This Review discusses the major advances and changes made over the past 3 years to our understanding of chimeric antigen receptor (CAR) T cell efficacy and safety. Recently, the field has gained insight into how various molecular modules of the CAR influence signalling and function. We report on mechanisms of toxicity and resistance as well as novel engineering and pharmaceutical interventions to overcome these challenges. Looking forward, we discuss new targets and indications for CAR T cell therapy expected to reach the clinic in the next 1–2 years. We also consider some new studies that have implications for the future of CAR T cell therapies, including changes to manufacturing, allogeneic products and drug-regulatable CAR T cells. This Review outlines the major advances that have been made to the efficacy and safety of chimeric antigen receptor (CAR) T cell therapies over the past 3 years and looks to new findings that will have consequences for the future of this immunotherapy.

Recent advances and discoveries in the mechanisms and functions of CAR T cells.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG. 

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GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas

Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies

https://www.cell.com/cell/pdf/S0092-8674(20)30263-4.pdf

The Emerging Landscape of Immune Cell Therapies.

https://www.nature.com/articles/s41586-022-04489-4.pdf

Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.This article is highlighted in the In This Issue feature, p. 627.

https://cancerdiscovery.aacrjournals.org/content/candisc/10/5/702.full.pdf

Tuning the Antigen Density Requirement for CAR T-cell Activity.

The formation of a hollow lumen in a formerly solid mass of cells is a key developmental process whose dysregulation leads to diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by experiments in the mouse blastocyst. However, lumens formed in other tissues adopt irregular shapes with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen shape in all cases. Here we use live-cell imaging to study the physical mechanism of lumen formation in Madin-Darby Canine Kidney cell spheroids, a canonical cell-culture model for lumenogenesis. We find that in this system, lumen shape reflects basic geometrical considerations tied to the establishment of apico-basal polarity. A physical model incorporating both cell geometry and intraluminal pressure can account for our observations as well as cases in which pressure plays a dominant role. The formation of a hollow lumen surrounded by cells is a key developmental process that sets the shape of tissues and organs. Here, the authors show how the combined influence of geometric constraints imposed by cell packing and osmotic pressure can generate the diverse range in lumen shapes observed in different tissues.

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Physical basis for the determination of lumen shape in a simple epithelium.

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion. High-performance engineered myosins robustly change speed or direction in response to an optical signal. In living cells, these motors localize to the tips of protrusions when illuminated and deliver molecular cargos.

Optical control of fast and processive engineered myosins in vitro and in living cells.

Optical Control of Fast and Processive Engineered Myosins In Vitro and in Living Cells

A continuous sheet of epithelial cells surrounding a hollow opening, or lumen, defines the basic topology of numerous organs. De novo lumen formation is a central feature of embryonic development whose dysregulation leads to congenital and acquired diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by recent experiments in the mouse blastocyst. High luminal pressure should produce lumen surfaces that bow outwards toward the surrounding cells. However, lumens formed in other embryonic tissues adopt highly irregular shapes, with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen growth in all cases. We used three-dimensional live-cell imaging to study the physical mechanism of lumen formation in Madin Darby Canine Kidney (MDCK) cell spheroids, a canonical cell-culture model for lumenogenesis. Our experiments revealed that neither lumen pressure nor the actomyosin cytoskeleton were required to maintain lumen shape or stability. Instead, we find that, in our model system, lumen shape results from simple geometrical factors tied to the establishment of apico-basal polarity. A quantitative physical model that incorporates cell geometry, cortical tension, and intraluminal pressure can account for our observations as well as cases in which pressure indeed plays a dominant role. Our results thus support a unifying physical mechanism for the formation of luminal openings in a variety of physiological contexts.

/pdf/physical-basis-of-lumen-shape-and-stability-in-a-simple-2j9xivql85.pdf

Vipul T. Vachharajani

Papers

Tuning the Antigen Density Requirement for CAR T-cell Activity.

Physical basis for the determination of lumen shape in a simple epithelium.

Optical control of fast and processive engineered myosins in vitro and in living cells.

Optical Control of Fast and Processive Engineered Myosins In Vitro and in Living Cells

Physical basis of lumen shape and stability in a simple epithelium