P023 | EFFICACY OF BISPECIFIC ANTIBODIES MOSUNETUZUMAB AND GLOFITAMAB IN B-CELL LYMPHOMA CELLS IS NOT AFFECTED BY THE RESISTANCE TO RITUXIMAB, OBINUTUZUMAB AND POLATUZUMAB.

Introduction: Monoclonal antibodies (mAbs) targeting CD20 and CD79b are key (pivotal) in treating B-cell non-Hodgkin lymphoma (B-NHL). However, the mechanisms of cross-resistance to anti-CD20 mAbs, anti-CD79b antibody-drug conjugate Polatuzumab vedotin (Pola), and bispecific antibodies (BiAbs) Mosunetuzumab (Mosu) and Glofitamab (Glofit) remain unclear. Since many patients receiving BiAbs have prior exposure to anti-CD20 mAbs or Pola, we developed resistant B-NHL cell lines to investigate cross-resistance in sequential treatments. Methods: Parental DOHH-2 cells (transformed follicular NHL; DMSZ) were exposed to increasing concentrations of the anti-CD20 mAbs Rituximab (RTX) and Obinutuzumab (OBI) and Pola. Cells regrowing at the highest concentrations were cloned as D-RTX-R (40 μg/mL), D-OBI-R (256 μg/mL), and D-Pola-R (40 μg/mL) and cultured with continuous drug pressure for ≥9 months. Surface antigen expression was evaluated by flow cytometry. Cytotoxicity (direct, CDC, ADCC, and BiAbs-mediated) was measured using calcein-AM assays. Peripheral blood mononuclear cells (PBMCs) from healthy donors, along with NK cells and CD3+ T-cells isolated using anti-CD56/CD3 beads (Miltenyi), were used as effector cells. Results: D-RTX-R, D-OBI-R, and D-Pola-R had IC50 values >100-fold higher for mAb-induced direct cell death versus parental cells. D-RTX-R and D-OBI-R cells exhibited significantly reduced CD20 and CD79b levels, while D-Pola-R cells showed increased surface CD20 (Fig.). RTX- and OBI-mediated CDC and ADCC were markedly impaired in D-RTX-R and D-OBI-R cells (PBMC and NK cells ± IL2), but both mAbs effectively induced CDC and ADCC in D-Pola-R cells, with efficacy comparable or higher than parental DOHH-2. Despite significantly reduced CD20 levels, acquired resistance to RTX and OBI did not affect the ability of Glofit to promote the cytotoxicity of purified CD3+ cells, probably due to its 2:1 (CD20:CD3) configuration, whereas Mosu was less effective in D-OBI-R cells. CD3+ cell-mediated efficacy of both Mosu and Glofit was greater in D-Pola-R cells, likely due to their higher CD20 expression. Conclusion: Our data indicate that RTX and OBI effectively trigger CDC/ADCC in Pola-resistant cells. Resistance to RTX or Pola does not compromise the cytotoxicity of Mosu or Glofit. The stronger BiAb efficacy in D-Pola-R cells supports the rationale for combining Pola with Glofit or Mosu in B-NHL, for both chemonaïve and anti-CD20 pretreated patients.