Wendell A. Lim

SUMMARY: Synaptic assemblies that form at cell-cell junctions are often organized by multivalent scaffold proteins with condensate forming properties. An emerging hypothesis is that these scaffolds provide a thresholding mechanism whereby small increases in the density of cell-cell recognition receptors can trigger all-or-none assembly selectively at the correct partner cell interface. We test this hypothesis by engineering synthetic scaffolded synapses. Coarse-grained simulations suggest that properly-tuned scaffold interactions can yield a sharp condensation threshold. Guided by these simulations, we designed modular scaffold proteins consisting of PDZ domains and intrinsically disordered regions (IDRs) of different molecular grammars. Within a specific region of parameter space, controlled by valency, receptor binding affinity and IDR-driven condensation, the synthetic scaffold proteins exhibit switch-like condensation behaviors, where co-condensates of receptor and scaffold selectively form only at interfaces with the proper partner cell. Notably, orthogonal receptor-scaffold pairs enable a single receiver cell to form multiple distinct interfaces, each associated with a different partner cell. We demonstrate that these synthetic synapse assemblies can recruit different client proteins to enhance intercellular signaling, orient cell division, or sculpt complex synaptic morphologies. Our results illustrate how the ultrasensitive condensation properties of modular scaffold proteins can be harnessed to create novel and highly specific cell-cell interfaces.

Diffuse midline glioma (DMG) is a lethal pediatric brain tumor with limited therapeutic options and a poor prognosis. The success of chimeric antigen receptor (CAR) T cell therapy requires both improving T cell tumor infiltration and persistence and reducing the risk of on-target/off-tumor toxicity. Here, we adapted and advanced a brain-restricted CAR T cell platform previously validated in glioblastoma to address the anatomical and biological challenges of DMG. We engineered a T cell circuit in which a brain-specific synthetic Notch (synNotch) receptor recognizing brevican (BCAN) induced CAR expression against EphA2 or IL-13Rα2 (“B-SYNC”), thereby restricting CAR expression to the central nervous system (CNS). In immunodeficient DMG models, a single infusion of intravenous (iv) or intracerebroventricular (icv) B-SYNC circuit T cells led to tumor infiltration, sustained persistence, antitumor activity, and extended survival. An alternative BCAN-primed CAR targeting B7-H3 or IL-13Rα2 (“B-SYNC–B/I”) similarly improved therapeutic efficacy when delivered intravenously. However, icv administration triggered fatal neurotoxicity because of low-level B7-H3 expression in nontumor CNS regions, indicating that rapid CNS exposure via icv delivery can amplify off-tumor toxicity. In contrast, in an immunocompetent syngeneic DMG model, both iv and icv delivery of a murinized BCAN-gated B7-H3 CAR (mB-SYNC–B7-H3) achieved durable tumor clearance and resistance to rechallenge without any detectable neurotoxicity. Mechanistically, synNotch-primed CAR T cells exhibited reduced exhaustion, enhanced activation, and enrichment of naïve and stem cell memory–like subsets. These findings demonstrate that brain-sensing synNotch CAR T circuits represent a generalizable and translatable strategy for targeting multiple CNS malignancies, including DMG.

Chemokines orchestrate the migration of immune cells throughout the body, but harnessing chemokine signaling to engineer targeted cell trafficking is challenging because of their overlapping interactions and broad expression patterns. Here we used structure-guided protein design to engineer orthogonal chemokine-receptor pairs with minimal crosstalk to native systems. T cells engineered with an ortho-receptor selectively infiltrated tissues producing the ortho-chemokine, in vitro and in vivo. By engineering T cells that both sense and secrete the ortho-chemokine, we generated collective swarming behavior in which pioneer cells attract follower cells via an ortho-chemokine gradient self-organized by positive feedback. By providing orthogonal genetic control over both the generation and sensing of chemokine gradients, this system enables the construction of autonomous spatial targeting programs in engineered cells.

T cell proliferative capacity and persistence determine the therapeutic efficacy of chimeric antigen receptor (CAR) T cells. However, strategies to externally enhance CAR-T cell expansion without genetic rewiring are lacking. Here, we engineer CAREp, programmable DNA-scaffolded PLGA microparticles displaying CAR-targeting antigens and CD28-costimulatory antibodies, to repeatedly stimulate human CD8⁺ CAR-T cells ex vivo. CAREp sustains a-EGFR CAR-T cell expansion for over 100 days across both 4-1BBζ and CD28ζ constructs, achieving up to 10¹⁸-fold cumulative expansion and surpassing tumor-cell or CD3/CD28-Dynabeads stimulation. Expanded cells retain effector function and mitochondrial fitness while exhibiting clonal enrichment, initially preserved memory-associated progenitor states, delayed exhaustion, and transiently activated telomerase delaying telomere attrition. Early transcriptomic responses show coordinated activation of DNA repair, chromatin remodeling, telomere maintenance, and mitochondrial function while restricting differentiation-associated signaling programs, mirroring long-term functional outcomes. These findings demonstrate that nanoscale ligand organization synchronizes acute CAR-T signaling with durable proliferative and metabolic states.

Figure S8. In vitro and in vivo activity of low a7inity scFv-guided CAR (KD = 3910 nM).

Figure S13. SKOV3 ovarian tumor volume measurements of individual mice treated with T cells expressing constitutive or inducible Sb1205 anti-HER2 CAR (related to Figure 5B and C).

Figure S3. Antigen expression and HER2 knock-down in a panel of cancer cell lines and additional Jurkat T cell reporter activation (NFAT-GFP) assays in co-culture with 293T* (HER2 KD) cells transfected with tumor associated antigens (related to Figure 2C, 2D.

Figure S5. Cytotoxic activity of Sb CARs expressed in human primary CD8+ T cells (related to Figure 2E)

Figure S9. Tumor volume measurement of individual mice treated with T cells expressing Sb1205 anti-HER2 CAR (related to Figure 4C and E).

Nucleotide sequences of constructs used in the study