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

Abstract

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.

Interaction of the Factor H Family Proteins FHR-1 and FHR-5 With DNA and Dead Cells: Implications for the Regulation of Complement Activation and Opsonization

“…FHR proteins were shown to enhance complement activation and C3 deposition on surfaces, including altered host surfaces such as those of necrotic cells ( 35 – 37 , 40 , 48 , 57 ) and tumor cells ( 81 ), but also on microbes ( 33 , 82 , 83 ). Here, we showed that FHR-1 and FHR-5 enhance complement activation on necrotic cells via their interactions with mCRP and PTX3.…”

A Family Affair: Addressing the Challenges of Factor H and the Related Proteins

“…The study by Hebecker and Józsi was the first to challenge the paradigm, demonstrating that, by binding C3b, FHR-4 in fact enhances alternative pathway activation ( 85 ), a mechanism which was also suggested previously by Närkiö-Mäkelä et al ( 96 ). This property of FHR-4 was recently exploited to overcome complement resistance of HER-2 positive tumor cells by applying FHR-4 based immunoconjugates ( 97 ). Studies by Tortajada et al and Goicoechea et al soon followed and described another mechanism, namely de-regulation by FHRs through competition with FH.…”

Identification of CFHR4 Associated With Poor Prognosis of Hepatocellular Carcinoma

“…CFHR4 as a rst-line epileptic drug phenytoin hypersensitivity prognostic marker, the sensitivity is 16%, while the speci city is 97% [38]. The above results suggest that FHR4 plays a potential role in the disease occurrence and development and may represent a novel therapeutic molecule [19].…”

“…As an amphiphilic protein, CFHR4 exists in human plasma and triglyceride-rich lipoproteins in the monomer or dimer form [14,15] and can play a role as a component of lipoproteins [16]. CFHR4 consists of a short common repetitive sequence (SCR) domain, which binds Creactive protein through its N-terminal SCR, activates the classical complement pathway, regulates complement-mediated foreign antigen processing [17], and enhances the regulation of phagocytic microorganisms and damaged host cells [18][19][20]. Studies have shown that CFHR4 shows abnormalities in various diseases, which may be connected to disease incidence and progression.…”