Generation of scalable off-the-shelf iNKT therapies

Invariant natural killer T cells (iNKTs) are rare T lymphocytes, emerging as an off-the-shelf platform for the treatment of blood and solid tumors, severe viral infections, and graft-versus-host disease. These features hold promise for improving global and equitable access to advanced cellular immunotherapies. However, limited scalability and persistence of donor-derived iNKT products hinder worldwide impact. We set out to establish large-scale iNKT off-the-shelf products capable of extended in vivo persistence, including enhanced tumor-redirected iNKTs engineered with chimeric antigen receptors (CAR-iNKT). Methods. To investigate clinically relevant methods to expand large-scale off-the-shelf products, we tested: 1) activating beads coated with alphaCD3/alphaCD28 antibodies; 2) alpha-galactosylceramide (alphaGC)-loaded artificial antigen-presenting cells (aAPCs), generated by engineering K562 cells expressing CD80/CD83/CD86/41BBL with the iNKT-specific antigen CD1d; and 3) cell-derived nanoparticles (CDNPs, Synecta T1), a feeder-free platform engineered to display membrane-bound OKT3, co-stimulatory ligands, and IL-7/IL-15 cytokines in a scalable, closed-system format, providing key aAPC-like stimulation cues without using live cell lines. To determine the resulting therapeutic potential, we assessed iNKT purity, yield, memory/exhaustion phenotype, and anti-tumor efficacy via in vitro 4h killing and cytokine assays and in vivo NSG xenografts. Results. All activation systems produced pure iNKT products. aAPCs induced the highest iNKT yield, as expected from antigen-specific CD1d-alphaGC stimulation. CDNPs were as effective as alphaCD3/alphaCD28 beads in sustaining proliferation after repeated stimulations. Phenotypically, aAPCs induced the highest CD62L levels, a known predictor of persistence, whereas CDNPs exhibited the most favorable effector profile, including the lowest LAG-3 and TIM-3 expression. CDNPs also showed the strongest in vitro cytotoxicity against tumor targets, with up to 60% killing as compared to aAPCs 25% and alphaCD3/alphaCD28 beads 35%. In vivo , aAPC-manufactured CAR-iNKTs persisted in osteosarcoma-bearing NSG xenografts for 90 days after a single infusion - the longest persistence reported in such a model - associated with complete and durable tumor eradication. Bead-expanded CAR-iNKTs eradicated tumors but were not detected at comparable late time points. Conclusions. We established clinically scalable iNKT/CAR-iNKT manufacturing workflows yielding high-purity cells. All products exhibited potent anti-tumor activity, but aAPC-expanded CAR-iNKTs resulted in exceptional persistence in vivo , superior to bead-expanded cells. The CDNP platform provides a clinically translatable, feeder-free activator that achieves superior functional potency, reduced exhaustion, and regulatory readiness (FDA-reviewed DMF), enabling scalable off-the-shelf iNKT/CAR-iNKT therapy for a broad range of applications.