Vaccinal antibodies: Fc antibody engineering to improve the antiviral antibody response and induce vaccine-like effects

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic highlights the urgent clinical need for efficient virus therapies and vaccines. Although the functional importance of antibodies is indisputable in viral infections, there are still significant unmet needs that require vast improvements in antibody-based therapeutics. The IgG Fc domain can be engineered to produce antibodies with tailored and potent responses that will meet these clinical demands. Engaging Fc receptors (FcRs) to perform effector functions as cytotoxicity, phagocytosis, complement activation, intracellular neutralization and controlling antibody persistence. Furthermore, it produces vaccine-like effects by activating signals to stimulate T-cell responses, have proven to be required for protection, as neutralization alone does not off the full protection capacity of antibodies. This review highlights antiviral Fc functions and FcRs’ contributions in linking innate and adaptive immunity against viral threats. Moreover, it provides the latest Fc engineering strategies to improve the safety and efficacy of human antiviral antibodies and vaccines.

KEYWORDS:

• Antibody engineering
• Fc effector functions
• antibody recycling
• antibody intracellular neutralization
• antibody antiviral responses
• T-cell responses
• vaccines

Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused significant morbidity and mortality worldwide. The urgent need to develop effective therapies and vaccines during COVID-19 pandemic has led to intense research efforts to overcome the limitations of current clinical treatment options. Although past and some of the current therapies have failed to show promising results, antibody-based treatments offer a significant benefit for SARS-COV-2 patients with an 80% reduction in hospitalization and disease progression rates.^1–3

After a viral infection, the human immune response generates antiviral antibodies to control viral spread in addition to preventing and providing long-term protection against reinfection.⁴ IgG antibodies are the most abundant immunoglobulin present in plasma and have been widely studied and applied in various clinical settings. Their characteristic features, such as their long half-life that is produced by their interactions with the neonatal Fc receptor (FcRn), neutralization potency and mediating Fc effector functions that result from their interactions with FcγRs and the complement system, have made them of particular interest.⁵

The four isotypes of human IgG are IgG1, IgG2, IgG3 and IgG4. These isotypes differ in their structural and functional properties, such as their binding affinities and interactions with immune effector cells, which affect their functional outcomes. It has been suggested that during natural infection, IgG isotypes have specific roles and differ in their responses to particular pathogens, antigens and epitopes.⁵

In viral infections, IgG1 and IgG3 are the most commonly induced isotypes, and they have the highest binding affinity for FcγRs, complement activation and potent effector functions, such as cellular cytotoxicity and the phagocytosis response.^6,7 Due to the short half-life of IgG3, the most preferred isotype for therapeutic activity is IgG1.⁸ In addition, this isotype is the most frequently applied to human antiviral antibodies.⁹

As shown in Figure 1, human antibodies are characterized by a Y-shaped structure. They consist of two fragment antigen-binding domains (Fabs) and a fragment crystallizable domain (Fc). The two domains are connected at their hinge regions, and both have important functions.¹⁰ When fighting viruses, the Fab domains mainly mediate virus neutralization, while the Fc domain activates a wide range of FcRs, which are expressed on vital effector immune cells and produce potent effects.¹¹ This activation triggers the elimination of viruses and the killing of infected cells by Fc effector functions, which include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC).¹²

Figure 1. The structural insights of the complete structure of IgG antibody. (A) Heavy chains are in blue, while light chains are in green and red. Glycosylation location is in white and light orange. PDB entry 1IGY. (B) surface IgG structure by domains: variable fragment (Fv), antigen-binding fragment (Fab) and fragment crystallizable region (Fc). PDB entry 1IGY. (C) IgG Fc domains: CH2 in green and CH3 in blue. Glycosylation site in light gray. PDB entry 1FC1.

Interestingly, antibodies can facilitate immune signals that stimulate T-cell responses by interacting with major immune antigen-presenting cells (APCs) to produce vaccine-like effects.^13–15 However, the use of antibodies and the number of approved antiviral monoclonal antibodies (mAbs) to combat viral threats are still limited.¹⁶ There are multiple reasons for this limited use, including a poor understanding of the actual mechanism of antibodies’ modes of action once injected into patients, the effect of different binding affinities among Fc and FcRs and the effect of antibodies’ serum concentrations required to reach the optimal level.¹⁷

The role of Fc-mediated activities in viral infections has been well studied and investigated,^18,19 but its clinical application remains a challenge. In the absence of human-augmented natural antibody responses,²⁰ several obstacles remain, including the specific contribution of different FcRs in viral infection and recovery, the involved signaling pathways and key receptors, the cell types that impact antiviral activity, the diversity of FcγRs across different species and the structural complexity of the different receptors that interact with the Fc domain.^21–23 Moreover, a better understanding of the selective engagement of antibody effector functions that mediate a protective response is required before using the antibody Fc domain to provide protection by inducing a T-cell response.^9,24,25

Antibody engineering is used to minimize and overcome some limitations and improve therapeutic outcomes.²⁰ Antibody Fc domain engineering has been extensively used to address the limitations that restrain antibody functionality for human therapy. This type of engineering can modulate the effector functions and circulation half-life and also optimizes the biophysical and biochemical properties of antibodies.^26–28 The new generation of therapeutic antibodies is heavily engineered with a main focus on the Fc region.²⁹

This review article aims to summarize advances in IgG Fc domain engineering to present the most effective antiviral responses that can lead to long-term viral protection. It also intends to highlight the mechanism of actions and signaling pathways of IgG Fc and its different receptors (FcRs) that trigger effector functions. Furthermore, the article aims to enhance Fc domain performance by providing the latest strategies and technologies being used to improve the overall response of human antiviral antibodies. The reader will gain knowledge about how to tailor the antibody response by altering its Fc domain and how selective activation or inhibition could result in protective activity.

An overview of IgG FcRs

FcRs are expressed in leukocytes, such as neutrophils, eosinophils, monocytes, macrophages, dendritic cells, natural killer (NK) cells, B-cells and T-cells; FcγRIIB on multipotent CD8 +³⁰ and FcγRIIIA are expressed in subpopulations of activated CD4 + T-cells.³¹ There are two main types of FcRs in humans: classical and non-classical FcRs. Classical type I FcγRs (FcγRI) are broadly classified as activation and inhibition receptors based on the presence of immunoreceptor tyrosine-based activation motif or immunoreceptor tyrosine-based inhibitory motif intracellular domains. Activating FcγRs include FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA and FcγIIIB. FcγRIIB is the inhibitory receptor for FcγRs.²⁵ Most of the effector leukocytes co-express both activitory and inhibitory FcγR; this cellular feature provides a sufficient balance and also allows regulation between receptors.³²

Although all the signaling events that activate FcγRs are similar, the specific biological outcomes for the effector functions are quite diverse.²⁵ The non-classical type of human FcRs involves C-type lectin receptors, such as DC-SIGN and FcεRII (CD23). Other non-classical FcRs include FcRn and tripartite motif-containing-21 (TRIM-21).³³ These receptors have distinctions in their structures, binding interfaces, ligand specificity and functions compared to type I FcγRs.³⁴ The final outcomes of these receptors’ interactions are influenced by several factors, such as the antibody isotype, the FcR type, the glycosylation state, the conformational state of the Ig Fc domain and the involvement of other signaling molecules.³⁵

Fc: FcγR’s effector functions

Antibody-dependent cellular phagocytosis (ADCP)

ADCP is a significant Fc effector function for clearing IgG-opsonized pathogens and also plays a role in antigen presentation.³⁶ This process is mediated by phagocytic cells (such as neutrophils and macrophages) and is initiated by interacting with FcγR’s receptors.³⁷ FcγRIIA is the prime mediator involved in ADCP to combat different viruses or virus-infected cells.³⁸ However, the signaling pathway for FcγR cross-linking in phagocytes is activated by the phosphorylation of Src family tyrosine kinases, which triggers the activation of other kinases (such as Akt, p70S6 and inositol 3-kinases).^39,40

Next, antibody-opsonized viruses are guided to lysosomes; thus, antigens are being processed into peptides .⁴¹ The cell type is a crucial factor that determines the outcome of phagocytosis. To elaborate, when macrophages drive the antibody phagocytosis process, it results in improved pathogen destruction and enhanced antigen presentation.³⁶ Conversely, when plasmacytoid dendritic cells drive the antibody phagocytosis process, they secrete IFN-α,⁴² which is considered a major antimicrobial defense mechanism.^43,44

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC is one of the major IgG Fc-dependent effector functions. It plays an important role in antiviral immunity and the clearance of viral infections and is usually mediated by NK cells; recent studies have also implicated neutrophils as mediators.⁴⁵ To induce an efficient ADCC response, an antibody’s Fc domain interacts with NK cells (Fc receptor-bearing cells) that express FcγRIIIA, which results in the production of cytotoxic granules, perforins and granzymes.⁴⁶ In viral infections, perforins create pores in the virus-infected cell’s surface, facilitating the entry of granzymes to cleave death substrates and drive infected cells into apoptosis.⁴⁷

Complement-dependent cytotoxicity (CDC)

Complement activation, which leads to virolysis and direct cell lysis, is considered one of the key effector functions for fighting viral infections. Antibodies are capable of triggering complement activation. This mechanism contributes to pathogen elimination in two ways: either directly via CDC or indirectly via phagocytic clearance.¹² The classical complement activation pathway is initiated when C1q binds to the Fc CH2 domain of antibodies bound to virus-infected cells.^48,49 Upon binding, the proteolytic cascade is activated, which leads to the release of anaphylatoxins (such as C3a and C5a) and the formation of the membrane attack complex (MAC), which forms pores that cause the lysis of target cells.⁵⁰

Triggering Fc effector functions to induce antiviral responses and vaccine-like effects by linking innate and adaptive immunity

Antigen presenting cells (DC): FcγRs

To activate a T-cell response, three signals are required: Signal 1 is the interaction between T-cell receptors (TCRs) and MHC I or MHC II, which present the antigen peptide. Signal 2 involves the expression of co-stimulatory molecules, and signal 3 is characterized by the release of anti-inflammatory cytokines.¹³ By engaging FcγR activation in APCs as DC, antibodies can induce T-cell responses by binding to immune complexes and triggering antigen processing for their presentation to CD4+ and CD8 + T-cells.^15,51 A unique feature of DCs is that they express both the activitory FcγRIIA and the inhibitory FcγRIIB receptor to maintain a balanced regulation of DC maturation.⁵²

The selective engagement of FcγRIIA in DC stimulates protective CD8 + T-cell responses and promotes long-term protection against viral infections.²² Upon the cross-linking of FcγRs with DC via FcγRIIA receptors, several biological effects take place that trigger all components required to activate T-cell responses for antigen processing and presentation.⁵³ Moreover, there is also an increase in the expression of co-stimulatory molecules,^13,54 such as CD80, CD86⁵⁵ and CD40.^55,56

Cytokine production due to the activation of FcγRs stimulates host antiviral immunity,⁵⁷ leading to the delivery of monocytes and DCs to the infection site to clear viral infections.⁵⁸ Examples of pro-inflammatory cytokines are tumor necrosis factor alpha⁵⁹ and interleukin-6.⁶⁰ Further evidence to support vaccine-like T-cell responses mediated by FcγR signaling in DC was offered by DiLillo and Ravetch,¹⁵ who investigated the mechanisms of inducing anti-tumor vaccinal effects in lymphoma patients treated with Rituximab. Their findings showed improved therapeutic outcomes via specific anti-idiotype T-cell responses that developed after mAb treatment. DiLillo and Ravetch¹⁵ utilized FcγR-humanized mice and observed that the selective engagement of hFcγRIIA with DCs stimulated long-term immunity and generated a potent vaccine-like effect. However, further research and studies are needed to understand the role of FcγRs in producing potent and directed T-cell responses in viral infections and to determine how the modulation of cytokines can shape the properties of FcγRs during viral infections.⁵⁷

TRIM-21: Fc

In viral infections, antibodies can be carried inside a cell’s cytosol, where they bind to TRIM-21.⁶¹ The intracellular Fc receptor TRIM-21 is activated when it binds to the Fc region of virus-associated antibodies and drives the formed immune complex into rapid proteasomal degradation. This mechanism is known as antibody-dependent intracellular neutralization (ADIN).⁶² ADIN that is mediated by TRIM-21 performs a dual effector mechanism inside cells; it eliminates viruses and also prevents their replication through proteasomal degradation.⁶² In addition, ADIN triggers innate signaling pathways to produce proinflammatory cytokines, such as NF-kB, AP-1, IRF3, IRF5 and IRF7, thus generating an antiviral state.⁶³ ADIN stimulation against incoming virions results in infection blocking,⁶¹ while the targeting of specific proteins leads to selective depletion, which is also called TRIM-Away.⁶⁴

Furthermore, activating the TRIM-21 Fc receptor mediates immune protection by inducing a cytotoxic T-cell killing response against viral infections. Caddy, Vaysburd⁶⁵ used the lymphocytic choriomeningitis virus (LCMV) as a model system to analyze the role of non-neutralizing anti-nucleoprotein (N) antibodies in providing protection by stimulating TRIM-21 interaction. After forming the phagocytosed antibody-N-protein immune complex, it was imported into the cytosol where it was recognized and detected by TRIM-21, which triggers ubiquitination and proteasomal degradation of the immune complex.⁶⁵ The results showed that TRIM-21 drives cross-presentation to display the proteasome-derived peptides for MHC class I and activates CD8 + T-cells, which may have future implications for designing vaccines.⁶⁵

Viruses continue to develop complicated pathways to evade antibody responses, such as mutating to escape from neutralizing antibodies. For example, the human immunodeficiency virus (HIV) mutates its viral neutralizing determinants to evade the responses of neutralizing antibodies during infection.⁶⁶ Such issues highlight the importance of the role of antibody cellular neutralization and other Fc effector functions in enhancing the protective efficacy of neutralizing and of N antibodies.

FcRn: Fc

FcRn plays a vital role in controlling antibodies’ persistence in the circulation;⁶⁷ it mediates virus neutralization⁶⁸ and facilitates antigen presentation to drive T-cell responses.⁶⁹ As a pH-dependent receptor, FcRn protects antibodies from lysosomal degradation by engulfing the antibody in acidic media (pH: 6–6.5) and then recycling it back into circulation, leading to a long half-life for antibodies.⁷⁰

Bai, Ye⁶⁸ studied the intracellular neutralizing activity of an influenza hemagglutinin-specific monoclonal antibody (Y8-mAb), which is characterized by mediating viral neutralization at acidic pHs only. The results of a mice model indicated a significant decrease in viral replication, pulmonary virus titers and viral inflammation in the lungs.⁶⁸ The mechanisms of Y8-mAb, which caused a reduction in viral replication, may be due to the interruption in viral fusion that occurs at an acidic endosomal pH, which blocks the interaction, while FcRn facilities the ordination of this interaction by organizing IgG in the endosome and also may boost the IgG endosomal concentration to block viral binding.⁶⁸

Moreover, Qiao, Kobayashi⁷¹ proposed that a further function of FcRn may be antibody-mediated antigen presentation in dendritic cells. Baker, Rath⁷² showed that FcRn enhances CD8 + T-cell responses and plays a significant role in the mucosal immune system. Due to these characteristic protection features, FcRn has become an attractive target for enhancing protective humoral immunity, pharmacological interventions and vaccination strategies.⁷³

Vaccines induce Fc effector functions

There is a significant body of evidence that describes the importance of Fc effector functions in infectious disease control and protection; therefore, designing vaccines to induce optimum Fc effector functions has become a promising strategy for vaccine development.^4,74

Vaccines are currently being evaluated to induce neutralizing antibodies with Fc effector functions, as with the ChAdOx1 nCov-19 vaccine⁷⁵ and the RTS, S/AS01 malaria vaccine.^76,77 Designing vaccine adjuvants to stimulate Fc effectors is a key strategy. Coler, Day⁷⁸ designed the ID93 Tuberculosis (TB) vaccine and used ID93 with GLA-SE, which is a fusion protein TB vaccine candidate combined with a toll-like receptor- 4 (TLR-4) agonist adjuvant. The results from the first human trial revealed the beneficial antibody Fc effector functions of ADCC and ADCP along with supported safety and immunogenicity profiles.⁷⁸ In addition, it was now possible to design immunogens to produce both a neutralizing and Fc effector response. A key factor to consider here is the steric hindrance effect caused by antibodies that target particular epitopes, as they may cause structural constraints that affect FcγR binding.⁷⁴

To elaborate further, Bournazos, DiLillo⁷⁹ characterized anti-Ebola virus (EBOV) antibodies that target glycoprotein epitopes based on their FcγR dependence. They found that epitopes located on glycoproteins induced optimal in vivo antiviral activity by mediating Fc effector activity. In contrast, epitopes that target membrane-mediated Fab activities and neutralization without depending on Fc–FcγR interactions to confer activity have also been described.⁷⁹

This particular area of research has recently gained attention with the publication of actual clinical applications, such as the second dose of the ChAdOx1 nCov-19 vaccine, which was able to induce effector functions via antibody-dependent neutrophil/monocyte phagocytosis, complement activation and NK cells activation.⁷⁵

Several factors may impact the quality of vaccines to induce Fc effector functions, including the route of immunization and the required number of immunizations, along with the minimum and maximum therapeutic dose.^74,80 Additional vaccines that have induced Fc effector functions are listed in Table 1.

Table 1. Human vaccines that induced Fc effector functions and produced protection or reduced the infection risk. Abbreviations: Human cytomegalovirus (HCMV)

Infectious agent	Vaccine	Fc Effector Functions	Reference
SARS-COV-2	ChAdOx1 nCov-19	Antibody-dependent neutrophil/monocyte phagocytosis, complement activation and NK cells activation	^Citation75
	mRNA BNT162b2	Antibody-dependent cellular cytotoxicity (ADCC)	^Citation81
Malaria	RTS, S/AS01	Engagement of FcγRIIA, Antibody-mediated phagocytosis (ADCP)	^Citation82
Tuberculosis	ID93 + GLA-SE	Antibody-dependent phagocytosis (ADCP) NK cell activation Antibody-dependent cellular cytotoxicity (ADCC)	^Citation78
HCMV	gB/MF59	Antibody-dependent cellular cytotoxicity (ADCC) Antibody-dependent cellular phagocytosis (ADCP)	^Citation83
HIV	RV144	Antibody-dependent complement activation Antibody-dependent cell-mediated cytotoxicity (ADCC)	^Citation84–86

However, further work is needed to address the obstacles and the limitations in this area, such as assay standardization, understanding the molecular mechanism that drives these Fc effector functions in vaccination and how to translate the promising findings of non-human models to humans.⁷⁴ Fc engineering strategies are described below with recent examples to improve the link between innate and adaptive immunity.

Fc antibody engineering to produce potent antiviral responses and vaccine-like effects

Fc antibody engineering for improved IgG effector functions

Optimizing the binding affinity of human FcγRs is a critical feature of therapeutic antibody functions. Increasing the binding affinity of Fc to activate selected receptors of FcγRs is a well-established engineering approach. This approach is important, as most FcγRs have a low affinity for their ligand (>1 μM), except for FcγRI.⁸⁷

Fc engineering for improved ADCC

NK-mediated ADCC is influential in tailoring the efficacy and potency of therapeutic antibodies. ADCC can by modified in two ways: amino acid mutations and glyco-engineering. FcγRIIIA is the key receptor involved in mediating this interaction, and it has been targeted in many studies that aimed to enhance the binding affinity of FcγRIIIA to develop antibodies with a potent ADCC response .⁸⁸

Stavenhagen, Gorlatov⁸⁹ employed yeast surface display techniques to identify five mutations in a novel Fc, F243L/R292P/Y300L/V305I/P396L (LPLIL), which improve FcγRIIIA’s binding affinity. Introducing these mutations into the IgG1 Fc domain has dramatically enhanced FcγRIII binding, and ADCC activity was boosted by up to 100 times.⁸⁹ Clinically, LPLIL was utilized in Margetuximab (Fc-optimized anti-HER2 mAb).⁹⁰ It showed decreased FcγRIIB binding, stimulated macrophages and activated HER2 antigen presentation.^91,92 The Fc-optimized Margetuximab has reached phase-III clinical trials, and promising results have been presented by Rugo, Im.⁹³

Shields, Namenuk⁹⁴ used alanine scanning mutagenesis to identify three Fc variants (S298A, E333A and K334A), which are known as AAA. AAA mutations increased IgG1’s binding affinity for FcγRIIIA while at the same time decreasing its affinity for binding to FcγRIIB. This alteration resulted in potent ADCC activity mediated by human NK cells. The same research team also identified selected Fc mutations E333A, K334A and A339T, which selectively enhance the binding to FcγRIIIA.⁹⁴ When AAA Fc mutations were inserted in Trastuzumab (used for breast cancer treatment), a 50–100-time enhanced killing of Her2+ cells was demonstrated as a result of the improved FcγRIIIA binding.⁹⁴

Fc glycoengineering to improve ADCC

All human IgG molecules have one conserved glycosylation site at N297.⁹⁵ Consequently, the attached glycans at N297 in Fc are significant for effective ADCC responses.^96,97 Since human sera contain fucosylated IgGs,⁹⁸ engineered afucosylated antibodies created by removing the Fc N-glycan fucose residues can increase the ADCC response by improving the binding affinity of FcγRIIIA.^99,100 Shields, Lai¹⁰¹ found that antibodies deficient in fucose had a 50-fold increased binding affinity for Fc FcγRIIIA, which resulted in an improved ADCC response. In addition, a synergistic effect was observed when combining the afucosylated antibodies with the enhanced ADCC IgG1 Fc variant.¹⁰¹ Mogamulizumab (afucosylated mAb for mycosis fungoides [MF]) is an example of an approved afucosylated antibody with a potent ADCC response.^102,103

Two technologies have been applied to engineer antibodies with reduced fucosylation: GlycoMab® technology¹⁰⁴ and Potelligent® technology.¹⁰⁵ However, antibody afucosylation remains a widely accepted glycoengineering strategy for improving ADCC activity¹⁰⁵ and producing enhanced antiviral activity.¹⁰⁶

Fc engineering for improved ADCP

Macrophage-mediated ADCP is crucial for designing antiviral antibodies with an effective antiviral response. FcγRIIA is commonly used by macrophages in ADCP and has been the focus of previous antibody research.^107–109

Richards, Karki¹¹⁰ screened up to 900 Fc variants to study the effects of different binding affinities of FcγR and the effect of inserting enhancement mutations. That study concluded that the G236A mutation (GA) alone improved the binding affinity of selective FcγRIIA by six to seven times in both alleles, which led to a strong ADCP response (FcγRIIA-dependent phagocytosis). This Fc mutation increased the binding affinity of FcγRIIA without affecting FcγRIIB but also decreased the IgG1 binding affinity. This issue was resolved by inserting the following combination of three mutations: G236A, S239D and I332E. This solution improved the phagocytosis of macrophages by up to 70 times and enhanced the FcγRIIIA-mediated ADCC of antibody-coated target cells by up to 31 times.¹¹⁰ Consequently, the GA mutation was inserted into the humanized IgG1 anti-EpCAM antibody, and elevated ADCP activity was indicated compared to the wild-type IgG.¹¹⁰

Lazar, Dang¹¹¹ used both computational design algorithms and high-throughput screening methods to design optimized Fc mutations. They identified three Fc mutants (S239D, A330L, I332E [DLE]) with an enhanced binding affinity for FcγRIIIA. As expected, the ADCC response increased when DLE was inserted into MEDI-522 (anti-integrin antibody). In addition, inserting DLE into Rituximab resulted in an enhanced ADCP response.¹¹¹

Smith, DiLillo¹¹² used FcγR humanized mice to selectively enhance the binding affinities of FcγRIIA and FcγRIIIA. They generated Fc mutations that consisted of G236A/S239D/A330L/I332E (GASDALIE).¹¹³ Accordingly, FcγRIIA was improved by 25 times, and FcγRIIIA was enhanced by 30 times.¹¹² Although these mutations increased the binding affinity of FcγRIIA, the effects of ADCP have yet to be determined in humans. Only a limited number of studies have attempted to engineer FcγRIIA for antiviral therapies, and several molecular mechanisms have yet to be identified.

Fc engineering for improved CDC

The interaction of C1q with the Fc domain is a required step for initiating CDC pathways and leading to potent complement fixation and direct virolysis.¹¹⁴ This was a targeted strategy for engineering antibodies to produce potent CDC responses.¹¹⁵

Moore, Chen¹¹⁶ designed and screened 38 Fc mutations to investigate their ability to produce effective CDC activity. They observed that the triplet mutation in S267E, H268F and S324T (EFT) had boosted C1q binding by 47-fold. As a result, the CDC activity increased 6.9-fold. However, the EFT mutation also led to low ADCP and ADCC activity as well as an unwanted increased binding affinity to the inhibitory FcγRIIB.

Adding the ADCC-enhancing mutations G236A and I332E resolved this issue. As a result, the ADCC functionality was restored to be similar to the wild-type IgG1.¹¹⁶ Therefore, removing S267E from the EFT mutations may reduce the FcγRIIB binding affinity and lower the CDC response. Thus, this study showed the importance of balancing effector functions to reach the desired outcome. Furthermore, Idusogie, Wong¹¹⁷ utilized site-directed mutagenesis to construct Rituximab mutants to enhance the drug’s CDC activity. They found that using the double Fc mutations K326A and E333A produced an improved binding affinity of Rituximab to CIq and thus enhanced the CDC response by 50% compared to wild-type Rituximab.¹¹⁷

Hexamerization using HexaBody technology is another strategy for enhancing C1q to produce potent CDC.⁴⁹ The characteristic feature of this approach is the targeting of Fc-Fc engagement to form organized antibody hexamers using E345R, E430G and S440Y (RGY) or the single mutation E345R. The formed antibody hexamers that activate the complement cascade only when the antigen has been bound to the targeted cell, which leads to more effective CDC compared to the IgG1 wild type.¹¹⁸ Schutze, Petry¹¹⁹ inserted the E345R HexaBody mutation into chimeric llama/human heavy-chain antibodies (hcAbs) to analyze its effect on CD38-expressing cells. The research team observed an improvement in CDC potency due to the formation of hexamers on the cell surface.¹¹⁹ These effector functions were combined for selective activation when required via the collaborative work of many scientists (Table 2).

Table 2. The effect of Fc engineering by inserting mutations. Mutation named as S298A means Serine was mutated to Alanine. Residues numbering is based on EU numbering system

	Fc mutations	IgG isotype	Effector Function	Mechanism of Action (MOA)	Reference
Fc mutations for enhanced	S298A/E333A/K334A (AAA)	IgG1	ADCC	↑ FcγRIIIA	^Citation94
effector functions	S239D/A330L/I332E (DLE)	IgG1	ADCC	↑ FcγRIIIA	^Citation111
	F243L/R292P/Y300L/V305I/P396L (LPLIL)	IgG1	ADCC	↑ FcγRIIIA, FcγRIIA	^Citation89
	L235V/F243L/R292P /Y300L/P396L (VLPLL)	IgG1	ADCC	↑ FcγRIIIA, FcγRIIA	^Citation90
	G236A/A330L/I332E (GAALIE)	IgG	ADCC	↑ FcγRIIA, FcγRIIIA	^Citation120,Citation121
	G236A/S239D/A330L /I332E (GASDALIE)	IgG	ADCC	↑ FcγRIIIA, FcγRIIA	^Citation113
	In one heavy chain: L234Y/L235Q/G236W/S239M/H268D/D270E/S298A In the opposing heavy chain: D270E/K326D/A330M/K334E	Asymmetrically engineered antibody Fc variant (Asym-mAb1)	ADCC	↑ FcγRIIIA, FcRIIA	^Citation122
	G236A	IgG1	ADCP	↑ FcγRIIA	^Citation110
	G236A/S239D/I332E (ADE)	IgG1	ADCC ADCP	↑ FcγRIIIA, FcγRIIA	^Citation110
	S239D/I332E (DE)	IgG1	ADCC ADCP	↑ FcγRIIIA	^Citation111
	S267E/H268F/S324T (EFT)	IgG	CDC ↓ADCC, ↓ADCP	↑ C1q affinity ↓ FcγRIIIA	^Citation116
	H268F/S324T (FT)	IgG	CDC ↑ ADCC, ↑ADCP	↑ C1q affinity ↑ FcγRIIA ↑ FcγRIIIA	^Citation116
	E345R/E430G/S440Y (RGY)	IgG	CDC	↑ C1q affinity Hexamerization	^Citation49,Citation123
	E345R	IgG	CDC	↑ C1q affinity Hexamerization	^Citation49
	K326W/E333S	IgG	CDC	↑ C1q affinity	^Citation117
Fc mutations for enhanced serum half-life	M252Y/S254T/T256E (YTE)	IgG1	Extended half-life	↑ FcRn affinity at pH 6	^Citation124
	M428L/N434S (LS)	IgG			^Citation125
	N434A	IgG			^Citation94

Effector-enhanced antiviral antibodies such as Elipovimab (used for HIV) are heavily Fc engineered. The FcγR binding-enhancing Fc mutations G236A, S239D, A330L, I332E, M428L and N434S were inserted to improve the Elipovimab binding affinity to FcγRIIIA, FcγRIIA and FcγRIIB.³² Furthermore, VIR-3434 (mAb for chronic hepatitis B virus [HBV] infections) is Fc-engineered as well. The Fc mutations G236A, A330L, I332E, M428L and N434S were introduced to enhance VIR-3434’s binding affinity for FcγRIIIA and FcγRIIA while reducing its binding to FcγRIIB.³² The clinical results of both these studies, which are currently underway, are urgently needed to determine their safety and effectiveness.¹²⁶

To this end, all these studies suggest that Fc engineering for optimal binding affinity and selective specificity to FcγRs stand out as a prospective and practical approach for enhancing the clinical efficacy and potency of antiviral mAbs.

The Fc engineering strategy for enhanced intracellular neutralization

The use of ADIN mediated by TRIM-21 to degrade invading viruses before they replicate has refocused interests on designing effective antibody-antiviral responses with vaccinal effects.¹²⁷ Ng, Kaliaperumal¹²⁸ engineered the IgG1 Fc domain to increase its binding affinity for TRIM-21 by 100 times. Their research group used phage display technology to identify five mutations that may enhance binding affinity: H433T, N434R, Y436F, S440I and T256P. Their results showed that using engineered Fc with a high binding affinity for TRIM-21 can facilitate antigen cross-presentation, which leads to better human DC maturation and induces an antigen-specific CD8 T-cell response.¹²⁸ In addition to these effects, the TRIM-21 Fc receptor affects the release of IFN-γ and other proinflammatory cytokines.¹²⁹ This finding suggests that the TRIM-21 Fc receptor approach might be considered for use in DC-derived vaccines.¹²⁸ TRIM-21 also collaborates with the complement system to prevent cellular infection using different protective antiviral mechanisms, such as sensing a complement-coated virus and thus triggering viral degradation and elimination.^{61,63,130,131}

TRIM-AWAY

TRIM-AWAY is a novel protein degradation technology used to target specific intracellular proteins tagged for degradation by an antibody.¹³² This strategy delivers antibodies into cells and facilitates the study of protein functions without prior modifications.^64,132 Zeng, Santos¹³³ used this technology to selectively degrade disease-causing proteins by TRIM-21-nanobody chimeras. Such studies highlight the possibility of using TRIM-AWAY technology to specifically target selective proteins for degradation, either as a research tool or for therapy.¹³³

Fc engineering to modulate antibody half-life circulation

Fc engineering to tailor the IgG half-life via modulating the interaction between FcRn and IgG has been broadly used to improve the clinical potential of antibodies.⁶⁷ To extend the serum persistence of IgG antibodies, the IgG-FcRn interaction has been altered by inserting Fc point mutations.¹³⁴

Dall’Acqua, Woods¹³⁵ used rationally designed libraries and phage displays to study the effects of IgG1 Fc-FcRn binding affinities. They identified the three Fc mutations M252Y/S254T/T256E (YTE). YTE was inserted into the anti-respiratory syncytial virus (RSV) antibody (Motavizumab-YTE), which enhanced the binding affinity by 10 times. Motavizumab-YTE was safe and well tolerated by patients and demonstrated an increased serum half-life up to 70–100 days and a clearance rate that was decreased by 71–86%¹³⁶ (ClinicalTrials.gov registration no. NCT00578682). In addition, Zalevsky, Chamberlain¹²⁵ combined rational design methods with high-throughput protein screening methods to discover Fc variants with a high binding affinity for human FcRn. Their research team identified the double Fc mutations M428L/N434S (LS). IgG1 LS enhanced the binding affinity for FcRn by 11 times in vitro.

Recently, Sotrovimab, a SARS-COV-2 engineered Fc antibody, has been granted Emergency Use Authorization (EUA) by the United States Food and Drug Administration.¹³⁷ The Fc domain of Sotrovimab was engineered by inserting the Fc mutations M428L and N434S (LS) to extend its circulation half-life, and it influenced the disease progression in COVID-19 patients with an 85% reduction in hospitalizations.^138,139 Other antiviral antibodies Elipovimab and VRC01LS are currently in phase-1 human clinical trials for HIV; both contain enhanced serum persistence mutations.¹²⁶

Fc fusion engineering for antigen binding

Using the full antibody structure of Fc has limitations due to its poor penetration into targeted tissues and weak binding affinity for certain surface molecules, such as viral glycoproteins.¹⁴⁰ Utilizing the Fc fragment instead of the full antibody structure to target viral antigen protein fusion or even the virus itself¹⁴¹ represents a promising approach for generating novel antigen-binding strategies.^142,143

Immunoadhesion is a strategy used in designing antibody-like molecules using the Fc domain for protein fusion with targeted epitopes.¹⁴¹ Consequently, this process produces a combination of two functions (dual effects) in a single well-designed molecule: a neutralizing effect and vaccine-like effects.¹⁴¹ Kruse¹⁴¹ designed an Fc-angiotensin-converting enzyme 2 (Fc-ACE2) fusion to be used as a therapy against future coronavirus pandemics, acute respiratory distress syndrome or other viruses that use ACE2 to infect cells.

Zhang, Zhou¹⁴⁴ developed an IgG Fc fused with RSV glycoprotein F (F-Fc) to be used as a potential vaccine candidate against RSV. That study reported the induction of immunization effects and a reduction in lung injuries caused by the RSV infection.¹⁴⁴ In addition, when the MPL adjuvant was added, neutralizing antibodies were generated after immunization with the F-Fc complex.¹⁴⁴

Creating accurately oriented antibody assemblies with controlled valency is a necessary and currently unfulfilled criterion that will be required to enhance antibody performance and potency.¹⁴⁵ Divine, Dang¹⁴⁵ designed nanocages that were composed of two components. The first is an antibody or Fc fusion, and the second is an Fc-binding homo-oligomer designed to direct the assembly of nanocages. The results of this approach showed improved antibody signaling to produce CD40 activation and a T-cell response.¹⁴⁵ In addition, the viral neutralization potency for a SARS-COV-2 pseudovirus was also enhanced.¹⁴⁵

Fc engineering for improved vaccine-like effects

Fc engineering can be utilized to produce strong vaccine-like effects through several methods, such as enhancing selective Fc-FcγR interactions, glycoengineering and improving FcRn interactions to regulate the antibody’s serum half-life.^69,146–149

Pelegrin, Naranjo-Gomez⁹ reviewed the possible improvements for mAb-based antiviral therapies for producing vaccine-like effects. They were particularly interested in identifying the cellular and molecular mechanisms involved in the induction of vaccine-like effects during antiviral mAb treatment. Direct mechanisms are actively involved, such as ADCC, ADCP, CDC and antibody-dependent cell-mediated virus inhibition (ADCVI).^18,150,151 Indirect mechanisms that stimulate adaptive immunity were observed when mAb therapies were combined, which enhanced and strengthened the Fc effector functions and targeted the infected cells.⁹ Bournazos and Ravetch⁵² explained the immunomodulatory mechanisms of the FcγRs that are involved in T-cell immunity. This process is similar to how human DCs express both FcγRIIA and FcγRIIB and maintain a balance between them to produce effective T-cell responses, such as antigen presentation and stimulating co-stimulatory molecules, which result in long-lasting protection. The three Fc mutations G236A/A330L/I332E (GAALIE)¹²¹ exhibit an increased binding affinity for the selective activated human FcγRs (FcγRIIA and FcγRIIIA) but reduced binding with the inhibitory FcγRIIB receptor.¹²¹

Upon further analyses, Bournazos, Corti²² found that GAALIE Fc mutations induced the activation of T-cell responses for both CD8+ and CD4 + T-cells, which provide protection against infections. Additionally, when inserting LS Fc mutations, GAALIE-LS demonstrated a longer half-life and superior protection with a 5.5-fold enhanced antiviral potency in vivo.²²

Clinically, VIR-7832 (a neutralizing COVID-19 antibody developed by Vir Biotechnology) has been designed to include GAALIE Fc mutations.¹⁵² This GAALIE-engineered antibody is currently in phase I/II clinical trials (NCT04746183).

Recently, Ullah, Prevost¹⁵³ applied live bioluminescence imaging (BLI) to analyze the actual effects of antibodies in prophylaxis and therapy, by using SARS-CoV-2-nanoluciferase infected mice model (K18-hACE2). In their study, authors signified that Fc effector functions were required for ideal efficacy and protection, in agreement with other studies.^4,80,154 They engineered Fc domain by inserting GASDALIE mutations for improved FcγRs binding affinity. They observed that the GASDALIE enhanced Fc variant had provided almost complete protection, engaging FcγRs and activating immune cells as neutrophils, monocytes and NKs. Furthermore, reduced viral load, decreased virus dissemination and spreading in addition to reduction of inflammation and cytokine-storm like phenotype.¹⁵³

Liu, Xu¹⁵⁵ used the receptor-binding domain (RBD) of the spike protein of SARS-COV-2 to design an Fc-based vaccine candidate for COVID-19 (SARS-COV-2 RBD-Fc). This recombinant subunit vaccine contains the RBD of SARS-COV-2 S1 conjugated to the human IgG Fc domain via the C-terminus of the SARS-COV-2 RBD. The results of the immunization of mice with SARS-COV-2 RBD-Fc indicated the presence of RBD-specific antibodies at high titers and also demonstrated virus neutralization. The Fc domain in this vaccine design acts as an immunopotentiator for enhancing vaccine immunogenicity by interacting with FcRs on the APC.^156–158 This novel vaccine candidate developed by Liu, Xu¹⁵⁵ demonstrated effective neutralization for the SARS-COV-2 D614G mutation (located outside the RBD) that had raised concerns by being more infectious compared to other mutations.¹⁵⁵ These results highlight the possibility of using the Fc domain in vaccine design to induce a long-term neutralizing antibody response and broad-spectrum vaccine-like effects to prevent and protect against viruses and their mutants.¹⁵⁵

Conclusion and recommendations

Newly emerging human infectious diseases caused by viral infections pose a serious threat to public health and global economies, as seen during the COVID-19 pandemic. This ongoing event highlights the urgent need to find effective therapeutics and protective vaccines to address the global crisis. With few therapeutic options available, antibody-based therapy has proven to be an effective strategy to meet these needs. It has long been thought that antibody neutralization is the main function of Fc effectors, and recent studies have reinforced the significant roles mediated by Fc effector functions for treating and preventing viral infections.⁴ As seen in the work of Winkler, Gilchuk,¹⁵⁹ intact Fc effector functions are required to mediate antibody protection in neutralizing human antibodies against SARS-COV-2. Vaccines that elicit Fc effector functions have become a target of vaccine development, as observed with the ChAdOx1 nCov-19 vaccine⁷⁵ and the RTS, S/AS01 malaria vaccine.^76,77 In addition, antibodies obtained from infected patients are attracting further research focus. For example, SARS-COV-2 neutralizing IgG1 antibody (SC31), which was initially isolated from a convalescent COVID-19 patient, had therapeutic efficacy when administered to hamsters infected with SARS-COV-2.⁸⁰ SC31 has reduced the incidence of severe disease progression and also decreased the viral load and presence of lung lesions in hamsters; its effectiveness was the result of its dual functionality: neutralizing the viral infection by blocking the binding receptor and mediating Fc effector functions, and inducing an IFNγ-driven antiviral response for maximal therapeutic efficacy.⁸⁰ It is also noteworthy that SC31 showed no evidence of antibody-dependent enhancement (ADE); human trials will soon begin for COVID-19 patients.⁸⁰ SC31 and all other examples mentioned in this review are antibodies that have been discovered and developed against viral infections to confirm their real therapeutic potential. However, there is still an unmet clinical need to improve antibody therapies. Fc engineering strategies have already proven to be a successful approach to enhance the clinical effectiveness and improve the functional selectivity and safety profile efficiency of antibodies.¹⁶⁰

The newest direction of research in this area is moving toward understanding the precise mechanisms involved in activating selective FcR and signaling pathways to produce a potent, tailored and protective antiviral response, as seen by the Fc antibody engineering work of Bournazos, Corti.²² In addition, it is also of interest to mimic the normal response of protective antibodies by maintaining a balance between the neutralization capacity and the required level of binding affinities to produce a sufficient level of effector functions and synergetic control interactions with adaptive immunity.⁸⁷ Although the basics of Fcγ receptors are well established, further work will be required to understand the specific contributions of FcRs in producing broad, long-term antiviral protection. Additionally, understanding the nature of the interaction between an antibody with a virus or the surface structural proteins of an infected cell is important to identify key factors that influence FcRs’ accessibility and the conformational changes that occur upon activation.

In regards to the vaccinal effect derived from T-cell activation, more data are needed to understand the immunological mechanisms required to stimulate DC activation via Fc-FcγR binding, FcRn antigen presentation and the intracellular neutralization mediated by TRIM-21 to address viral threats. Vaccines that induce Fc effector functions should be evaluated to optimize their efficacy and safety using well-constructed standardized assays to understand how neutralizing antibody activity can be improved by combining it with FcR engagement and also how Fc effector functions contribute to the neutralization breadth of antibodies.⁷⁴

Critically, as evidenced in this review, Fc engineering has shown great potential to meet clinical demands, but careful consideration must be taken when selecting the best approach to engineer the antibody Fc domain. Choosing the right approach to engineer the antibody Fc domain will depend on the desired outcomes, such as improving binding affinities and selectivity, enhancing effector function potency or even combining engineering strategies to produce tailored synergistic effects with safe developability profiles. However, in some cases, engineering Fc to have better binding properties may cause unwanted effects, such as cell destruction due to FcγR saturation level differences. Therefore, caution and further study will be required to identify the proper level of activation to understand the complex concerns required when using Fc engineering or the intact Fc domain.¹⁶¹

Goulet and Atkins¹⁶² recently reviewed the general factors to consider in antibody design and development, but further research is still necessary to identify the factors and properties that should be avoided when designing effective antiviral antibodies with vaccine-like effects to prevent unwanted immunogenicity and other harmful side effects. Safety is at the top of these considerations, and several strategies have been proposed to enhance antibody safety and prevent ADE using Fc engineering.¹⁶³ One strategy used to decrease ADE activity is to replace IgG subclasses.¹⁶⁴ IgG4 was selected due to its lowered ability to recruit effector functions (i.e., its low affinity for C1q and FcγRs).¹⁶⁵

Muhammed¹⁶⁶ engineered an ACE2-IgG4-Fc fusion to avoid FcR binding while retaining viral neutralization and showed an increased binding affinity for both the SARS-COV-2 RBD and spike protein. Moreover, a stabilizing point mutation (S228P) located in the hinge was inserted to ensure the stability of the ACE2-IgG4-Fc fusion protein.¹⁶⁷ This approach has not yet been tested in humans, but the research team suggested that engineering the Fc domain to avoid FcR binding and to utilize IgG4 may have clinical applications for preventing ADE while administering therapy against viral infections.¹⁶⁶

Another proposed strategy is minimizing or abrogating the binding of FcRs by inserting key point mutations. Wang, Peng¹⁶⁸ developed an Fc engineered neutralizing antibody against SARS-COV-2 called MW05. MW05 was engineered by introducing the Fc mutations L234A and L235A (LALA) to abrogate the ADE activity mediated by FcγRIIB. A prophylactic and therapeutic response was observed after a single dose of MW05/LALA. Their study also demonstrated that LALA Fc mutations were able to eliminate ADE and minimize the risk of Fc-mediated acute lung injury in vivo.¹⁶⁸

Further clinical trials with large number of patients are needed to investigate the effectiveness of published research; these results will help generate safe, potent and tailored Fc optimized antibodies with antiviral and vaccine-like responses and will also produce effective vaccines that confer long-lasting immunity. Overall, Fc antibody engineering will play a vital role in developing future next-generation antiviral antibody therapeutics and vaccines for infectious diseases in general with a particular interest against emerging viral infections.

Acknowledgments

The author would like to thank Prof Hani Faidah (Faculty of Medicine., Umm Al-Qura University), Dr Saud Almaslmani (Orthopaedic surgeon., Faculty of Medicine., Umm Al-Qura University) and Dr Faz Chowdhury (Chairman and CEO., Nemaura Medical Inc) for their critical reviews.

Disclosure statement

The author declares that she has no conflict of interest related to this study.

Additional information

Funding

This work received no specific grant from any funding agency.