Triple combination of glofitamab with CD19-CD28 and CD19–4-1BBL deepens and prolongs antitumor responses in vivo. (A) PBMC-derived T cells from a healthy donor were stimulated with glofitamab and a dose titration of CD19-CD28. Graphs show 4-1BB expression of CD4⁺ and CD8⁺ T cells after 48 hours, assessed via flow cytometry. Shown are mean + SEM of technical triplicates. (B) Cytokine release of splenocytes derived from a patient with BCL after 72 hours of stimulation with 25 pM glofitamab and/or CD19-CD28 or CD19–4-1BBL at 0.5 or 1 nM. Cytokines were assessed via cytokine bead array. Bars show means and dots indicate individual values of technical duplicates. (C) Experimental design of in vivo efficacy study in OCI-Ly18 tumor-bearing humanized NSG mice. Twelve mice per group received vehicle (histidine buffer) or obinutuzumab pretreatment (Gpt, 30 mg/kg), followed by weekly injections of glofitamab (5 mg/kg) alone or in combination with CD19-CD28 (1 mg/kg), CD19–4-1BBL (1 mg/kg) according to the depicted scheme. Costimulatory antibodies were injected simultaneously with glofitamab. (D-E) Tumor volumes assessed via caliper. Each line represents tumor volume over time in 1 mouse.