# Eliciting or silencing complement activation: strategies for optimizing monoclonal antibodies functions

Axelle Amen, Nicole Thielens, Chantal Dumestre-Pérard, Giovanna Clavarino

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Axelle Amen, Nicole Thielens, Chantal Dumestre-Pérard, Giovanna Clavarino. Eliciting or silencing complement activation: strategies for optimizing monoclonal antibodies functions. Current Opinion in Immunology, 2025, 96, pp.102638. 10.1016/j.coi.2025.102638. hal-05244716

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Review

# Eliciting or silencing complement activation: strategies for optimizing monoclonal antibodies functions☆

Axelle Amen<fnref n="1" />, Nicole Thielens<fnref n="2" />, Chantal Dumestre-Pérard<fnref n="1" /> and
Giovanna Clavarino<fnref n="1" />

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updates

Tuning complement activity is essential to maximize therapeutic efficacy while minimizing off-target effects in monoclonal antibody (mAb) design. Complement-enhancing strategies are primarily applied to mAbs targeting pathogens, infected cells, tumors, or immunosuppressive cells within the tumor microenvironment. In contrast, complement-silencing strategies are more appropriate for mAbs used in autoimmune or inflammatory diseases, as well as in transplantation settings. This review highlights the symmetrical approaches available to either enhance or suppress complement activation: isotype or subtype selection, fragment crystallizable (Fc) region mutations, glycosylation engineering, and bispecific antibody design. These latter can either recruit C1q or block complement inhibitors to enhance activation or co-localize complement inhibitors. Additionally, Fc-deficient formats provide a straightforward way to eliminate complement-activating functions. As understanding of complement biology deepens and mAb engineering advances, especially with artificial intelligence-driven design, tailored modulation of complement activity will be central to next-generation mAb therapeutics.

## Addresses

Corresponding author: Amen, Axelle (aamen1@chu-grenoble.fr)

**Current Opinion in Immunology** 2025, 96:102638

This review comes from a themed issue on **Complement**

Edited by **Chantal Dumastre-Perard** and **Marie-Agnes Durey**

For complete overview of the section, please refer to the article collection, "Complement (2025)"

Available online 7 August 2025

https://doi.org/10.1016/j.coi.2025.102638

0952-7915/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Monoclonal antibodies (mAbs) have become a cornerstone of modern therapeutics, demonstrating remarkable efficacy in treating a wide range of diseases, including cancer, autoimmune disorders, and infectious diseases [1–4]. They can recognize antigens through their fragment antigen binding (Fab) sites and engage effector functions through the fragment crystallizable (Fc) region. Tuning the ability of mAbs to activate or silence the classical complement cascade can strongly modulate their mechanism of action and thus their therapeutic efficacy.

Upon C1q binding, initiation of the classical complement pathway leads to generation of pro-inflammatory anaphylatoxins C3a and C5a, increase of opsonization via C3b deposition, and cell lysis with the formation of the Membrane Attack Complex (MAC), this phenomenon being known as Complement Dependent Cytotoxicity (CDC) [5,6]. CDC contribution to mAb efficacy depends on several parameters, such as mAb structure, targeted antigen characteristics (its concentration, conformation as monomer or polymer, and orientation on cell membrane), expression of complement inhibitors at the targeted cell surface, as well as disease context [7].

While complement activation can be a potent mechanism for eliminating pathogens, cancer cells, or immunosuppressive cells within the tumor microenvironment [8,9], uncontrolled complement activation can also contribute to tissue damage and inflammation, as seen in autoimmune diseases, graft or sepsis settings [10–12] (Figure 1). This review will explore strategies for increasing or silencing complement activation in the context of mAb design and engineering. Finally, current perspectives in the field will be discussed.

## Increasing complement activation in monoclonal antibody design

Several strategies have been employed to increase complement activation through modifications of the mAb structure and can be used simultaneously to optimize mAb design (Figure 2).

<footnote n="1">¹Univ. Grenoble Alpes, CNRS, CEA, UMR5075, IBS, Laboratoire d'Immunologie CHU Grenoble Alpes, 38000 Grenoble, France</footnote>

<footnote n="2">²Univ. Grenoble Alpes, CNRS, CEA, UMR5075, IBS, Grenoble, France</footnote>

<footnote>* Given the role as Guest Editor, Chantal Dumastre-Perard had no involvement in the peer review of the article and has no access to information regarding its peer-review. Full responsibility for the editorial process of this article was delegated to Eric Gershwin.</footnote>
<figure><img src="image_4.png" /><figcaption>**Figure 1**</figcaption></figure>

Main pathological contexts where complement activation is either enhanced (left, red) or inhibited (right, green) through mAb design with representative mAb examples discussed in this review. *Created using BioRender.*

<figure><img src="image_5.png" /><figcaption>**Figure 2**</figcaption></figure>

mAb design strategies to promote complement activation. In light pink: bispecific mAbs, with one arm targeting the antigen and the other arm recruiting C1q or neutralizing regulatory proteins. In orange: amino acid mutations designed to either increase C1q fixation to its binding site or to promote mAbs oligomerization and finally C1q recruitment. (EFT ± AE corresponds to the combination of the following point mutations: S267E+H268F +S324T+G236A+I332E). In yellow: glyco-engineering with galactosylation or eventually afucosylation. In red: preferred isotypes for increased CDC: IgG1, IgG3, and IgM. Oligomerization/IgG3 and IgM isotypes are not at scale. *Created using BioRender.*
## Isotype and subtype selection

Human IgG isotypes and subclasses exhibit key structural differences — including variations in hinge region length, disulfide bond configuration, and glycosylation patterns — that significantly affect their capacity to bind C1q. While the pentameric (occasionally hexameric) architecture of IgM provides a highly avid platform for efficient C1q recruitment, monomeric IgG antibodies typically require multimerization to achieve comparable binding due to C1q hexameric structure [13]. Although recent progress has highlighted the theoretical advantages of IgM mAbs over their IgG counterparts in enhancing complement activation while targeting poorly immunogenic antigens (such as lipopolysaccharides, lipid A, gangliosides, proteolipids, and glycans), the clinical translation of these insights has been limited until now, as pointed out by Keyt et al [14]. Given the absence of approved monoclonal IgM therapies to date, this review will primarily address IgG mAbs. Among the IgG subclasses, IgG1 and IgG3 are more effective at activating the complement system than IgG2 and IgG4 [15,16]. Beyond their strong C1q binding, IgG1 mAbs are favored for their prolonged serum half-life and a combination of historical, manufacturing, and regulatory considerations, all factors that contribute to their dominance in the market, notably in antitumor and anti-infectious applications [17–19]. Among the 79 mAbs listed as available in the United States in 2020 in the excellent review by Lu et al [20], 52 were from the IgG1 isotype, corresponding to 66%. Example from unmodified IgG1 mAbs includes Avelumab, which targets Programmed Cell Death Ligand 1 (PD-L1) expressed on tumor cells [21].

## Amino acid mutations

Several amino acid substitutions in the CH2 and CH3 domains have been identified as able to increase the affinity for C1q [22]. An exhaustive list of amino acid mutations and their effects is available in the excellent review by Damelang et al [18]. Among them, the combination of point mutations S267E+H268F +S324T+G236A+I332E, known as 'EFT ± AE' was described by Moore et al [23] as able to increase affinity to C1q of an anti-CD20 mAb, enhancing CDC activity by 23 times.

In addition to enhancing C1q binding, certain amino acid mutations can promote IgG clustering on the target cell surface, thereby mimicking the C1q-binding platform characteristic of IgM [24–27]. For example, the E430G mutation, located at the CH2–CH3 interface, enhances Fc–Fc interactions and promotes IgG hexamerization upon antigen binding. This strategy has been shown to be promising in oncology, notably with HexaBody®-CD38 (GEN3014), a human anti-CD38 IgG1 incorporating the E430G mutation. In preclinical studies, HexaBody®-CD38 exhibited superior CDC

compared to Daratumumab, supporting its advancement into clinical trials (NCT04824794) [28]. HexaBodies targeting *Neisseria gonorrhoeae* showed increased C1q binding and deposition of C4 and C3 compared to wild-type mAbs, translating to improved efficacy in mice [29]. Similarly, a HexaBody against *Streptococcus pneumoniae* exhibited enhanced CDC across multiple serotypes *in vitro* and protected mice from severe pneumonia [30]. Alternatively, the HexElect® platform offers a novel strategy to enhance selectivity by requiring dual antigen recognition on the same cell and Fc hetero-oligomerization, enabling CDC selectively on highly specific cell types [31].

## Glycosylation engineering

Glycosylation patterns on the Fc region of mAbs significantly influence their capacity to recruit C1q. Antibodies typically carry N-linked glycans attached to asparagine (Asn) residues, while O-linked glycans, though less common, have only been reported in the hinge region of IgG3 and will not be further discussed [32]. The conserved N-linked glycan at position Asn297 in the Fc region is critical for maintaining Fc quaternary structure, and its modification can profoundly impact complement activation [33,34]. In particular, galactosylation of Fc glycans in IgG1 enhances both C1q binding and CDC and also promotes IgG hexamer formation [35]. While afucosylation is predominantly associated with enhanced antibody-dependent cellular cytotoxicity (ADCC) via increased affinity for FcγRIIIa, emerging evidence suggests it can also modulate CDC under specific conditions.

Notably, combining afucosylation with Fc-engineering strategies, such as introducing EFTAE mutations, has been shown to synergistically enhance both CDC and ADCC [36]. These findings underscore the potential of integrating glyco-engineering and protein-engineering approaches to optimize antibody effector functions. Moreover, glyco-engineering alone may represent a safer strategy to potentiate CDC, as it avoids the introduction of potentially immunogenic amino acid substitutions, making it particularly attractive for next-generation therapeutic antibody design.

## Bispecific antibodies

Bispecific antibodies (bsAbs) represent a promising strategy for harnessing the complement system to achieve targeted cell lysis [37]. Unlike conventional therapeutic antibodies, which depend on Fc-mediated interactions with C1q to initiate the classical complement pathway, bsAbs can be engineered to simultaneously bind a cell surface antigen and a component of the complement system, such as C1q. This dual specificity allows for the direct recruitment of C1q to the target cell, effectively bypassing limitations related to antigen density and membrane distribution that
typically constrain efficient C1q engagement and activation. In a study by Cruz et al. [38], multiple bsAbs were developed with one arm directed against C1q and the other against distinct antigens of bacterial or human origin. These antigens included IsdB and Psl, both abundantly expressed by *Staphylococcus aureus* and *Pseudomonas aeruginosa*, respectively; CD20, found on B cells; and glucocorticoid-induced tumor necrosis factor receptor (GITR), a co-stimulatory receptor overexpressed on activated T helper cells. All constructs demonstrated enhanced CDC, highlighting the therapeutic potential of bsAbs in both infectious or tumoral diseases.

Alternative bispecific formats have also been developed using nanobody-based constructs. Pedersen et al. [39] engineered bispecific molecules by fusing two single-domain nanobodies: one directed against C1q and the other targeting tumor-associated antigens, such as EGFR or CD38. These compact constructs demonstrated superior complement-dependent CDC compared to conventional IgG1 antibodies targeting the same

antigens, underscoring the potential of nanobody-based platforms for efficient complement recruitment.

Another strategy to enhance classical complement pathway activation involves co-targeting membrane-bound complement regulatory proteins to neutralize their functions, such as CD46 (membrane cofactor protein), CD55 (decay accelerating factor) and CD59 (MAC-inhibitory protein), which are frequently overexpressed by tumor cells as a mechanism of immune evasion. In a study by Macor et al. [40], two bsAbs were engineered to simultaneously bind CD20 and either CD55 or CD59. These constructs effectively inhibited tumor growth and prevented lymphoma development in murine models, demonstrating the therapeutic potential of overcoming local complement resistance.

## Complement silencing in monoclonal antibody design

Silencing or minimizing complement activation is desirable to avoid side effects such as inflammation, tissue damage, lysis of bystander cells and can be achieved

<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

mAb design strategies to avoid complement activation. In light green: bispecific mAbs, with one arm targeting the antigen and the other arm recruiting regulatory proteins. In gray: amino acid mutations to decrease C1q binding. (LALA ± PG corresponds to the combination of the following point mutations: L234A, L235A, P329A, G237A). In green: glyco-engineering with agalactosylation or eventually sialylation. In turquoise: preferred isotypes for decreased CDC: IgG4 and IgG2. In blue: alternative fragments. Fab: fragment antigen binding, scFv: single-chain fragment variable. Alternative fragments/IgG2 are not at scale. *Created using BioRender.*
using the strategies described below (Figure 3). Importantly, the extension of depleting mAbs originally developed for hematology, such as Rituximab, to autoimmune diseases raises important considerations regarding their mechanism of action. These mAbs often rely on CDC to eliminate target cells, which may inadvertently trigger excessive complement activation in already inflamed tissues. This dichotomy highlights the need for careful tailoring of complement functions in mAb design. Such approaches may help preserve therapeutic efficacy while minimizing the risk of exacerbating inflammation.

## Isotype selection

As seen previously, the IgG4 isotype is characterized by the lowest affinity for C1q, followed by IgG2. This property renders IgG4 and IgG2-based therapeutic antibodies particularly suitable for chronic inflammatory conditions where minimizing complement-mediated tissue damage is desirable. A prominent example is Eculizumab, a recombinant humanized mAb engineered with an IgG2/IgG4 hybrid Fc region. Eculizumab binds to complement component C5, inhibiting its cleavage into C5a and C5b, and consequently preventing the formation of MAC [41]. Eculizumab has demonstrated clinical efficacy in several complement-mediated disorders, including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and anti-aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder (NMOSD).

Another example is provided by anti-PD-1 monoclonal antibodies, such as pembrolizumab, nivolumab, cemiplimab, dostarlimab, and retifanlimab, all of which are designed with IgG4 isotype [42]. This choice aligns with the therapeutic objective of immune checkpoint inhibition — namely, to block PD-1 signaling without triggering cytotoxic depletion of PD-1-expressing T cells, these latter being essential for their antitumoral functions. Also, within the field of checkpoint blockade, isotype switching has also been used for anti-CTLA-4 mAbs. Tremelimumab was designed as an IgG2 antibody with reduced effector functions and CDC, aiming to limit Treg depletion and thereby reduce the autoimmune side effects and toxicity observed with ipilimumab, which was designed in the IgG1 format [43].

## Amino acid mutations

Specific amino acid mutations in the Fc region can also be introduced to reduce or abolish C1q binding [44]. Among the most widely characterized mutations is D270A, which minimally impacts overall Fc effector functions while reducing complement recruitment [45]; or K322A, a mutation sufficient, even alone, to abrogate C1q binding [46]. Combinatorial point mutations have also been developed, most notably the ‘LALA’ variant (L234A, L235A) as well as its extended version, ‘LALA-

PG’ (L234A, L235A, P329A, G237A), abrogating C1q binding and effector functions [47,48]. A relevant application of this strategy is demonstrated by aquaporumab (AQmab) targeting aquaporin-4 (AQP4), the principal autoantigen in seropositive NMOSD, hindering the binding of pathogenic anti-AQP4 autoantibodies. Engineered with the LALA mutations, AQmab offers a promising therapeutic approach for autoimmune conditions where tissue damage is mediated by complement activation rather than antigen binding by autoantibodies *per se* [49,50].

## Glycosylation engineering

As discussed above, N-linked glycosylation at Asn-297 in the Fc domain of IgG is essential for maintaining Fc structural integrity and enabling interactions with C1q. Site-directed mutagenesis at Asn-297 (substituting this residue with alanine, glycine, or aspartic acid) for glycosylation removal has been shown to severely impair C1q binding. In IgG1, such mutations can completely abolish CDC; in IgG3, they lead to a marked reduction in complement activation [51,52].

The glycan composition itself is also a major determinant of complement activation. Agalactosylated IgG exhibit diminished C1q affinity, though the exact mechanism remains under investigation. Finally, the role of Fc sialylation is even more nuanced. Some studies suggest that Fc sialylation suppresses pro-inflammatory effector functions, including complement activation, possibly through conformational changes or engagement of inhibitory receptors [53].

## Bispecific antibodies

In a study by Wang et al, a novel class of bsAbs has been developed to locally inhibit complement activation by recruiting endogenous regulatory proteins, such as Factor H or C4b-binding protein, to disease-relevant sites [54]. In addition, LALAPG mutations are introduced in the Fc region to prevent complement activation. This targeted strategy effectively blocks all major complement pathways (classical, lectin, and alternative) *in vitro* and *in situ*, protecting a range of cell types from complement-mediated damage. Importantly, this targeted approach offers distinct advantages over systemic complement inhibition, which is often associated with higher infection risk. By focusing complement inhibition to specific sites, these bsAbs reduce off-target effects positioning them as a promising therapeutic platform for complement-driven diseases, such as autoimmune disorders, transplant rejection, and ischemia-reperfusion injuries.

## Antibody fragments

Alternative mAb formats that lack the Fc region, such as Fab fragments, single-chain variable fragments, and nanobodies, are inherently unable to engage C1q and are particularly advantageous when the therapeutic goal is
limited to antigen binding and neutralization, especially
of soluble targets. A clinically relevant example is
Certolizumab pegol, a PEGylated Fab fragment of a
humanized anti-TNF-α antibody [55]. Due to its lack of
an Fc domain, Certolizumab does not activate the
complement system thereby reducing the risk of com-
plement-mediated inflammation, which is an important
consideration in the treatment of chronic inflammatory
conditions, such as rheumatoid arthritis and Crohn's
disease.

Perspectives

While this review has primarily focused on the interplay
between mAbs and the canonical serum functions of the
complement system, emerging research underscores the
significant role of the complosome — the intracellular
counterpart of complement — in regulating intracellular
processes, such as metabolism, gene transcription, au-
tophagy, and inflammation modulation, as reviewed in
the excellent article by Freiwald and Afzali [56]. Simi-
larly, recent research articles also described interactions
between complement, complosome, and the cGAS-
STING pathway, resulting in increased IFN-γ secretion
by host cells, particularly in autoimmune and autoin-
flammatory diseases [57]. This expanding understanding
may pave the way for new mAb-based therapies de-
signed to achieve more precise and context-dependent
immune modulation, in autoimmune and autoin-
flammatory contexts, where both systemic and cell-in-
trinsic complement functions are often disrupted.

*In vivo*, complement activation occurs alongside other
Fc-mediated effector mechanisms such as ADCC and
antibody-dependent cellular phagocytosis [58]. A better
prediction of the interplay between these effector re-
sponses is critical for optimizing therapeutic antibody
design. For instance, complement inhibition can, in
certain contexts, enhance ADCC by preventing com-
plement-mediated clearance or inhibition of natural
killer cells. To address this, the development of more
predictive models is essential, such as advanced *in vitro*
[59,60] or *in vivo* systems, including humanized mouse
models or transgenic animals expressing human com-
plement components [61]. However, translating findings
from animal models to humans remains challenging due
to interspecies differences in complement system com-
ponents, particularly in membrane-bound and soluble
regulatory proteins.

Conclusion

Through careful selection of antibody isotypes, sophis-
ticated Fc engineering, and precise glycan modifications,
it is possible to tailor the complement-activating prop-
erties of mAbs to match specific therapeutic goals. While
enhancing complement activation holds significant pro-
mises for improving antitumor and anti-infectious

disease efficacy, minimizing its activation is crucial for
avoiding unwanted inflammation and tissue damage in
autoimmune and other inflammatory conditions. In the
excellent annual review ‘Antibodies to watch’ [62],
which lists the most promising mAbs for the coming
year, the 2025 edition comprises numerous mAbs with
modifications either eliciting or silencing complement
activation, highlighting the promises of this strategy.

Finally, future advancements in our understanding of
complement biology, coupled with improved preclinical
models, will be essential to maximizing the potential of
complement modulation in therapeutic antibody design.
Innovative antibody engineering technologies will surely be
accelerated with the increased use of artificial intelligence
software [63], ultimately leading to more effective and safer
treatments for a wide range of human diseases.

**Declaration of Competing Interest**

The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.

**References and recommended reading**

Papers of particular interest, published within the period of review, have
been highlighted as:

• of special interest

• of outstanding interest

1. Zahavi D, Weiner L: Monoclonal antibodies in cancer therapy. Antibodies MDPI; 2020:1-20.

2. Mullard A: FDA approves 100th monoclonal antibody product. Nature Reviews. Drug Discovery NLM (Medline); 2021:491-495.

3. Desoubeaux G, Pelegrin M: Monoclonal antibodies in infectious diseases: new partners in the therapeutic arsenal. *Med/Sci* 2019, 35:1008-1013.

4. Goldberg BS, Ackerman ME: Antibody-mediated complement activation in pathology and protection. Immunology and Cell Biology John Wiley and Sons Inc.; 2020:305-317.

5. Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT: Complement system part I - molecular mechanisms of activation and regulation. Frontiers in Immunology Frontiers Research Foundation; 2015.

6. Merle NS, Noe R, Halbwachs-Mecarelli L, Fremeaux-Bacchi V, Roumenina LT: **Complement system part II: role in immunity.** *Complement System Part II Role Immunity Frontiers Immunology [Internet]* 2015, **6**:257(Available from), (https://hal.sorbonne-universite.fr/hal-01216398v1).

7. Weiner GJ: Building better monoclonal antibody-based therapeutics. Nature Reviews Cancer Nature Publishing Group; 2015:361-370.

8. Vacca F, Sala C, Rappuoli R: Monoclonal Antibodies for bacterial pathogens: mechanisms of action and engineering approaches for enhanced effector functions. Biomedicines MDPI; 2022.

9. Ovcinnikovs V, Dijkman K, Zom GG, Beurskens FJ, Trouw LA: Enhancing complement activation by therapeutic anti-tumor antibodies: Mechanisms, strategies, and engineering approaches. Seminars in Immunology Academic Press; 2025.

Excellent review where readers can notably find complementary in-
formation on the impacts of mAb valency and affinity, antigen spacing
and clustering, and tumor micro-environment on complement activa-
tion, all in the context of anti-tumoral mAbs.
10. Golay J, Taylor RP: The role of complement in the mechanism of action of therapeutic anti-cancer mabs. Antibodies MDPI; 2020:1-23.

11. Gunn BM, Alter G: Modulating antibody functionality in infectious disease and vaccination. Trends in Molecular Medicine Elsevier Ltd; 2016:969-982.

12. Lu LL, Suscovich TJ, Fortune SM, Alter G: Beyond binding: antibody effector functions in infectious diseases. Nature Reviews Immunology Nature Publishing Group; 2018:46-61.

13. Abendstein L, Dijkstra DJ, Tjokrodirijo RTN, van Veelen PA, Trouw LA, Hensbergen PJ, et al.: Complement is activated by elevated IgG3 hexameric platforms and deposits C4b onto distinct antibody domains. Nat Commun 2023, 14 4027.

14. Keyt BA, Baliga R, Sinclair AM, Carroll SF, Peterson MS: Structure, function, and therapeutic use of IgM antibodies. Antibodies MDPI; 2020:1-35.

15. Michaelsen TE, Sandlie I, Bratlie DB, Sandin RH, Ihle O: Structural difference in the complement activation site of human IgG1 and IgG3. Scand J Immunol 2009, 70:553-564.

16. Vidarsson G, Dekkers G, Rispens T: IgG subclasses and allotypes: from structure to effector functions. Front Immunol 2014, 5:520.

17. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H: Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 2008, 329:112-124.

18. Damelang T, Brinkhaus M, van Osch TLJ, Schuurman J, Labrijn AF, Rispens T, et al.: Impact of structural modifications of IgG antibodies on effector functions. Frontiers in Immunology Frontiers Media SA; 2023.

Excellent review providing a detailed, up-to-date overview of Fc modifications affecting effector functions such as complement activation, notably glycosylation patterns and amino acid mutations.

19. Carter PJ, Lazar GA: Next generation antibody drugs: pursuit of the “high-hanging fruit”. Nature Reviews Drug Discovery Nature Publishing Group; 2018:197-223.

20. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, et al.: Development of therapeutic antibodies for the treatment of diseases. J Biomed Sc BioMed Central Ltd.; 2020.

21. Chen Y, Pei Y, Luo J, Huang Z, Yu J, Meng X: Looking for the optimal PD-1/PD-L1 inhibitor in cancer treatment: a comparison in basic structure, function, and clinical practice. Frontiers in Immunology Frontiers Media S.A.; 2020.

22. Liu R, Oldham RJ, Teal E, Beers SA, Cragg MS: Fc-engineering for modulated effector functions—improving antibodies for cancer treatment. Antibodies MDPI; 2020:1-34.

23. Moore GL, Chen H, Karki S, Lazar GA: Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. MAbs 2010, 2:181-189.

24. Diebolder CA, Beurskens FJ, De Jong RN, Koning RI, Strumane K, Lindorfer MA, et al. Complement Is Activated by IgG Hexamers Assembled at the Cell Surface [Internet]. Available from: (https://www.science.org).

25. Strasser J, De Jong RN, Beurskens FJ, Schuurman J, Parren PWHI, Hinterdorfer P, et al.: Weak fragment crystallizable (Fc) domain interactions drive the dynamic assembly of igg oligomers upon antigen recognition. ACS Nano 2020, 14:2739-2750.

26. Strasser J, De Jong RN, Beurskens FJ, Wang G, Heck AJR, Schuurman J, et al.: Unraveling the macromolecular pathways of IgG oligomerization and complement activation on antigenic surfaces. Nano Lett 2019, 19:4787-4796.

27. van der Horst HJ, Mutis T: Enhancing Fc-mediated effector functions of monoclonal antibodies: the example of HexaBodies. Immunological Reviews John Wiley and Sons Inc; 2024:456-465.

An excellent review detailing the mechanisms of hexamerization, with a focus on the HexElect platform, a selective CDC activation technology not covered in the review due to space constraints. HexElect relies on

engineered antibody mutations that individually impair hexamerization but, when combined on cell surfaces, enable hetero-hexamer formation. This allows CDC to occur only in the presence of co-expressed antigens, ensuring targeted immune activation.

28. Hiemstra IH, Santegoets KCM, Janmaat ML, De Goeij BECG, Hagen WT, Van Dooremalen S, et al.: Preclinical Anti-Tumour Activity of HexaBody-CD38, a Next-Generation CD38 Antibody With Superior Complement-Dependent Cytotoxic Activity [Internet]. 2023. Available from: (www.thelancet.com).. HexaBody-CD38 contains the E430G mutation, promoting Fc hexamerization. In vitro, it showed potent CDC across various tumor cell types. In vivo, its anti-tumor activity surpassed Daratumumab in patient-derived xenograft models.

29. Gulati S, Beurskens FJ, de Kreuk BJ, Roza M, Zheng B, Deoliveira RB, et al.: Complement alone drives efficacy of a chimeric antigenococcal monoclonal antibody. PLoS Biol (6) 2019, 17:e3000323.

30. Aguinagalde Salazar L, Den Boer MA, Castenmiller SM, Zwarthoff SA, de Haas C, Aerts PC, et al.: Promoting Fc-Fc interactions between anti-capsular antibodies provides strong immune protection against Streptococcus pneumoniae. Elife 2023, 12:e80669.

A HexaBody carrying E430G and E345K mutations, designed to target a carbohydrate structure on the pneumococcal capsule, showed enhanced CDC against multiple serotypes and protected mice from severe pneumonia.

31. Oostindie SC, Rinaldi DA, Zom GG, Wester MJ, Paulet D, Al-Tamimi K, et al.: Logic-gated antibody pairs that selectively act on cells co-expressing two antigens. Nat Biotechnol 2022, 40:1509-1519.

32. Dekkers G, Treffers L, Plomp R, Bentlage AEH, Boer M de, Koeleman CAM, et al.: Decoding the human immunoglobulin G-glycan repertoire reveals a spectrum of Fc-receptor- and complement-mediated-effector activities. Front Immunol 2017, 8.

33. Subedi GP, Barb AW: The structural role of antibody N-glycosylation in receptor interactions. Structure 2015, 23:1573-1583.

34. Lee CH, Romain G, Yan W, Watanabe M, Charab W, Todorova B, et al.: IgG Fc domains that bind C1q but not effector Fc3 receptors delineate the importance of complement-mediated effector functions. Nat Immunol 2017, 18:889-898.

35. van Osch TLJ, Nouta J, Derksen NIL, van Mierlo G, van der Schoot CE, Wuhrer M, et al.: Fc galactosylation promotes hexamerization of human IgG1, leading to enhanced classical complement activation. J Immunol 2021, 207:1545-1554.

36. Wirt T, Rosskopf S, Rösner T, Eichholz KM, Kahrs A, Lutz S, et al.: An Fc double-engineered CD20 antibody with enhanced ability to trigger complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, [cited 2025 May 12], Transfus Med Hemotherapy [Internet] 2017, 44:292-300, https://doi.org/10.1159/000479978

37. Kontermann RE, Wing MG, Winter G: Complement recruitment using bispecific diabodies. Nat Biotechnol [Internet] (7) 1997, 15:629-631 [cited 2025 May 11] (https://www.nature.com/articles/nbt0797-629).

38. Cruz JW, Damko E, Modi B, Tu N, Meagher K, Voronina V, et al.: A novel bispecific antibody platform to direct complement activity for efficient lysis of target cells. Sci Rep 2019, 9.

39. Pedersen DV, Gytz H, Winkler MBL, Zarantonello A, Baumann N, Hansen AG, et al.: Bispecific complement engagers for targeted complement activation. J Immunol 2023, 211:403-413.

Bispecific Complement Engagers with one single domain targeting C1q and the other targeting either EGFR or CD38 demonstrated higher CDC than corresponding wild-type mAbs in certain cell types.

40. Macor P, Secco E, Mezzaroba N, Zorzet S, Durigutto P, Gaiotto T, et al.: Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice. Leukemia 2015, 29:406-414.
41. Rother RP, Rollins SA, Mojcik CF, Brodsky RA, Bell L: Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. *Nat Biotechnol* 2007, 25:1256-1264.

42. Martineau R, Susini S, Marabelle A: Fc effector function of immune checkpoint blocking antibodies in oncology. Immunological Reviews. John Wiley and Sons Inc; 2024.

43. Boulard P, Gouilleux-Gruart V, Watier H: Finding the right heavy chains for immunostimulatory antibodies. International Journal of Molecular Sciences MDPI; 2022.

44. Zhiqiang An, et al.: IgG2m4 an engineered antibody isotype with reduced Fc function. MAbs (6) 2009 1:572-579.

45. Shields RL, Namenuk AK, Hong K, Meng YG, Rae J, Briggs J, et al.: High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. *J Biol Chem* 2001, 276:6591-6604.

46. Thommesen JE, Michaelsen TE, Løset ge, Sandlie I, Brekke OH: Lysine 322 in the human IgG3C H 2 domain is crucial for antibody dependent complement activation [Internet]. *Mol Immunol* 2000, **37**:107827 (Available from), <https://www.elsevier.com/locate/molimm>.

47. Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong Y, Ultsch M, et al. : Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc [Internet]. 2000. Available from: <https://academic.oup.com/jimmunol/article/164/8/4178/8039404>.

48. Lo M, Kim HS, Tong RK, Bainbridge TW, Vernes JM, Zhang Y, et al.: Effector-attenuating substitutions that maintain antibody stability and reduce toxicity in mice. *J Biol Chem* 2017, **292**:3900-3908.

49. Duan T, Tradtrantip L, Phuan PW, Bennett JL, Verkman AS: Affinity-matured 'aquaporumab' anti-aquaporin-4 antibody for therapy of seropositive neuromyelitis optica spectrum disorders. *Neuropharmacology* 2020, **162**.

50. Prüss H: Autoantibodies in neurological disease. Nature Reviews Immunology Nature Research; 2021:798-813.

51. Hristodorov D, Fischer R, Linden L: With or without sugar? (A) glycosylation of therapeutic antibodies. *Mol Biotechnol* 2013, **54**:1056-1068.

52. Tao MH, Morrison SL: Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. *J Immunol* [Internet] 1989, **143**:2595-2601 [cited 2025 May 9] <https://pubmed.ncbi.nlm.nih.gov/2507634/>.

53. Quast I, Keller CW, Maurer MA, Giddens JP, Tackenberg B, Wang LX, et al.: Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity. *J Clin Investig* 2015, **125**:4160-4170.

54. Wang H, van de Bovenkamp FS, Dijkstra DJ, Abendstein L, **Borggreven NV**, Pool J, et al.: Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators. *Front Immunol* 2024, **15**. A one-of-a-kind bispecific antibody design capable of locally inhibiting complement activation across different cell types. The antibodies were engineered with one arm targeting either complement regulator factor H or C4b-binding protein, and the other arm targeting DNP, biotin, or HLA.

55. Sibilia J: Recombinant proteins or monoclonal antibodies: comparative properties and interest in rheumatoid arthritis. Medecine/Sciences Editions EDK; 2009:1033-1037.

56. Freiwald T, Afzali B: The secret life of complement: challenges and opportunities in exploring functions of the complosome in disease. Journal of Clinical Investigation American Society for Clinical Investigation; 2025.

57. Skopelja-Gardner S, An J, Elkon KB: Role of the cGAS-STING pathway in systemic and organ-specific diseases. Nature Reviews Nephrology Nature Research; 2022:558-572.

58. Crescioli S, Jatiani S, Moise L: With great power, comes great responsibility: the importance of broadly measuring Fc-mediated effector function early in the antibody development process. mAbs Taylor and Francis Ltd.; 2025.

59. Fischinger S, Fallon JK, Michell AR, Broge T, Suscovich TJ, Streeck H, et al.: A high-throughput, bead-based, antigen-specific assay to assess the ability of antibodies to induce complement activation. *J Immunol Methods* 2019, **473**.

60. Spencer DA, Goldberg BS, Pandey S, Ordonez T, Dufloo J, Barnette P, et al.: Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis. *Nat Commun* 2022, **13**.

61. Jackson HM, Foley KE, O'Rourke R, Stearns TM, Fathalla D, Morgan BP, et al.: A novel mouse model expressing human forms for complement receptors CR1 and CR2. *BMC Genet* 2020, **21**.

62. Crescioli S, Kaplon H, Wang L, Visweswaraiah J, Kapoor V, **Reichert JM**: *Antibodies to watch in 2025*. MAbs [Internet] 2025, **17**. Available from: <https://www.tandfonline.com/doi/full/10.1080/19420862.2024.2443538>.

A comprehensive and up-to-date list of commercialized mAbs and those in development, providing detailed information on their structure and design, including modifications intended to enhance or reduce complement recruitment.

63. Buchanan A, Bennett E, Croasdale-Wood R, Evers A, Fennell B, Furtmann N, et al.: How to think about designing smart antibodies in the age of genAI: integrating biology, technology, and experience. MAbs [Internet] 2025, **17**:2490790Available from: <http://www.ncbi.nlm.nih.gov/pubmed/40211100>.