PM54重塑肿瘤微环境以增强检查点阻断疗效

Background : PM54, a next-generation synthetic ecteinascidin, combines enhanced DNA-minor-groove binding with dual activity: direct cytotoxicity and immune modulation through immunogenic cell death and innate signalling cascades. This study aimed to characterize the immunomodulatory effects of PM54 alone and in combination with PD-1/PD-L1 blockade in in vitro and humanized tumor models. Methods : PBMCs from healthy donor were pre-stimulated with anti-CD3/CD28 complex and coculture for 96h with MDA-MB-231 (high PD-L1) pre-treated with PM54 and IOs (anti-PD1/PD-L1). Number of resulting target cells was analysed by flow cytometry. NXG mice engrafted with human hematopoietic CD34+ cells (Hu-CD34 + ) were xenografted with MDA-MB-231 xenografts. Randomly allocated mice (n≥4/group) were treated with placebo, PM54 (0.6 mg/kg, i.v., Days 0, 14) ± atezolizumab (10 mg/kg, i.p., twice weekly). On day 28, animals were euthanized, and tumors weighed and analyzed by multiparametric flow cytometry and RNA-seq. Results : In vitro coculture experiments showed an increase in the immune mediated cytotoxicity of target cells upon combination of lower doses of PM54 with atezolizumab, sensitizing cells to immunotherapy. On day 28, treatments of Hu-CD34 + bearing MDA-MB-231 tumors induced a statistically significant tumor reduction (vs placebo: 988.4 mm 3 ). The median of tumor volume (mm 3 ) was 305.8 (p=0.0159) for PM54, 715.6 (p=0.0317) for atezolizumab and 199.7 (p=0.0286) for the combination. Of note, statistically significant tumor reduction was also recorded in the combination vs PM54 or atezolizumab (in both, p=0.0159). In humanized MDA-MB-231, PM54 + atezolizumab expanded CD3⁺ cells (32.2% vs 9.3%), increased CD8⁺ cells (44.7% vs 19.8%), reduced Tregs (10.0% vs 42.8%), modestly increased cytotoxic NK cells, and downregulated PD-L1 on tumor and immune compartments. Transcriptomics analysis demonstrated that the combination arm renders the highest number of deregulated genes (1404 up and 981 down genes vs 537 and 908 up genes or 278 and 552 down genes in PD-L1 o PM54 treatment, respectively). Among them, GSEA showed an increase upon treatment or combination in MHC genes, IFNg and IFNalpha response, and NK activity and TCR signaling, leading to an enhance inflammatory response. Moreover, CIBERSORT analysis of immune populations complemented cytometry evaluation with an increase in CD8 T cells, Tfh, activated DCs and NK cells and M1/M2 ratio increase upon treatment and combination. Conclusions: PM54 is a dual action immunochemotherapeutic that drives ICD, increases allogenic immune cytotoxicity of tumoral cells and, in vivo, augments effector CD8⁺ responses, diminishes Tregs, and reprograms suppressive myeloid cells. These immunologic effects synergize with PD-1/PD-L1 blockade, supporting development of PM54-based combinations for transcriptionally driven, immune-cold malignancies.