Epcoritamab Induces in Vitro-Derived Terminally Differentiated Exhausted T Cells to Kill B Cells

Background: Exhausted T cells (T_ex) are defined by reduced effector function, yet retain responsiveness to certain immunotherapies including checkpoint inhibitors. Epcoritamab, a bispecific antibody targeting CD3 on T cells and CD20 on B cells, engages and redirects T cells to kill malignant B cells in relapsed/refractory (R/R) diffuse large B-cell lymphoma and R/R follicular lymphoma. This study evaluated the ability of epcoritamab to activate in vitro-derived T_ex cells to target and kill CD20-expressing B cells.

Methods: CD3⁺ T cells and CD20⁺ B cells were isolated from healthy donors. T cells underwent 2 cycles of stimulation during a 120-hour incubation in vitro using anti-CD3/CD28 antibodies to mimic chronic T-cell activation and induce T-cell exhaustion. Non-exhausted or in vitro-derived T_ex cells were co-cultured with B cells at a 2:1 ratio in an allogeneic mixed lymphocyte reaction. Cell activation, functional status, and cytotoxic activity were assessed using flow cytometry and cytokine profiling panels. Peripheral blood mononuclear cells (PBMCs) collected at baseline from patients with aggressive non-Hodgkin lymphoma (aNHL) enrolled in the EPCORE NHL-1 study, commercially sourced PBMCs from apparently healthy donors, and commercially sourced PBMCs from patients with multiple solid tumor types were profiled using a T-cell immune-phenotyping panel to characterize peripheral immune fitness.

Results: Patients from EPCORE NHL-1 with R/R aNHL had increased levels of peripheral T cells expressing markers associated with reduced cell function compared with healthy donors and patients with solid tumors. To investigate whether epcoritamab can activate T_ex cells, an in vitro T_ex-cell assay was utilized. Following repeated CD3/CD28 co-stimulation, in vitro-derived T_ex cells showed increased surface expression of PD-1, TIM-3, and LAG-3, with a concomitant hyporesponsive cytokine response (IFNγ/TNFα/IL-2) and reduced proliferation (Ki67) associated with T-cell dysfunction. T_ex cells also exhibited a gene expression profile similar to terminally differentiated T_ex cells with elevated levels of HAVCR2 and lower levels of TCF7, a gene highly expressed in precursor T_ex cells and downregulated as T_ex cells differentiate. Epcoritamab treatment in vitro significantly enhanced the cytotoxic activity of these T_ex cells against CD20-expressing B cells, induced IFN-γ secretion, and promoted T_ex-cell activation, demonstrating the capacity of T_ex cells to kill in a dose-dependent manner. Unexpectedly, T_ex cells showed more rapid B-cell depletion compared with non-exhausted T cells when treated with epcoritamab. Although the T_ex cells induced superior B-cell killing at 24 hours, greater T_ex-cell death was also observed over 96 hours compared with non-exhausted T cells, suggesting T_ex cells may be rapidly depleted after killing target cells.

Conclusion: Patients from EPCORE NHL-1 with R/R aNHL showed elevated levels of markers associated with exhaustion at baseline. An in vitro T_ex-cell assay demonstrated epcoritamab can effectively harness the cytotoxic potential of in vitro-derived T_ex cells to target and eliminate CD20-expressing B cells. Ongoing studies are investigating the capacity of T_ex cells to kill tumor cells in vitro. Taken together, these findings highlight the therapeutic potential of epcoritamab in inducing T_ex cell-mediated killing for the treatment of B-cell malignancies.

Lin:Genmab: Current Employment, Current equity holder in publicly-traded company. Spires:Genmab: Current Employment, Current equity holder in publicly-traded company. Gale:Genmab: Current Employment, Current equity holder in publicly-traded company. Cheadle:Genmab: Current Employment, Current equity holder in publicly-traded company. Casneuf:Genmab: Current Employment, Current equity holder in publicly-traded company. Masters:Genmab: Current Employment, Current equity holder in publicly-traded company. Grimm:Genmab: Current Employment, Current equity holder in publicly-traded company. Jabado:Genmab: Current Employment, Current equity holder in publicly-traded company. Ahmadi:Genmab: Current Employment, Current equity holder in publicly-traded company. Fereshteh:Genmab: Current Employment, Current equity holder in publicly-traded company. Jure-Kunkel:Genmab: Current Employment, Current equity holder in publicly-traded company. Steele:Janssen: Other: owns Janssen stock; AbbVie: Other: owns AbbVie stock; Genmab: Current Employment, Other: owns Genmab stock. Blum:Genmab: Current Employment, Current equity holder in publicly-traded company.