GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE312nnn/GSE312032/primaryOK200TranscriptomicsHomo sapiensExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE312032GEOGSEfalseEnhancing the iNKT cell immunotherapy platform by combining optimised CAR endodomains with novel iNKT engagersiNKT cells are emerging as a highly promising cellular immunotherapy platform for the treatment of cancer. Their distinct biological features including their stereotypical CD1d-restricted TCR and ability to swiftly bridge and activate innate and adoptive T cell immunity underpin their potential for off-the-shelf therapy and pleiotropic anti-tumor effects. Accordingly, chimeric antigen receptor (CAR)-iNKT outperform CAR-T counterparts in preclinical models of blood cancers including myeloma and solid tumors. However, optimal designs of CARs that would maximise the activity of CAR-iNKT against myeloma and their combination with orthogonal therapeutic modalities that would expand their anti-myeloma potency have not been investigated. Here, we find that underpinned by enhanced avidity, amongst five different 2nd and 3rd generation CAR endodomains, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity in vivo. Increased avidity and anti-myeloma activity of CD28z CAR-iNKT is dependent on a novel cross talk between plexin D1 (PLXND1) on CAR-iNKT and semaphorin 4A (SEMA4A) on myeloma cells in vivo as well as in vitro. PLXND1-dependent avidity also underpins higher anti-myeloma activity of CAR-iNKT over CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated BCMA iNKT-specific engagers and selected a high affinity and efficacy design which exerts significant anti-myeloma activity in vitro and in vivo in conjunction with adoptively transferred iNKT. Finally, combined, dual target therapy with FCRL5 CD28z CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, we provide mechanism-based, proof-of-principle that a novel, optimised iNKT-based, dual-targeting combinatorial therapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies.2026/07/07GSE312032GSM9336636GSM9336635GSM9336634GSM9336633GSM9336639GSM9336638GSM9336637GSM9336641GSM933664034284312032Homo sapiens