In depth characterization of autologous infused bhv-1100 armored memory-like NK cells in MRD+ multiple myeloma patients receiving autologous stem cell transplant Free

Introduction/Background: Minimal residual disease (MRD) negative complete response portends better prognosis in multiple myeloma (MM) pts. Autologous stem cell transplant (ASCT) is a well-established consolidation approach. BHV-1100, an Antibody Recruiting Molecule (ARM), engages CD38 on MM cells and CD16 (via IgG) on natural killer (NK) cells to promote antibody-dependent cell cytotoxicity (ADCC) without NK fratricide. Cytokine-induced memory-like (CIML) NK cells have shown promise in myeloid malignancies, but their role in MM remains underexplored.

Methods: We conducted a prospective, open-label, single-arm, Phase 1 trial to evaluate the safety and immune effects of autologous CIML NK cells pre-coated ex vivo with BHV-1100 in MRD+ MM patients (pts) undergoing ASCT after first or second remission (NCT04634435). NK cells were collected via non-mobilized leukapheresis, activated overnight with IL-12/15/18, coated with BHV-1100, and infused after melphalan conditioning at least 24 hours prior to stem cell infusion. Seven pts (6M, 1F, median age 57 years) were enrolled in the study. All pts received ASCT in first remission. 2 pts received Daratumumab-Lenalidomide-Bortezomib-Dexamethasone (DaraRVD) and 5 pts RVD as induction therapy. Building on previously reported expansion and cytotoxicity data from infused NK cells, we present in-depth immune profiling of peripheral blood (PB) via 10X scRNA sequencing, O-link serum profiling and BM (BM) profiling via Multiparameter immunofluorescence imaging (MIFI) and flow cytometry.

Results: CIML NK cells were manufactured at 100% success rate in the first 5 pts, with an infusion dose of 5-10x10⁶ cells/kg-bw. In two pts receiving prior DARA treatment target dose was not reached (1.63x10^6 and 4.3x10⁶/kg-bw cells). Prior to ASCT, 5 pts were MRD+ sCR and 2 in VGPR. All pts improved response, including two MRD- CR at 30 days post ASCT. At most recent follow up 1 pt is deceased due to sepsis while in MRD- sCR and 6 pts are alive. We observed a 3.5-fold expansion of NK cells in the PB at D+14 (42.32% vs 12% lymphocytes (LYM)) until D+60 (15% LYM) in the first 5 pts. In the 2 DARA pre-treated pts NK cells reached 6% LYM vs <2% at screening (SRN). PB NK cells expressed genes associated with activation and cytotoxicity, including GZMB, PRF1, GNLY, and RANTES(Patient 1-4). Compared to baseline, NK cells at D+60 displayed upregulation of genes involved in immune response-activating signal transduction, leukocyte activation, and cytokine production (p=0.000001), supporting CIML NK cells to boost the NK cell compartment in MM. There was significant enrichment of interferon (IFN) alpha and gamma signaling pathways (p= 0.001, p=0.0042, respectively) in NK cells at D+28 compared to D+60, with elevated expression of IFN-stimulated genes IFITM3, CST3, and XCL2, suggesting IFN-driven activation early post-infusion. MIFI of BM biopsies revealed increased percentage of Grz+CD56+CD3- cells at D+28 compared to SRN (15% vs 4.8% CD56+ cells, p = 0.03). There was a reduction in total CD3+ cells (6.7 vs 3.67%, p = 0.0173), however an increase in GrzB expressing CD3+ cells (38.45% vs 15.54%, p = 0.0173), suggesting activation of both NK and T cells in the BM. Flow cytometry of BM aspirates showed a significant reduction in BAFF+ (45.58 vs 3.1%, p = 0.004) CD6+ (65 vs 31%, p=0.033) and CD166+ (71.62 vs 40.2%, p = 0.03) B cells between SRN and D+28. Non-statistically significant changes were noted in T cells (increased effector memory CD8+ (6% vs 16% CD8+ T cells) and effector memory conventional T cells (18.5 vs 28.5%)). Profiling of serum revealed elevations in FLT3LG (2832.59 pg/mL), HGF (1352.52 pg/mL), and IL-18 (732.1 pg/mL) between D+7 and +28, coinciding with peak NK cell numbers and stem cell engraftment (neutrophil count). At D+28 elevations in VEGFA (872.1 pg/mL), CXCL11 (1058.9pg/mL) and TNFSF12 (710.2 pg/mL) can be seen which persisted through D+60, reflecting sustained immune activation without cytokine release syndrome; there was no elevated IL-6, or IFN-g and IL-10 was only elevated at D+14.

Conclusion: This study provides detailed immune characterization of autologous, BHV-1100-coated CIML NK cells in MM pts undergoing ASCT in first remission. Findings suggest robust NK cell expansion, BM infiltration, and cytotoxic activity, with favorable cytokine profiles. These data support the potential of ARM-enhanced CIML NK therapy as an immunologic bridge in MM and warrant further investigation.