bioRxiv2024
BackgroundDirecting selective complement activation towards tumor cells is an attractive strategy to promote their elimination. We have generated Complement-activating Multimeric immunotherapeutic compleXes (CoMiX) that selectively stimulate the alternative pathway using Factor H Related protein 4 (FHR4) or the classical complement pathways using triple Fc dimers on HER2-expressing tumor cells. MethodsWe used the C4bp C-terminal--/{beta}-chain multimerising scaffolds to generate CoMiX-FHR4 and CoMiX-Fc with 2 different VHH anti-HER2, VHH(T) and VHH(P), recognising trastuzumab-or pertuzumab-competing HER2 epitopes, respectively: FHR4/VHH(T), FHR4/VHH(P), VHH(T)/Fc, VHH(P)/Fc. The different CoMiX were compared in vitro for C3b and C5b9 depositions, complement-dependent cytotoxicity, and their ability to activate NK cells and phagocytosis by macrophages using one-way ANOVA and post-hoc Tukeys tests. We further explored their therapeutic efficacy in vivo on human BT474 breast cancer xenografts established in NUDE mice, when used individually or in combination, as compared to trastuzumab or pertuzumab. ResultsFHR4/VHH(T) and FHR4/VHH(P) led to the highest C3b and C5b9 depositions and CDC, both individually and in combinations on BT474 tumor cells (p< 0.0001) surpassing the very low complement activating capacity of trastuzumab and pertuzumab. CoMiX-Fc showed NK cell activation and complement-mediated BT474 phagocytosis by M2 macrophages. In the xenograft model, CoMiX-FHR4 molecules reduced the tumor volume by a factor of 7.33 compared to the PBS control. VHH(T)/Fc had no effect on tumor growth, while VHH(P)/Fc led to a 2.75-times tumor volume reduction that was higher than pertuzumab (p< 0.01). Trastuzumab and its combination with pertuzumab remained the most potent regimen, alone or in combination, to completely inhibit tumor growth. CoMiX-FHR4, CoMiX-Fc and C3b deposition were visualized as soon as one hour after injection resulting in a massive homogeneous complement deposit 6 hours after injection. Interestingly, CoMiX-FHR4 significantly reduced the growth of trastuzumab-resistant cancer cells in contrast to trastuzumab and induced a large NK cell infiltration into the tumor. ConclusionsCoMiX-FHR4 and CoMiX VHH(P)/Fc significantly inhibit tumor growth through complement activation, NK cells infiltration, and phagocytosis by macrophages. CoMiX-FHR4 proteins delay xenograft growth of BT474 cells resistant to trastuzumab and could thus be an attractive approach when resistance to antibody emerges. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSComplement activation represents a substantial part of the overall biological activity of few therapeutic antibodies used in cancer immunotherapy. Factor H-related protein 4 can activate complement by serving as a platform for the assembly of alternative pathway C3 convertase by competing with factor H for C3b binding. We previously showed that multimeric recombinant proteins displaying the FHR4 complement effector moiety and a nanobody anti-HER2 targeting moiety selectively direct the activation of the complement alternative pathway on HER2-expressing tumor cells, leading to subsequent cell destruction through direct cell lysis or through the activation of host effector cells. What this study addsWe used in the current work a novel complement-directed tumor cell distruction strategy in vivo. We showed that CoMiX-FHR4 and CoMiX-Fc (based on triple Fc dimers), targeting HER2-positive breast tumor cells, inhibit tumor growth in a model of BT474 xenograft in NUDE mice by stimulating complement activation, BT474 death, NK cell activation, and phagocytosis of tumor cells by macrophages. CoMiX-FHR4 remain efficient in xenografts of BT474 cells resistant to trastuzumab. How this study might affect research, practice or policyWe demonstrate for the first time that directed complement activation on tumor cells is an alternative to therapeutic antibodies which is particularly promising when resistance to standard-of-care treatment occurs.