Immunodepletion of MDSC By AMV564, a Novel Tetravalent Bispecific CD33/CD3 T Cell Engager Restores Immune Homeostasis in MDS in Vitro

Myelodysplastic syndromes (MDS) are age-dependent and genetically diverse hematopoietic neoplasms associated with inflammation, cytopenias, myeloid dysplasia and risk for acute myeloid leukemia (AML) progression. Mounting evidence indicates that emergence of somatic gene mutations arising in hematopoietic stem and progenitor cells (HSPC) with age plays an important role in MDS pathogenesis. Our investigations indicate that sustained activation of innate immune response directs inflammation in the bone marrow (BM) microenvironment that contributes to hematopoietic and immune impairment through local generation of inflammatory cytokines, expansion of regulatory T- cells, and up-regulation and activation of pattern recognition receptors. Critical cellular effectors are the inflammation-associated immature myeloid cells termed myeloid-derived suppressor cells (MDSCs). Human MDSCs, distinguished phenotypically by CD33^high and HLA-DR⁻Lin⁻, expand and are activated in cancers. We previously reported that MDSCs are profoundly expanded in the local BM microenvironment of MDS where they contribute to MDS hematopoietic BM failure. MDSCs suppress T-cell responses and impair CD4⁺/CD8⁺ function, suggesting that this may be a key mechanism fostering clonal escape from anti-tumor immune response. Indeed, the MDSC population size limits the benefit of immune checkpoint antagonists in the treatment of solid tumors and effective strategies to deplete MDSCs are lacking. Therefore, we hypothesized the high CD33 surface density in MDSCs can be exploited for selective immune-depletion by the T-cell engager AMV564, a novel CD33/CD3 tetravalent bispecific antibody that recognizes both CD33 and CD3, currently in clinical development for AML (NCT03144245). Depletion of MDSCs may restore immune function, hematopoiesis and improve sensitivity to checkpoint inhibitors. Primary BM mononuclear cells (BMMNC) from 15 MDS patients were treated with AMV564 or isotype IgG control for 5-7 days in vitro . AMV564 treatment of MDS BMMNC eliminated CD33⁺ MDSCs in a dose-dependent manner and expanded CD4⁺ and CD8⁺ T-cells compared to controls (P ≤0.001). Proliferation of CD4⁺ and CD8⁺ T-cells, measured by Brdu incorporation, more than doubled with AMV564 treatment (P ≤0.001). IFNγ, a T-cell activation biomarker, dramatically increased in AMV564 treated cells indicating T-cells are fully activated by AMV564 at concentrations as low as 0.7 ng/ml (P ≤0.01). Moreover, depletion of MDSCs and immunological restoration was accompanied by significant improvement in colony-forming capacity after AMV564 treatment (P ≤0.01). Checkpoint inhibition alone with 10 µg/ml of anti-PD1 antibody did not result in reduction of MDSCs and T-cell activation in MDS BMMNCs, demonstrating that anti-PD1 alone is insufficient to appropriately activate T-cells or modulate MDSC cell number. Interestingly, combined treatment of MDS BMMNC with anti-PD1 and AMV564 yielded more than additive T-cell activation and increased colony forming capacity (P ≤0.01), indicating the potential to augment MDS-specific immune responses. Our findings demonstrate that AMV564 treatment selectively depletes MDSCs to reactivate T lymphocytes by improving both quantity and quality of immune responses leading to the improvement of hematopoiesis. These preclinical data provide a strong rationale for clinical investigation of this innovative approach in patients with MDS.