BPS2026 – Discovery and characterization of an inhibitory anti-CD19 nanobody

CD19 is a B cell co-receptor exclusively found on the surface of B cells and is an important target for antibody and CAR T cell therapy for autoimmunity and B cell malignancies. To define new strategies for modulating this complex, we immunized a llama with a CD19-CD81 fusion construct and generated a yeast display library. Fluorescence-activated cell sorting (FACS) resulted in the identification of a high-affinity CD19-CD81 binder, “CD001”. While previously reported anti-CD19 antibodies have limited effects on B cell activation, CD001 has the unique property of attenuating BCR signaling. B cells treated with CD001 exhibit a significantly attenuated intracellular calcium response upon BCR stimulation. To understand why CD001 has this unique property, we used cryo-electron microscopy (cryo-EM) to characterize the CD001 epitope. CD001 binds to a distinct epitope within a flexible extracellular loop on the CD19 ectodomain. Structure-guided mutagenesis of amino acid residues within the complementary determining regions (CDRs) of CD001 confirmed the binding epitope identified in our cryo-EM structure. We are currently working to understand why CD001 has inhibitory effects on B-cell activation. We hypothesized that CD001 disrupts CD19 interactions involved in signal amplification. To test this, we used peroxidase-catalyzed proximity labeling coupled to mass spectrometry to assess if CD001 treatment alters the proteins recruited to CD19 upon B-cell activation. These experiments revealed distinct spatiotemporal remodeling of the CD19 proximal proteins upon CD001 treatment compared to treatment with other CD19 antibodies. Together, these results identify CD001 as a novel, high-affinity CD19-targeting nanobody. CD001 binds to a distinct epitope compared to other CD19 antibodies and has unique functional properties. As future directions, we will engineer a second generation CD001 coupled with cytokine receptor targeting chimeras (kineTAC) and access the ability of CD001-kineTAC to maintain inhibition through kineTAC recycling.