Enhancing immunotherapy efficacy in myeloma with a novel dual-action mitochondrial toxic peptide that overcomes resistance and induces immunogenic cell death.

Abstract Background T cell-engaging (TCE) immunotherapies have significantly improved outcomes in multiple myeloma (MM), but primary and acquired resistance limit their success. One key mechanism involves upregulation of PD-L1 on MM cells and PD-1 on T cells, leading to immune evasion and T cell exhaustion. Blocking PD-1/PD-L1 alone has limited clinical activity. We developed a novel dual-action therapeutic peptide, PP-k, that binds PD-L1 to block its interaction with PD-1 on T cells and delivers a mitochondrial-targeting proapoptotic peptide(klaklak)2, inducing direct toxic to PD-L1-expressing tumor cells. We explored the efficacy of PP-k in overcoming resistance to T cell engagers. Methods Standard MM cell lines (RPMI-8226 and H929), along with Teclistamab-resistant OPM2 and JJN3 cell lines (selected via prolonged Teclistamab exposure, an approved BCMA×CD3 bispecific antibody for relapsed MM), and their parental counterparts, were used. Cytotoxicity of PP-k was assessed using MTS assays. PP-k, Teclistamab, or their combination were evaluated in co-cultures of MM cells and healthy donor CD3⁺ T cells. For real-time monitoring, some MM cells were engineered to express GFP and secreted luciferase. Tumor cell viability was assessed by luciferase activity in conditioned media and flow cytometry, which was also used for T cell profiling. T cell activation was further measured by IFN-γ and Granzyme B secretion using ELISA. PP-k efficacy was also tested in a co-culture model with MM cells with mouse calvaria. In-vivo efficacy of PP-k was tested using NSG mice, subcutaneously implanted with human MM cells treated with PP-k (20 mg/kg, i.p.,5 doses). Results PP-k exhibited IC50 values of 8–15 µM across all cell lines. Notably, Teclistamab-resistant cells (OPM-2 and JJN3-TCE) retained PD-L1 expression levels comparable to sensitive lines and showed similar sensitivity to PP-k. In co-cultures, PP-k synergized with Teclistamab at below IC50 doses, enhancing CD8⁺ T cell activation and increasing IFN-γ/Granzyme B secretion. In a bone-mimetic coculture model (H929 cells + mouse calvaria + T cells), PP-k alone induced significant tumor elimination, underscoring its efficacy in a tumor microenvironment context. Strikingly, in T cell co-cultures with Teclistamab-resistant OPM-2-TCE and JJN3-TCE cells (which had low BCMA and were unresponsive to 0.03125-0.0625 nM Teclistamab), the combination of PP-k + Teclistamab overcame resistance, achieving near-complete tumor cell killing. While PP-k alone did not alter T cell profiles, it amplified Teclistamab-driven T cell activation. In vivo, PP-k monotherapy reduced H929 tumor burden in NSG mice. Mechanistic studies revealed that PP-k induced mitochondrial disruption and immunogenic cell death, evidenced by ATP release and calreticulin exposure. Conclusion PP-K enhances T cell-mediated killing of MM cells by blocking PD-1/PD-L1 suppression while directly inducing mitochondrial toxicity in tumor cells. It overcomes Teclistamab resistance in BCMA-low models and shows efficacy in bone-mimetic and xenograft systems. By combining immune checkpoint blockade with direct tumor cytotoxicity, PP-K represents a promising dual-function therapeutic strategy to overcome resistance and improve T cell-engaging therapies in multiple myeloma.

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