relapsed or Refractory Non-Hodgkin Lymphoma.

To address antigen escape and loss of T-cell functionality, we report a phase I clinical trial (NCT04007029) evaluating autologous naive and memory T (TN/MEM) cells engineered to express a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR; CART19/20) for patients with relapsed/refractory non-Hodgkin lymphoma (NHL), with safety as the primary endpoint. Ten patients were treated with 36 × 106 to 165 × 106 CART19/20 cells. No patient experienced neurotoxicity of any grade or over grade 1 cytokine release syndrome. One case of dose-limiting toxicity (persistent cytopenia) was observed. Nine of 10 patients achieved objective response [90% overall response rate (ORR)], with seven achieving complete remission [70% complete responses (CR) rate]. One patient relapsed after 18 months in CR but returned to CR after receiving a second dose of CART19/20 cells. Median progression-free survival was 18 months and median overall survival was not reached with a 17-month median follow-up. In conclusion, CART19/20 TN/MEM cells are safe and effective in patients with relapsed/refractory NHL, with durable responses achieved at low dosage levels. SIGNIFICANCE: Autologous CD19/CD20 bispecific CAR-T cell therapy generated from TN/MEM cells for patients with NHL is safe (no neurotoxicity, maximum grade 1 cytokine release syndrome) and demonstrates strong efficacy (90% ORR, 70% CR rate) in a first-in-human, phase I dose-escalation trial. This article is highlighted in the In This Issue feature, p. 517.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

Distinct population and single-neuron selectivity for executive and episodic processing in human dorsal posterior cingulate.

Posterior cingulate cortex (PCC) is an enigmatic region implicated in psychiatric and neurological disease, yet its role in cognition remains unclear. Human studies link PCC to episodic memory and default mode network (DMN), while findings from the non-human primate emphasize executive processes more associated with the cognitive control network (CCN) in humans. We hypothesized this difference reflects an important functional division between dorsal (executive) and ventral (episodic) PCC. To test this, we utilized human intracranial recordings of population and single unit activity targeting dorsal PCC during an alternated executive/episodic processing task. Dorsal PCC population responses were significantly enhanced for executive, compared to episodic, task conditions, consistent with the CCN. Single unit recordings, however, revealed four distinct functional types with unique executive (CCN) or episodic (DMN) response profiles. Our findings provide critical electrophysiological data from human PCC, bridging incongruent views within and across species, furthering our understanding of PCC function.

Engineering Next-Generation CAR-T Cells: Overcoming Tumor Hypoxia and Metabolism.

T cells engineered to express chimeric antigen receptors (CARs) have shown remarkable success in treating B-cell malignancies, reflected by multiple US Food and Drug Administration-approved CAR-T cell products currently on the market. However, various obstacles have thus far limited the use of approved products and constrained the efficacy of CAR-T cell therapy against solid tumors. Overcoming these obstacles will necessitate multidimensional CAR-T cell engineering approaches and better understanding of the intricate tumor microenvironment (TME). Key challenges include treatment-related toxicity, antigen escape and heterogeneity, and the highly immunosuppressive profile of the TME. Notably, the hypoxic and nutrient-deprived nature of the TME severely attenuates CAR-T cell fitness and efficacy, highlighting the need for more sophisticated engineering strategies. In this review, we examine recent advances in protein- and cell-engineering strategies to improve CAR-T cell safety and efficacy, with an emphasis on overcoming immunosuppression induced by tumor metabolism and hypoxia.

Engineering Principles for Synthetic Biology Circuits in Cancer Immunotherapy.

Recent advances in biomolecular engineering have led to novel cancer immunotherapies with sophisticated programmed functions, including chimeric antigen receptor (CAR) T cells that bind tumor-associated antigens (TAA) to direct coordinated immune responses. Extensive engineering efforts have been made to program not only CAR specificity, but also downstream pathways that activate molecular responses. Collectively, these efforts can be conceptualized as an immunotherapy circuit: TAAs bind the CAR as input signals; intracellular signaling cascades process the binding interactions into transcriptional and translational events; and those events program effector output functions. More simply, this sequence may be abstracted as input, processing, and output. In this review, we discuss the increasingly complex scene of synthetic-biology solutions in cancer immunotherapy and summarize recent work within the framework of immunotherapy circuits. In doing so, a toolbox of basic modular circuits may be established as a foundation upon which sophisticated solutions can be constructed to meet more complex problems.See related article on p. 5.

T cells to fix a broken heart.

In vivo engineered T cells provide a promising approach to treat cardiac diseases.

Killer fatigue: Transition to NK-cell-like phenotype is a signature of CAR-T cell exhaustion.

Exhaustion of chimeric antigen receptor (CAR)-T cells hinders their therapeutic efficacy, especially in treating solid tumors. In this issue of Cell, Good et al. develop an in vitro model of antigen-driven CAR-T cell exhaustion to characterize signatures of dysfunction, including a transition to a natural killer (NK)-like phenotype, and suggest new gene targets to prevent exhaustion.

Imagery-based strategies for memory for associations.

Cued recall of word pairs is improved by asking participants to combine items in an interactive image. Meanwhile, interactive images facilitate serial-recall (Link Method), but even better when each item is imagined alongside a previously learned peg-word (Peg List Method). We asked if a peg system could support memory for pairs, hypothesising it would outperform interactive imagery. Tested with cued recall, five study strategies were manipulated between-subjects, across two experiments: (1) Both words linked to one peg; (2) Each word linked to a different peg; (3) Peg list method but studying as a serial list; (4) Interactive imagery (within-pairs); (5) Link Method. Participants were able to apply peg-list strategies to pairs, as anticipated by mathematical modelling. Error-patterns spoke to mathematical models; peg lists exhibited distance-based confusability, characteristic of positional-coding models, and errors tended to preserve within-pair position, even for inter-item associative strategies, suggesting models of association should incorporate position. However, the peg list strategies came with a speed-accuracy tradeoff and did not challenge the superiority of the interactive imagery strategy. Without extensive practice with peg list strategies, interactive imagery remains superior for associations. Peg strategies may excel instead in tasks that primarily test serial order or with extensive training.

Stability of ripple events during task engagement in human hippocampus.

High-frequency activity bursts in the hippocampus, known as ripples, are thought to support memory consolidation during "offline" states, such as sleep. Recently, human hippocampal ripples have been observed during "online" episodic memory tasks. It remains unclear whether similar ripple activity occurs during other cognitive states, including different types of episodic memory. However, identifying genuine ripple events in the human hippocampus is challenging. To address these questions, spectro-temporal ripple identification was applied to human hippocampal recordings across a variety of cognitive tasks. Overall, ripple attributes were stable across tasks of visual perception and associative memory, with mean rates lower than offline states of rest and sleep. In contrast, while more complex visual attention tasks did not modulate ripple attributes, rates were enhanced for more complex autobiographical memory conditions. Therefore, hippocampal ripples reliably occur across cognitive states but are specifically enhanced during offline states and complex memory processes, consistent with a role in consolidation.