Effect of Obexelimab on the Production of Anti-Erythrocyte Autoantibodies in Vitro By Mitogen Stimulated Direct Anti-Globulin Test (MS-DAT)

Background: Autoimmune hemolytic anemia (AIHA) is a rare disease with an incidence of 0.8 to 3/100,000 people per year and is caused by an autoimmune attack against erythrocyte antigens. AIHA are classified as “warm” (wAIHA) or “cold” forms (CAD), according to the thermal amplitude of the autoantibody and based on the direct anti-globulin test. AIHA displays a multifactorial pathogenesis (genetic, environmental, and miscellaneous factors) contributing to tolerance breakdown. Several mechanisms, such as autoantibody production, complement activation and monocyte/macrophage phagocytosis are implicated in extra-/intra-vascular hemolysis with bone marrow compensation to address anemia. Several new drugs targeting B-cells, plasma cells, complement, and phagocytosis are in clinical trials. Obexelimab is a bifunctional, non-depleting humanized monoclonal antibody that binds CD19 and FcγRIIb to inhibit B cells, plasmablasts, and CD19-expressing plasma cells. FcγRIIb serves as an antibody-sensing down-regulator of humoral immunity that is naturally engaged by immune complexes for BCR-driven proliferation, cytokine secretion, differentiation and antigen presentation to T cells.

Aim: To evaluate in vitro effects of obexelimab on anti-RBC autoantibodies production in a cohort of wAIHA patients at a single tertiary hematologic center. We further aimed to compare the in vitro effect of obexelimab to rituximab.

Methods: Heparinized blood samples from 10 patients with active wAIHA (Hb<10 g/dL, LDH>1.5xULN) were diluted 1:6 with RPMI 1640 and cultured, either unstimulated or stimulated with 2 µg/mL phytohemagglutinin (PHA), for 48 hours and 1% pokeweed (PWM) for 7 days. These samples were incubated with obexelimab (5, 15, 45, 135 and 405 μg/mL), rituximab (comparator, 200 and 400 μg/mL) or IgG isotype (neutral, 5, 15, 405 μg/mL). The amount of anti-RBC-bound IgG was evaluated by the competitive immunoenzymatic assay MS-DAT. The positivity for MS-DAT was defined as a value exceeding 150 ng/mL, the mean of PHA, PMA (phorbol-12-myristate-13-acetate), and PWM-stimulated cultures plus 3 SD of 210 healthy blood donors.

Results:

In unstimulated cultures, obexelimab effectively reduced the production of anti-RBC autoantibodies in vitro with a median percentage of reduction of 13% and a maximal reduction at 15 and 405 μg/mL (26% and 27% versus baseline, respectively). Rituximab also markedly reduced anti-RBC autoantibody production in vitro as previously published (median percentage of reduction of 10%), while the isotype control had no effect as expected. In cultures stimulated with PHA, obexelimab reduced the production of anti-RBC autoantibodies by 8% and 6% at 15 and 405 μg/mL, respectively. Rituximab reduced anti-RBC autoantibody production in vitro by 30%, while the isotype control had no effect. In PWM-stimulated cultures, obexelimab markedly reduced the production of anti-RBC autoantibodies by 20% and 35% at 15 and 405 μg/mL, respectively. Rituximab reduced anti-RBC autoantibody production in vitro by 40%, while the isotype control had no effect.

Conclusion: These preliminary results demonstrate an immunomodulatory effect of obexelimab on the production of anti-erythrocyte autoantibodies in vitro either in unstimulated or mitogen-stimulated wAIHA patient whole blood cultures. This work supports the ongoing Phase 2 study, evaluating the efficacy and safety of obexelimab in patients with wAIHA.

Fattizzo:Alexion: Consultancy; Roche: Consultancy, Other: travel to congress; Novartis: Consultancy; Samsung: Speakers Bureau; Sobi: Speakers Bureau; Janssen: Consultancy; Agios: Research Funding; Zenas BioPharma: Research Funding. Matijevic:Zenas BioPharma: Current Employment. Youd:Zenas BioPharma: Current Employment. Feng:Zenas BioPharma: Current Employment. Barcellini:Alexion, AstraZeneca Rare Disease: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Consultancy; Sanofi: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.