Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis

Abstract

SummaryC5 plays a major role in complement activation; C5 convertase cleaves C5 into the pro‐inflammatory C5a, and C5b, the nidus for the formation of the lytic membrane attack complex. C5 is a major target for anti‐complement drugs, necessitating better methods for the study of C5 function. Purification of C5 is complicated; classical methods involve precipitation or pH shifts that result in functional loss and low yield. We here present a method for C5 purification using a novel anti‐C5 monoclonal antibody (mAb); RO7112689 (C5i mAb, SKY59), pH‐switch engineered to induce antibody–antigen dissociation in the acidic endosome (~ pH 5·5). RO7112689 was bound on an affinity column; applied serum was completely depleted of C5. Elution at pH 5 produced fully active C5 at 98% yield. The mAb also bound C5b in the C5b6 complex, preventing C5b6 binding to target membranes and enabling purification of C5b6 from activated serum. RO7112689 inhibited C5 in mouse serum and efficiently purified mouse C5. Used as capture, RO7112689 produced sensitive and specific assays for human and mouse C5. This novel antibody enables efficient production of intact, fully active, pure human and mouse C5, and quantification of C5 in these species. The demonstration that RO7112689 binds C5b6 adds an additional mechanism of membrane attack complex inhibition by this mAb.

Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

“…Three of these human C5‐blocking mAb were selected for further characterization; testing for inhibition in other species showed that two of the mAb, 7D4 and 4G2 (both against C5 α chain), inhibited C5 in rat, rabbit, guinea pig and mouse sera, whereas 10B6 (against C5 β chain) was human‐specific. RO7112689 bound the C5 β chain, as reported previously, and was a weak inhibitor of guinea pig and mouse C5 (Fig. ), whereas Eculizumab bound C5 α and is human‐specific .…”

“…CP haemolytic activity in serum was essentially absent at 2 hr post‐induction in 4G2‐treated animals but at later time‐points, residual haemolytic activity of approximately 35% of controls was detected (Fig. c), probably due to the high sensitivity of the haemolytic assay for residual C5 . As expected, serum from the untreated control animals retained full haemolytic activity across the time course.…”

“…Sandwich ELISA were used to confirm C5 binders to eliminate issues around denaturation and demonstrate separate epitopes for test mAb to RO7112689 used as capture. Standard curves were generated using in‐house proteins; C5, C5b6 and C6 purified as previously described.…”

“…The modified form of Eculizumab, ALXN1210, uses a similar pH switch technology to increase recycling efficiency and drug half‐life . Unlike Eculizumab, RO7112689 also binds C5b6, the intermediate product of MAC formation, and so inhibits reactive lysis . RO7112689 and Eculizumab were used in this study for comparison with the novel in‐house mAb.…”

The complement C5 inhibitor crovalimab in paroxysmal nocturnal hemoglobinuria

“…Crovalimab binds to the C5 b-chain and prevents cleavage of the wild-type and SNP C5 by the C5 convertase. In addition, crovalimab uniquely inhibits C5b6 deposition on membranes, 8,9 further limiting membrane attack complex-mediated tissue damage. SMART has led to limited C5 accumulation and increased C5 binding capacity in nonhuman primates.…”