FHR4‐based immunoconjugates direct complement‐dependent cytotoxicity and phagocytosis towards HER2‐positive cancer cells

• Published in: Molecular oncology Vol. 13; no. 12; pp. 2531 - 2553
• Main Authors: Seguin‐Devaux, Carole, Plesseria, Jean‐Marc, Verschueren, Charlène, Masquelier, Cécile, Iserentant, Gilles, Fullana, Marie, Józsi, Mihály, Cohen, Jacques H. M., Dervillez, Xavier
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01-12-2019; John Wiley and Sons Inc; Wiley
• Subjects: Alternative pathway; Binding sites; C4bp; Cancer; CDC; Cell activation; Cell surface; Complement activation; Complement component C3b; Complement factor H; Complement regulatory proteins; complement resistance; Cytotoxicity; Epitopes; ErbB-2 protein; Experiments; FHR4; Immunoconjugates; Immunoglobulins; Lymphoma; MAC; Macrophages; Membrane attack complex; Monoclonal antibodies; multimers; Ofatumumab; Pathogens; Pertuzumab; Phagocytosis; Protein C; Proteins; Targeted cancer therapy; Trastuzumab; Tumors; Belgium; Canada; Germany; Luxembourg; C4bp; multimers; CDC; FHR4; MAC; complement resistance
• ISSN: 1574-7891; 1878-0261
• DOI: 10.1002/1878-0261.12554

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.
We propose a new approach of complement‐mediated destructive tumour cell targeting by generating immunoconjugates harbouring multimeric (a) factor H‐related protein 4 (FHR4)‐complement effector functions with; (b) VHH anti‐HER2 targeting functions. The number of FHR4 valences dictates the efficacy of the multimers to selectively and locally activate complement alternative pathway on HER2‐tumour cell surface, leading to complement‐dependent cytotoxicity and complement‐dependent cell‐mediated phagocytosis.