TY - JOUR
T1 - FHR4-based immunoconjugates direct complement-dependent cytotoxicity and phagocytosis towards HER2-positive cancer cells
AU - Seguin-Devaux, Carole
AU - Plesseria, Jean Marc
AU - Verschueren, Charlène
AU - Masquelier, Cécile
AU - Iserentant, Gilles
AU - Fullana, Marie
AU - Józsi, Mihály
AU - Cohen, Jacques H.M.
AU - Dervillez, Xavier
N1 - Funding Information:
This work was supported by the Luxembourg Institute of Health, the Fondation Recherche sur le SIDA of Luxembourg and by the ?Soci?t? d?Acc?leration du Transfert de Technologie du Nord? (C-Switch Project).
Publisher Copyright:
© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b-binding protein C-terminal-α-/β-chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent-positive regulator of the AP, the human factor H-related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VHH targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab-recognising epitopes [VHH(T) or VHH(P)], respectively, were used as HER2 anchoring moieties. Optimised high-FHR4 valence heteromultimeric immunoconjugates [FHR4/VHH(T) or FHR4/VHH(P)] were selected by sequential cell cloning and a selective multistep His-Trap purification. Optimised FHR4-heteromultimeric immunoconjugates successfully overcame FH-mediated complement inhibition threshold, causing increased C3b deposition on SK-OV-3, BT474 and SK-BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement-dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane-anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement-dependent cell-mediated cytotoxicity. We showed that the degree of FHR4-multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH-mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady-state towards activation on tumour cell surface.
AB - Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b-binding protein C-terminal-α-/β-chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent-positive regulator of the AP, the human factor H-related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VHH targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab-recognising epitopes [VHH(T) or VHH(P)], respectively, were used as HER2 anchoring moieties. Optimised high-FHR4 valence heteromultimeric immunoconjugates [FHR4/VHH(T) or FHR4/VHH(P)] were selected by sequential cell cloning and a selective multistep His-Trap purification. Optimised FHR4-heteromultimeric immunoconjugates successfully overcame FH-mediated complement inhibition threshold, causing increased C3b deposition on SK-OV-3, BT474 and SK-BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement-dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane-anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement-dependent cell-mediated cytotoxicity. We showed that the degree of FHR4-multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH-mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady-state towards activation on tumour cell surface.
KW - C4bp
KW - CDC
KW - FHR4
KW - MAC
KW - complement resistance
KW - multimers
UR - http://www.scopus.com/inward/record.url?scp=85073985298&partnerID=8YFLogxK
U2 - 10.1002/1878-0261.12554
DO - 10.1002/1878-0261.12554
M3 - Article
C2 - 31365168
AN - SCOPUS:85073985298
SN - 1574-7891
VL - 13
SP - 2531
EP - 2553
JO - Molecular Oncology
JF - Molecular Oncology
IS - 12
ER -