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<p>Due to its central role in regulating adaptive immune responses through CD40 engagement, the CD40 ligand (CD40L) represents an attractive therapeutic target for inflammatory and autoimmune diseases. Here, CD40L-specific affibody molecules were generated by directed evolution using an <i>Escherichia coli</i> surface display platform. Two affibody candidates were identified that specifically recognized human CD40L on the surface of activated primary T cells. To exploit the homotrimeric architecture of CD40L, the affibody candidates were genetically fused to the NC1 domains of human collagen XV or XVIII, yielding well-defined homotrimeric constructs. The trimeric affibody formats were characterized with respect to oligomeric state, thermal stability, binding kinetics, cellular target engagement, and functional inhibition of CD40 signaling. NC1-mediated trimerization resulted in an approximately 100-fold improvement in apparent affinity, markedly enhanced binding to membrane-bound CD40L, and potent inhibition of CD40L-induced NF-κB activation in a reporter cell assay. Collectively, these results establish collagen XV and XVIII NC1 domains as effective trimerization modules for affibody molecules and highlight a strategy for engineering Fc-independent, multivalent antagonists targeting CD40L.</p> Graphical Abstract <p></p>

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Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年6月24日

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  • Cornelia Westerberg,
  • Chiara Sorini,
  • Mariam Al-Haddad,
  • Hanna Mehari,
  • Stefan Ståhl,
  • Maja Jagodic,
  • John Löfblom

摘要

Due to its central role in regulating adaptive immune responses through CD40 engagement, the CD40 ligand (CD40L) represents an attractive therapeutic target for inflammatory and autoimmune diseases. Here, CD40L-specific affibody molecules were generated by directed evolution using an Escherichia coli surface display platform. Two affibody candidates were identified that specifically recognized human CD40L on the surface of activated primary T cells. To exploit the homotrimeric architecture of CD40L, the affibody candidates were genetically fused to the NC1 domains of human collagen XV or XVIII, yielding well-defined homotrimeric constructs. The trimeric affibody formats were characterized with respect to oligomeric state, thermal stability, binding kinetics, cellular target engagement, and functional inhibition of CD40 signaling. NC1-mediated trimerization resulted in an approximately 100-fold improvement in apparent affinity, markedly enhanced binding to membrane-bound CD40L, and potent inhibition of CD40L-induced NF-κB activation in a reporter cell assay. Collectively, these results establish collagen XV and XVIII NC1 domains as effective trimerization modules for affibody molecules and highlight a strategy for engineering Fc-independent, multivalent antagonists targeting CD40L.

Graphical Abstract