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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.

Chemical screens across hundreds of cell lines have shown that the drug sensitivities of human cancers can vary by genotype or lineage. However, most drug discovery studies have relied on culture media that poorly reflect metabolite levels in human blood. Here, we perform drug screens in traditional and Human Plasma-Like Medium (HPLM). Sets of compounds that show conditional anticancer activity span different phases of global development and include non-oncology drugs. Comparisons of the synthetic and serum-derived components that comprise typical media trace sets of conditional phenotypes to nucleotide synthesis substrates. We also characterize a unique dual mechanism for brivudine, a compound approved for antiviral use. Brivudine selectively impairs cell growth in low folate conditions by targeting two enzymes involved in one-carbon metabolism. Cataloged gene essentiality data further suggest that conditional phenotypes for other compounds are linked to off-target effects. Our findings establish general strategies for identifying drug-nutrient interactions and mechanisms of action by exploiting conditional lethality in cancer cells. [ABSTRACT FROM AUTHOR]

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the GD2 disialoganglioside and chimeric antigen receptor modified T-cells targeting GD2 (GD2-CART) eradicate DMGs in preclinical models. Arm A of the Phase I trial NCT04196413 administered one IV dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline glioma at two dose levels (DL1=1e6/kg; DL2=3e6/kg) following lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) GD2-CART infusions (10-30e6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability, and identification of a maximally tolerated dose of IV GD2-CART. Secondary objectives included preliminary assessments of benefit. Thirteen patients enrolled and 11 received IV GD2-CART on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CART manufacturing was successful for all patients. No dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric tumor reductions (52%, 54%, 91% and 100%). One patient exhibited a complete response ongoing for >30 months since enrollment. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. Sequential IV followed by ICV GD2-CART induced tumor regressions and neurological improvements in patients with DIPG and sDMG. DL1 was established as the maximally tolerated IV GD2-CART dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.