IntroductionT cell activation requires T cell receptor (TCR) engagement by its specific ligand. This interaction initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation.MethodsWe employed TIRF microscopy to establish detailed spatial and kinetic relationships among these molecules in live Jurkat and murine primary T cells. We used anti-TCR (CD3) antibodies to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Using this model, we aimed to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events, and how these effects could affect the immune response.ResultsOur results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation.ConclusionThese results suggest that CD28 co- stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

Background One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 (“A2”) that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH).Methods A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2− HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking.Results A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the ‘on’ and ‘off’ cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2− tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity.Conclusions We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.

Chimeric antigen receptors (CAR) T cells have been successfully used to treat lymphoma, leukemia, and multiple myeloma, but adverse effects due to cytokine secretion, CAR-T cell exhaustion, and loss of target antigen have limited their potential. Furthermore, while CARs have been designed to harness T Cell Receptor (TCR) signaling, they are significantly less sensitive than TCRs, resulting in suboptimal signaling. We have developed novel Chimeric Adapter Proteins (CAPs) that are designed to trigger signaling downstream of the TCRζ chain. CAPs are chimeric molecules that contain adapter domains in tandem with the kinase domain of ZAP70, fused to an extracellular targeting domain. We hypothesized that CAPs would be more potent than CARs because kinetic proofreading steps that define the signaling threshold and the inhibitory regulation of upstream molecules are bypassed. Indeed, second generation CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19-28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking cancer treatment that has produced remarkable clinical efficacy for hematopoietic malignancies, yet “on-target off-tumor” toxicity due to lack of target specificity limits the therapeutic potential of CAR-T cells in solid tumors. We are developing dual CAR-T cells composed of a canonical activator CAR (aCAR), intended to elicit efficacy in solid tumors, and an inhibitory CAR (iCAR) designed to efficiently inhibit the aCAR activity in normal tissue and vital organs. The iCAR and aCAR scFvs of this system bind distinct cell-surface antigens that are widely co-expressed on the normal epithelial from which solid tumors originate. Upon antigen binding, signal propagation through iCAR cytoplasmic domain (iDomain) counteracts aCAR induction of CAR T-cell activation and killing; thus protecting normal tissue. Dual CAR-T cells are activated and kill when exposed to tumor cells that have lost iCAR target expression due to chromosomal loss-of-heterozygosity (LOH) which is common in all cancers. iCAR-targeted LOH generates absolute and irreversible tumor specificity therefore tumor-specific overexpression of the iCAR or aCAR targets is not required. We have carried out a comprehensive screen using human T-cells to identify the most effective inhibitory iDomains derived from cell-surface receptors that naturally inhibit or moderate immune cell activation. The cell-based assay suite developed for this screen consisted of cell lines that co-express iCAR and aCAR target at clinically relevant levels and isogenic partners that mimic LOH through CRISPR editing of the iCAR target. The expression of dual CARs in T cells as biscistronic constructs introduced with lentiviral vectors was successful in a limited number of cases, and it was necessary to develop alternative methods of iCAR aCAR co-expression to complete the screen. Combining aCAR transduction with iCAR mRNA Electroporation (T-REP) proved to be a useful method to disentangle iDomain potency and expression level. The screen identified new iDomains that may expand the potential of iCAR technology in developing strategies to treat solid tumors without compromising efficacy for safety. Citation Format: David Bassan, Jason Yi, Neta Chaim, Nir Bujanover, Sarit Tabak, Tanya Kim, Yael Lopesco, Leehee Weinberger, Kristina Vucci, Michael Weist, Caitlin Schnair, Gregor B. Adams, Orit Foord, Frank J. Calzone, Rick Kendall, Adi Sharbi-Yunger. Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2848.

Background Chimeric antigen receptor (CAR)-T cell therapy has shown incredible clinical success for hematopoietic malig-nancies, but for solid tumors is limited by “ on-target off-tumor ” toxicity to vital organs due to lack of target specific-ity. We have developed dual CAR-T cells consisting of a can-onical activating CAR (aCAR), intended to elicit efficacy in solid tumors, and an inhibitory CAR (iCAR) designed to effec-tively suppress aCAR activation in normal tissues. The iCAR and aCAR bind distinct cell-surface antigens that are widely co-expressed in normal tissues. The iCAR is allele specific, and targets an antigen that is commonly lost in cancer due to chromosomal loss-of heterozygosity (LOH). Therefore, whereas healthy cells express the iCAR antigen and are protected, tumors have irreversibly lost iCAR antigen expression via LOH and are killed. Methods this study developed dual CARs with an aCAR targeting Her2 and iCAR targeting HLA-A2. We screened bicistronic dual CARs in human PBMCs following lentiviral transduction against target cell lines that express normal of both paired with targets following of the HLA-A2 to In-vitro assays included Luciferase-based assays, live-imaging killing assays, In-vivo performed in NSG-mice inoculated with either the HLA-A2+ or HLA-A2 KO linker to generate dual CARs with high potency and tumor-specificity. These dual CARs were highly active against HLA-A2 KO targets ( “ tumor ” ) and inhibited against HLA-A2+ targets ( “ normal tissue ” ). Conclusions These results show that the iCAR enables the tre-mendous therapeutic potential of CAR-T therapy to transition to solid tumors while maintaining safety and tumor specificity.

Translation of the unprecedented efficacy of chimeric antigen receptor (CAR) T-cell technology into solid tumors requires a solution to the problem of on-target damage of vital organs. We have developed an inhibitory CAR platform (iCAR) that imposes self-regulation in CAR T-cells to restrict inappropriate activation against normal cells without compromising tumor efficacy. The system consists of an iCAR that is co-expressed with a conventional activating CAR (aCAR) designed to activate T-cells. The iCAR and aCAR scFvs bind distinct cell-surface antigens that are ubiquitously expressed across all solid tumor histologies and normal tissues. The iCAR is allele-specific whereas the aCAR is pan-allelic. The iCAR technology was validated in functional assays and NSG models with cancer cell lines that mimic loss-of-heterozygosity (LOH) which is common in most human cancers due to increased chromosomal instability. In this context, loss of iCAR target expression due to LOH generates irreversible target specificity. Tumor-specific overexpression of aCAR and iCAR targets, a common barrier to the clinical translation of potent agents targeting the cell-surface proteins is not required. We have shown using in vitro and in vivo models that CAR T-cell activation and target killing are fully inhibited by dual iCAR + aCAR antigen engagement. This outcome validates the concept of iCAR-mediated protection of normal cells in vital organs. In contrast, tumors that mimic iCAR target LOH through CRISPR editing were fully eradicated with aCAR engagement alone. T-cell activation, proliferation, and cytotoxic activity after exposure to target cells lacking iCAR target antigen was found to be quantitatively indistinguishable from the results obtained with single input aCAR CAR T-cells. These results suggest that iCAR technology can enable treatment of cancer patients with solid tumors using CAR T-cells that maximize potency and efficacy by mitigating on-target CRS, neurotoxicity, and inevitable organ damage. The restriction of CAR T activation to the local tumor environment by iCAR-mediated self-regulation directly addresses a fundamental technology gap that cannot be solved by CAR T dosing or aCAR modification alone. Citation Format: Michael R. Weist, Adi Sharbi-Yunger, Jason Yi, Caitlin Schnair, David Bassan, Tanya Kim, Sarit Tabak, Yael Lopesco, Leehee Weinberger, Nir Bujanover, Neta Chaim, Kristina Vucci, Orit Foord, Frank J. Calzone, Rick Kendall, Gregor B. Adams. Rewiring CAR T-cells for absolute solid tumor specificity and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2826.

We designed variant human TCRs composed of the full-length TCRα/β or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR α- and β-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the α/β-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.

UBE3A is a HECT (homologous to E6AP C-terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin-proteasome pathway. The UBE3Agene is of unique interest for its gene dosage-dependent effect in the developing brain: Precise deletion or null mutation of the maternal copy of UBE3A causes a severe intellectual disability known as Angelman syndrome; meanwhile, duplication or triplication of the gene region in which UBE3A resides is linked to a prevalent syndromic form of autism known as Dup15q syndrome. However, little is known about the effects of missense variants which cause a single amino acid change in the enzyme, and prediction of disease outcomes for a given variant remains a challenge. Here, we pose that investigating variants' effects on UBE3A functional activity levels is critical for predicting disease. In order to identify if precise mutations in UBE3A are sufficient to drive disease, we devised a high-throughput assay to screen the functional consequence of UBE3A missense variants. We screened over 150 variants and identified distinct functional classes of UBE3A mutants based on their effect on enzymatic activity. Importantly, we identified over a dozen novel gain-of-functionvariants that aberrantly hyperactivate UBE3A enzyme activity. Through collaborations with clinical centers, we confirm that individuals possessing hyperactivating UBE3A variants exhibited phenotypes that were distinguishable from Angelman. Mice carrying a specific hyperactivating mutation on the maternal allele exhibited aberrant motor and early communication defects, as well as microcephaly. Finally, we mapped the results of our screen to the UBE3A protein structure to reveal a previously-undefined allosteric regulatory exosite within the catalytic domain that we show to act as a charge-dependent regulator of enzymatic activity. We found additional HECT domain enzymes to possess disease-associated variants within their exosites, suggesting that exosite dysfunction is a common mechanism underlying a set of neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that deep structure-functional analysis of protein variants can uncover disease-relevant regulatory mechanisms.

Cell therapy is poised to play a larger role in medicine, most notably for immuno-oncology. Despite the recent success of CAR-T therapeutics in the treatment of blood tumors and the rapid progress toward improved versions of both CAR- and TCR-Ts, important analytical aspects of preclinical development and manufacturing of engineered T cells remain immature. One limiting factor is the absence of robust multivariate assays to disentangle key parameters related to function of engineered effector cells, especially in the peptide-MHC (pMHC) target realm, the natural ligand for TCRs. Here we describe an imaging-based primary T cell assay that addresses several of these limitations. To our knowledge, this assay is the first quantitative, high-content assay that separates the key functional parameters of time- and antigen-dependent T cell proliferation from cytotoxicity. We show that the assay sheds light on relevant biology of CAR- and TCR-T cells, including response kinetics and the influence of effector:target ratio.

Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.

The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.