Hexamerization-enhanced CD20 antibody mediates complement-dependent cytotoxicity in serum genetically deficient in C9

Area of Science:

• Immunology
• Cell Biology
• Hematology

Background:

• Chronic lymphocytic leukemia (CLL) cells often exhibit resistance to complement-dependent cytotoxicity (CDC).
• The terminal complement pathway, involving C9, is traditionally considered essential for effective CDC.
• Hexamer formation-enhanced monoclonal antibodies (mAbs) offer a potential strategy to overcome resistance.

Purpose of the Study:

• To investigate the efficacy of hexamer formation-enhanced CD20 mAb Hx-7D8 against patient-derived CLL cells.
• To determine the role of the terminal complement component C9 in Hx-7D8-mediated CDC.
• To elucidate the mechanism of cell death induced by Hx-7D8 in the absence of C9.

Main Methods:

• Analysis of CDC using normal human serum (NHS) and serum deficient in C9.
• Treatment of patient-derived CLL cells with Hx-7D8.
• Assessment of intracellular calcium (Ca2+) increases using FLUO-4.
• Evaluation of cell viability using TOPRO-3 staining.

Main Results:

• Hx-7D8 induced significant CDC, killing up to 80% of CLL cells, even in the complete absence of C9.
• Transient intracellular Ca2+ increases were observed during CDC in C9-deficient serum, persisting longer than expected.
• The retained Ca2+ signal correlated with TOPRO-3 staining, indicating Ca2+ influx as a key mediator of cell death.

Conclusions:

• Efficient antibody-mediated hexamer formation enables CDC through narrow C5b-8 pores, independent of C9.
• Small C5b-8 pores facilitate Ca2+ influx, which is the proximate cause of cell death during CDC.
• Hx-7D8 demonstrates potent anti-leukemic activity against CLL cells, suggesting its therapeutic potential.