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J. Biol. Chem., Vol. 279, Issue 16, 16727-16735, April 16, 2004

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Engineering an APRIL-specific B Cell Maturation Antigen^*

Darshana R. Patel, Heidi J. A. Wallweber, JianPing Yin, Stephanie K. Shriver, Scot A. Marsters, Nathaniel C. Gordon, Melissa A. Starovasnik, and Robert F. Kelley¶

From the Department of Protein Engineering and Department of Molecular Oncology, Genentech, Inc., South San Francisco, California 94080

B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC₅₀ = 8 ± 5 µM) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC₅₀ = 11 ± 3 nM). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr¹³, Ile²², Gln²⁵, and Arg²⁷) that could impart ligand specificity. Substitution of Tyr¹³ was tolerated for BAFF binding but not APRIL binding. Arg²⁷ was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC₅₀ = 350 and 700 nM, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC₅₀ > 100 µM), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC₅₀ = 12 nM, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.

Received for publication, November 10, 2003 , and in revised form, February 3, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Protein Engineering, Genentech, Inc., 1 DNA Way, MS 27, South San Francisco, CA 94080. Tel.: 650-225-2321; Fax: 650-225-3734; E-mail rk{at}gene.com.

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