Figure 1.
Design and functional evaluation of an affinity-optimized CD19-targeted CD28 agonist. (A) CD19-CD28 is composed of one CD19 binder and one CD28 binder. The Fc part is devoid of FcγR binding (huIgG1 PGLALA). Heterodimerization and correct assembly are achieved via knob into hole (kih) mutation and CrossMab technology. (B) Binding of CD19-CD28 affinity variants to human CD28 on CHO cells, genetically modified to overexpress CD28. To monitor unspecific binding interactions, a DP47 huIgG1 was included as negative control. Binding was assessed via flow cytometry. Dots show individual values of technical duplicates. (C) Luciferase activity in an IL-2 reporter assay with Jurkat IL-2 promoter cells after 6 hours of stimulation with increasing concentrations (0.5 pM to 200 nM) of CD19-CD28 and 10 nM glofitamab. NALM-6 cells served as target cells (E:T ratio 5:1). Dots show individual values of technical duplicates. (D) Experimental design of an in vivo biodistribution study. Non–tumor bearing humanized NSG mice (3 mice per group) were treated with vehicle (histidine buffer) or 5 mg/kg of untargeted, Alexa-Fluor-647-(AF647)-labeled CD28 affinity variants. (E-F) Blood and splenic T cells were analyzed for drug binding via flow cytometry. Bars show mean + standard error of the mean (SEM) of 3 animals per group and time point. Dots show values of individual mice. (G) Experimental design of in vivo efficacy study. Humanized NSG mice (9-10 mice per group) were subcutaneously (s.c.) injected with 1 × 106 NALM-6 lymphoma cells in 1 flank. After 18 days, mice were treated IV with vehicle (histidine buffer), 0.15 mg/kg glofitamab, and 1 mg/kg CD19-CD28 variants according to the depicted timeline. (H) Tumor volumes shown as mean + SEM of 9 to 10 mice per group. Significance was calculated using an unpaired, 2-tailed Student t test. ∗∗P < .01. (I) Immunohistochemical analysis of CD8+ T-cell infiltration in tumors on study day 56. Upper row, ×2 original magnification. Lower row, ×20 original magnification. Images were captured with a VS120 virtual slide microscope (Olympus) and analyzed with Tissue Studio software (Definiens) for cell quantification. E:T, effector to target; glofit., glofitamab.

Design and functional evaluation of an affinity-optimized CD19-targeted CD28 agonist. (A) CD19-CD28 is composed of one CD19 binder and one CD28 binder. The Fc part is devoid of FcγR binding (huIgG1 PGLALA). Heterodimerization and correct assembly are achieved via knob into hole (kih) mutation and CrossMab technology. (B) Binding of CD19-CD28 affinity variants to human CD28 on CHO cells, genetically modified to overexpress CD28. To monitor unspecific binding interactions, a DP47 huIgG1 was included as negative control. Binding was assessed via flow cytometry. Dots show individual values of technical duplicates. (C) Luciferase activity in an IL-2 reporter assay with Jurkat IL-2 promoter cells after 6 hours of stimulation with increasing concentrations (0.5 pM to 200 nM) of CD19-CD28 and 10 nM glofitamab. NALM-6 cells served as target cells (E:T ratio 5:1). Dots show individual values of technical duplicates. (D) Experimental design of an in vivo biodistribution study. Non–tumor bearing humanized NSG mice (3 mice per group) were treated with vehicle (histidine buffer) or 5 mg/kg of untargeted, Alexa-Fluor-647-(AF647)-labeled CD28 affinity variants. (E-F) Blood and splenic T cells were analyzed for drug binding via flow cytometry. Bars show mean + standard error of the mean (SEM) of 3 animals per group and time point. Dots show values of individual mice. (G) Experimental design of in vivo efficacy study. Humanized NSG mice (9-10 mice per group) were subcutaneously (s.c.) injected with 1 × 106 NALM-6 lymphoma cells in 1 flank. After 18 days, mice were treated IV with vehicle (histidine buffer), 0.15 mg/kg glofitamab, and 1 mg/kg CD19-CD28 variants according to the depicted timeline. (H) Tumor volumes shown as mean + SEM of 9 to 10 mice per group. Significance was calculated using an unpaired, 2-tailed Student t test. ∗∗P < .01. (I) Immunohistochemical analysis of CD8+ T-cell infiltration in tumors on study day 56. Upper row, ×2 original magnification. Lower row, ×20 original magnification. Images were captured with a VS120 virtual slide microscope (Olympus) and analyzed with Tissue Studio software (Definiens) for cell quantification. E:T, effector to target; glofit., glofitamab.

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