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Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Immunoglobulin G (IgG) antibodies can form ordered hexamers upon binding cell-surface antigens.
  • Fc:Fc interactions in IgG hexamers initiate the classical complement pathway via C1q binding.
  • Engineered point mutations can enhance IgG hexamer formation and complement-dependent cytotoxicity (CDC).

Purpose of the Study:

  • To investigate the complement-dependent cytotoxicity (CDC) potential of engineered hexamer formation-enhanced (HexaBody) monoclonal antibodies (mAbs).
  • To examine the role of specific complement components in the CDC activity of HexaBody mAbs, alemtuzumab (anti-CD52), and mAb W6/32 (anti-HLA).
  • To gain insight into the enhanced efficacy of these mAbs in promoting CDC.

Main Methods:

  • Flow cytometry, high-resolution digital imaging, and four-color confocal microscopy were employed.
  • Experiments utilized B cell lines and primary chronic lymphocytic leukemia cells.
  • Sera depleted of single complement components (C1-C9) were used to assess CDC activity, alongside neutralization studies with an anti-C9 mAb.

Main Results:

  • HexaBody CD20 and CD38 mAbs demonstrated faster and more robust CDC compared to wild-type counterparts.
  • HexaBody mAbs, alemtuzumab, and mAb W6/32 showed significant CDC activity in sera lacking individual complement components C6 to C9.
  • Neutralization studies confirmed that C9 is essential for CDC activity against cell lines.

Conclusions:

  • Engineered HexaBody mAbs exhibit potent CDC activity by efficiently forming hexamers and activating the complement cascade.
  • These mAbs, along with highly avid binders like alemtuzumab and W6/32, effectively focus activated complement components (C3b, C9) on the cell surface.
  • Enhanced CDC efficacy is achieved with a low threshold of Membrane Attack Complex (MAC) binding, providing a deeper understanding of their mechanism.