Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas

Abstract

APRIL (A proliferation-inducing ligand) is a TNF family member that binds two TNF receptor family members, TACI and BCMA. It shares these receptors with the closely related TNF family member, B-cell activating factor (BAFF). Contrary to BAFF, APRIL binds heparan sulfate proteoglycans (HSPGs), which regulates cross-linking of APRIL and efficient signaling. APRIL was originally identified as a growth promoter of solid tumors, and more recent evidence defines APRIL also as an important survival factor in several human B-cell malignancies, such as chronic lymphocytic leukemia (CLL). To target APRIL therapeutically, we developed two anti–human APRIL antibodies (hAPRIL.01A and hAPRIL.03A) that block APRIL binding to BCMA and TACI. Their antagonistic properties are unique when compared with a series of commercially available monoclonal anti–human APRIL antibodies as they prevent in vitro proliferation and IgA production of APRIL-reactive B cells. In addition, they effectively impair the CLL-like phenotype of aging APRIL transgenic mice and, more importantly, block APRIL binding to human B-cell lymphomas and prevent the survival effect induced by APRIL. We therefore conclude that these antibodies have potential for further development as therapeutics to target APRIL-dependent survival in B-cell malignancies.

The Design and Characterization of Receptor-selective APRIL Variants

“…Following incubation, cells were cooled on ice to halt endocytosis; treated for 1 min with either acid solution (0.154 M NaCl, pH 2, to strip off surface-exposed antibody) or PBS, 1% BSA as a control; and then analyzed by FACS for the presence of the remaining PE label. The efficacy of the acid stripping was tested and optimized previously (32). All APRIL receptor staining on lymphoma cells (mouse and human) were performed after incubation with FcR blocking reagent (Miltenyi Biotech, Leiden, The Netherlands).…”

“…1E). R231A, previously shown by us to be a mutation that leads to loss of both TACI and BCMA binding, but not binding to heparan sulfate proteoglycans, was included as a negative control (14,32). Binding to human BCMA-Fc was retained by all variants predicted to selectively bind BCMA (Fig.…”

“…This is a validated approach that has been used previously (14,32). Protein expression was quantified by Western blot using an anti-FLAG tag antibody ( Fig.…”

“…whether the R206E variant would also be unable to stimulate endogenous WT TACI, we used a receptor internalization assay (32). We chose the mouse A20 cell line that has been shown to express high amounts of TACI (10,32).…”

“…We chose the mouse A20 cell line that has been shown to express high amounts of TACI (10,32). Treatment with WT APRIL for 90 min at 37°C triggered TACI internalization, as shown by the high PE signal retained after acid treatment, which marks the antibody that was internalized, together with the receptor (Fig.…”