Relapsed and refractory multiple myeloma (RRMM) represents a persistent and difficult clinical problem. The majority of patients ultimately experience disease recurrence after standard therapies and have very few remaining treatment alternatives. Although BCMA CAR-T cell therapy has delivered impressive clinical responses in selected cases, its broader application is constrained by dependence on patient-derived cells, lengthy manufacturing periods, elevated costs, and significant immune-mediated toxicities. These barriers have generated a strong need for alternative immunotherapies that are safer, easier to obtain, and can be administered more promptly. Umbilical cord blood mononuclear cells (CBMC) were grown in co-culture with X-ray-irradiated bone marrow mononuclear cells (BMMC) sourced from RRMM patients, supplemented with a specific set of cytokines, to create a new immune effector cell product suitable for RRMM treatment. Phenotypic profiling and functional properties were examined, with particular focus on MHC-restricted and MHC-unrestricted killing pathways, using flow cytometric marker analysis, cytokine quantification, in vitro tumor lysis assays, and receptor-blocking studies. In vivo antitumor potency was tested in immunodeficient mouse xenograft models bearing MM.1S and RPMI-8226 human myeloma cells. Through this tumor-priming approach, we reliably expanded CD8+ NKT-like cells that displayed potent tumor-killing activity and markedly increased cytokine release when challenged with myeloma cell lines and freshly isolated RRMM patient cells. These tumor-primed CD8+ NKT-like cells (TPNC) mediated cytotoxicity via dual mechanisms: MHC-independent pathways dependent on LFA-1 and DNAM-1, as well as MHC-restricted, TCR-driven target recognition. TPNC efficiently assembled immunological synapses, rapidly redirected cytotoxic granules toward targets, and was capable of sequential killing of multiple tumor cells. In xenograft models, administration of TPNC led to a substantial reduction in tumor burden, markedly extended survival times, and detectable persistence in peripheral blood without apparent adverse effects. Applying the identical priming protocol to acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) also yielded functional TPNC with strong lytic activity. Notably, TPNC killed primary ALL cells at levels comparable to anti-CD19 CAR-NK cells. TPNC constitute an innovative cytotoxic lymphocyte population generated by tumor antigen-driven priming. Their capacity for both innate-like and adaptive-like target recognition, combined with versatile effector functions and an encouraging safety profile, suggests they could serve as a practical, scalable, and patient-specific platform for immunotherapy across multiple hematologic malignancies.
