Figure 2.
Sequential single‐cell sorting allowed the selection of cell clones expressing multimers with enhanced FHR4 mean valence and improved AP activation efficacy. (A) FHR4/VHH(T) trifunctional heteromultimer‐containing supernatants from clones F10.B1 (FHR4H) and those after three sequential cell sortings (2E3, 2E9 and IA3, respectively) showed increased FHR4 mean valence which correlates with an increase of MFI for C3b of a factor 2.32 between FHR4H and IA3. (B) Surface plasmon resonance analysis showed higher C3b binding specificity for FHR4 between the 1st (clone 2E3) and the 3rd (clone IA3) cell sorting. Sensorgrams for 2E3 (left) and IA3 (right) are depicted. (C) C3b binding increased of a factor 1.35 between captured 2E3 and IA3 multimers (58 and 78 RU for 2E3 and IA3, respectively) was observed which correlates with increase mean FHR4 valence within multimers. (D) C3b activation takes place according to a bimodal reaction only for the high FHR4 IA3 clone but not the low‐FHR4 A1 clone. At same multimer densities on SK‐OV‐3 cells (right graph), only FHR4 densities from clone IA3 – but not from clone A1 – (left graph) succeeded in overcoming the CIT (middle graph), the critical point where the competition of FHR4 with FH for C3b is strong enough to inhibit FH and to subsequently engage efficient AP‐positive amplification loop, leading to a linear increase of C3b deposition (phase 2 in the middle graph).