Review
Boosting half-life and effector functions of therapeutic antibodies by
Fc-engineering: An interaction-function review
Marcela Helena Gambim Fonseca
⁎
, Gilvan Pessoa Furtado, Marcus Rafael Lobo Bezerra,
Larissa Queiroz Pontes, Carla Freire Celedonio Fernandes
Fundação Oswaldo Cruz, Fiocruz Ceará, Eusébio, CE 61760-000, Brazil
a b s t r a c t
a r t i c l e
i n f o
Article history: Received 9 April 2018
Received in revised form 20 July 2018 Accepted 21 July 2018
Available online 21 July 2018
Due mainly to their high level of affinity and specificity, therapeutic monoclonal antibodies (mAbs) have been frequently selected as treatment for cancer, autoimmune or chronic inflammatory diseases. Despite the increas-ing number of mAbs and related products in the biopharmaceutical market, they are still expensive, can cause undesired side effects, and eventually cause resistance. Antibody engineering, which emerged to overcome lim-itations faced by mAb therapy, has supported the development of modified mAbs for immunotherapy. As part of this approach, researchers have invested in obtaining antibody fragments, as well as in Fc region modifications, since interactions with Fc receptors influence an antibody's half-life and mechanism of action. Thus, Fc engineer-ing results in antibodies with more desirable characteristics and functions for which they are intended, createngineer-ing
“fit-for-purpose”antibodies with reduced side effects. Furthermore, aglycosylated antibodies, produced in bacte-rial cultivation, have been an alternative to create new effector functional human immunotherapeutics, while re-ducing mAb therapy costs. This review highlights some features that enhance mAb performance, related to the improvement of antibody half-life and effector responses by both Fc-engineering and glycoengineering.
© 2018 Elsevier B.V. All rights reserved.
Keywords: Antibody Fc region Fc-engineering Fc gamma receptor Monoclonal antibody Neonatal Fc receptor Serum half-life
Contents
1. Introduction . . . 306
2. Fc-FcRn interaction and strategies to modulate antibody half-lives . . . 307
3. Fc-FcγR interaction and strategies to improve antibody effector functions. . . 308
4. The nature of carbohydrates on the Fc region: their role in antibody structure, effector functions and glycoengineering . . . 309
5. Conclusion and future perspectives . . . 310
Contributors . . . 310
Declarations of interest . . . 310
Acknowledgements . . . 310
Conflict of interest . . . 310
References. . . 310
1. Introduction
Since thefirst monoclonal antibody (Orthoclone - muromonab), approved in 1986 for preventing kidney transplant rejection, mAbs have been introduced into the market and used in the treatment of sev-eral diseases, such as cancer, autoimmune, cardiovascular, infectious, and chronic inflammatory diseases. About 74 therapeutic mAbs and
related products (antibody fragments, antibody-drug conjugates, Fc-fusion proteins) have been approved by the FDA. These products ac-count for more than half of sales related to biopharmaceuticals [1].
The great majority of approved mAbs belongs to the immunoglobu-lin G (IgG) class, mainly IgG1. Each one consists of two heavy chains, with approximately 50 kDa each, formed by one variable (VH) and three constant (CH1, CH2, CH3) domains, and two light chains, with about 25 kDa each, comprised of one variable (VL) and one constant (CL) domain. VL and VH domains, together with CL and CH1 constitute the antigen binding site (Fab), responsible for antibody affinity/ ⁎ Corresponding author at: Rua São José S/N, Precabura, Eusébio, CE 61760-000, Brazil.
E-mail address:marcela.gambim@fiocruz.br(M.H.G. Fonseca).
https://doi.org/10.1016/j.ijbiomac.2018.07.141
0141-8130/© 2018 Elsevier B.V. All rights reserved.
Contents lists available atScienceDirect
International Journal of Biological Macromolecules
specificity. CH2 and CH3 constitute the crystallizable fragment (Fc re-gion), responsible for antibody serum half-life via the neonatal Fc recep-tor (FcRn), and effecrecep-tor functions via Fc gamma receprecep-tors (FcɣRs), or C1q complement protein binding [2]. It is important to note that the CH2 domain of each heavy chain isN-glycosylated, which can influence the structure, function and pharmacokinetics of mAbs [3].
Even though IgG subclasses (IgG1, IgG2, IgG3and IgG4) shareN90% sequence identity in their heavy chains, differences in amino acid se-quences have been identified in sites that bind to FcRn, FcɣR, and C1q. This explains changes in serum half-life and effector functions among IgG subclasses [2].
Knowledge of IgG molecular organization and its structure together with DNA recombinant technology have enabled a large number of modifications in antibody structure and function. Hence, development of antibodies with improved half-lives and/or effector functions could be performed by modifying the Fc region, by substituting out key amino acid residues that participate in the interaction with FcRn or FcɣR receptors, or even modifying glycan structures (Fig. 1).
This review summarizes different approaches used for Fc-engineering aiming to improve pharmacokinetic and pharmacody-namic parameters of engineered mAbs.
2. Fc-FcRn interaction and strategies to modulate antibody half-lives
In 1964, the existence of a receptor involved in“IgG protection”was hypothesized in order to explain IgGs' extended half-life, when com-pared to other immunoglobulin classes [4]. This receptor, also called the neonatal Fc receptor (FcRn), was identified in the placenta, which is responsible for transporting maternal IgGs to the fetus [5,6], and transporting IgGs from a mother's milk to neonate across the intestinal epithelium [7,8]. Besides the placenta and intestinal epithelium, FcRn is expressed in numerous adult tissues including the mammary gland, vascular endothelium, kidney, lung, brain, skin, eyes, genital system and hematopoietic cells, such as monocytes, macrophages, dendritic, and B cells [9,10]. To date, it is known that FcRn function is not limited to IgG transcytosis, but that it also participates in the IgG and albumin recycling processes [11,12].
Although plasma proteins are generally endocytosed and hydro-lyzed in lysosomes, IgGs, when endocytosed, bind to FcRn expressed
in endosomes, at a specific pH (b6.5), protecting them against degra-dation [10]. When the IgG-FcRn complex returns to the cell surface, IgG dissociates from FcRn at a neutral pH, and is released back into circulation [13,14]. Thus, interaction between IgG and FcRn is one of the critical features in determining mAb efficacy. While the half-life of IgG1, IgG2and IgG4isotypes is about 21 days, IgG3, which does not bind efficiently to FcRn, presents a half-life of approxi-mately 7 days [14].
It is important to note that interaction between the Fc region and FcRn occurs in the CH2-CH3 junction. Key Fc residues involved in bind-ing to FcRn have been identified (Table 1). Histidine residues (H310, H435 and H436) have a special role, seeming to act as pH sensors [2]. These amino acids become positively charged at a pH≤6.5, which re-sults in the formation of salt bridges with acidic FcRn residues (Fc H310/FcRn E117, Fc H435/FcRn E132, and Fc H436/FcRn D137). At pH≥7.0, histidine residues are deprotonated and FcRn is released from Fc binding [15]. I253 and S254 residues, both in the CH2 domain, and Y436, in the CH3 domain, were also identified as critical to human IgG1: FcRn binding [13,16].
Structural analysis confirmed that one Fc homodimer can simulta-neously interact with two FcRn molecules with high affinity [17]. The 2:1 binding ratio between FcRn and IgG seems to be critical for efficient binding, recycling and serum persistence of IgG [13]. Other factors that may influence antibody serum half-life include endocytosis rates, anti-body stability and post-translational modifications. Rituximab (anti-CD20) and Trastuzumab (anti human epidermal growth factor receptor 2-HER2), for example, present short half-lives because they are endocytosed through their antigen receptor and degraded [10]. Further-more, studies have shown that oxidation at M252 in CH2 and M428 in CH3 can decrease Fc-FcRn binding and lead to IgG degradation in lyso-somes [18,19].
Extending mAb half-life can be a strategy to maintain antibody con-centration in the serum, capable of triggering clinical responses and avoiding repeated injections. In order to improve antibody half-life, dif-ferent Fc engineering approaches have been used. Therefore, several studies have proposed mutations in amino acid residues at the CH2-CH3 domain interface, to increase antibody affinity for FcRn at an acidic pH. Consequently, mAb degradation via a lysosomal route may be prevented, contributing to its return to blood circulation [10,14].
Fig. 1.Main Fc regions for IgG improvement. Three Fc regions, prone to modifications, would enhance antibody efficacy. (A) Glycosylation sites. Carbohydrate pattern, which differs depending on the expression platform used, affects conformational stability of IgGs, as well as Fc binding to other molecules. (B) FcRn binding site, and (C) FcγR binding site. Mutagenesis in both regions have been approached to enhance antibody activity. All three regions combined are able to activate and recruit immune system cells and molecules. IgG structure–PDB Access Number: 1IGY.
Ghetie et al. [20] modified three amino acid residues around the FcRn-Fc binding interface (T252/T254/T256) and selected, by Phage Display, variants with increased affinity for mouse FcRn. Mutations on residues at the same positions (M252Y/S254T/T256E) in Motavizumab, a humanized IgG1mAb anti-respiratory syncytial virus (RSV), resulted in ~10-fold increase affinity for FcRn at pH 6.0, and an increased anti-body half-life in healthy adults [21]. Furthermore, engineering variants (M428L/N434S) of IgG1Bevacizumab (anti-vascular endothelial growth factor - VEGF) and Cetuximab (anti-epidermal growth factor - EGFR) also showed better affinity for human FcRn and half-life in human FcRn transgenic mice and monkeys [22]. Aiming to improve FcRn bind-ing, Shen et al. [23] engineered two IgG1antibodies (Alirocumab and Evolocumab) against proprotein convertase subtilisin/kexin type 9 (PCKS9), an enzyme involved in plasma cholesterol metabolism. Simi-larly, mutations introduced in the Fc region (M428L/N434S) of anti-oncostatin M mAb improved antibody half-life [24]. Besides that, muta-tions (M252Y/S254T/T256E) in the Fc region of MEDI4893, an antibody used to neutralize alpha-toxin, aStaphylococcus aureusvirulence factor, prolonged antibody half-life 4-fold [25].
Fc engineering has also been used to increase therapeutic protein half-life. As described, Fc-fusion enables interaction with FcRn increas-ing the protein's half-life by recyclincreas-ing and inhibitincreas-ing lysosomal degrada-tion. Etanercept, alefacept, and abatacept are examples of Fc-fusion proteins approved for clinical use. These proteins are composed of an extracellular portion of membrane receptor linked to the Fc region of human IgG1. The receptor portions of etanercept, alefacept and abatacept are, respectively, the extracellular ligand-binding portion of the human TNF receptor (TNFR), the extracellular CD2-binding portion of human leukocyte function-associated antigen-3 (LFA-3) and the ex-tracellular domain of human cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [26].
Recent studies have brought forward the possibility of fusing pro-teins to monomeric Fc, with one set of CH2 and CH3 domains, as an al-ternative approach aimed at simplifying product development. Erythropoietin, coagulation factor IX, and interferon are examples of proteins fused to one arm of the Fc. One of the challenges of monomeric Fc is that it presents weak binding to FcRn. Thus, efforts have been made to develop monomeric Fc fusion proteins with FcRn binding affinity comparable to that of dimeric Fc fusion molecules [27].
On the other hand, antibody half-life reduction can be useful to avoid background and systemic exposure to radiolabeled probes during diag-nostic imaging; to reduce IgG levels in antibody-mediated diseases, as well as to induce a rapid clearance of IgG-toxin or IgG-drug complexes. In this regard, antibody engineering has been used to decrease Fc-FcRn interaction [28]. Abdegs (antibodies that enhance IgG degradation) are Fc-engineered antibodies that bind to FcRn with high affinity and re-duced pH dependence. They are able to interact with FcRn at both acidic and neutral pH presenting a competitive advantage for FcRn-binding compared to endogenous IgG isotypes, accelerating wild type IgG deg-radation [10]. Swiercz and colleagues [29] reported a reduction in back-ground and improvement of contrast during Positron Emission
Tomography (PET), performed after injection of radiolabeled Pertuzumab (HER2-specific antibody), administered 8 h later by the de-livery of an Abdeg, in mice bearing HER2 overexpressing tumors.
3. Fc-FcγR interaction and strategies to improve antibody effector
functions
mAbs can act in different ways to promote their pharmacological ac-tion. They can bind to soluble antigens, preventing them from commu-nicating with their receptors, e.g., Certolizumab, Infliximab and Adalimumab (anti-TNF-alpha) or Bevacizumab (anti-VEGF); and to re-ceptors, inhibiting their interaction with a ligand, e.g., Cetuximab and Panitumumab (anti-EGFR), Trastuzumab (anti-HER2) or Basiliximab and Daclizumab (anti-IL2 receptors) [14].
After interaction with their antigens, antibodies can also interact in various modes with immune system cells through their Fc region. Bind-ing of antibodies to antigens on the surface of the target cell opsonizes them. The Fc region of IgG is detectable by FcγRs expressed on the phagocytes leading to phagocytosis and death of the target cell, a re-sponse known as Antibody Dependent Cellular Phagocytosis (ADCP). Besides that, the Fc region of mAb can interact with the protein C1q and triggers the classical complement cascade, which results in the for-mation of a membrane attack complex and target cell lysis, a phenome-non described as Complement Dependent Cytotoxicity (CDC). Also, the classical complement cascade generates soluble proteins, like C3b, that opsonize target cells and induce phagocytosis by macrophages, neutro-phils, and dendritic cells, since they have complement receptors. Finally, the Fc region of IgG interacts with FcγRs leading to antibody-dependent cell-mediated cytotoxicity (ADCC) [17]. ADCC is an important mecha-nism of action of mAbs, and NK cells play a key role in this scenario. En-gagement of FcγRs expressed on NK by crosslinking with mAb recruits and activates these cells leading to secretion of lytic granules containing perforin and granzymes. Perforin generates pores in target cell mem-branes, whereas granzymes enter the cytoplasm leading to apoptosis. Also, the Fas/FasL interaction is established resulting in apoptotic cell death, mediated by caspase activation [14].
In humans, the FcγR family is composed of three classes of receptors: high-affinity IgG receptors, FcγRI (CD64), which binds to the Fc region with KD10−8
–10−9M, and low-af
finity IgG receptors, FcγRII (CD32), with KD~10−7M, and Fc
γRIII (CD16), with the lowest affinity KD 10−5M [17]. Fc
γRI is expressed on monocytes, macrophages, dendritic cells and can be induced in neutrophils. There are many isoforms of FcγRII distributed differently in hematopoietic cells. FcγRIIa and FcγRIIc are predominantly expressed on phagocytic cells (neutrophils, mono-cytes, and macrophages), whereas FcγRIIb is mainly expressed on B lymphocytes. FcγRIII has two isoforms: FcγRIIIa, expressed on macro-phages, natural killer (NK) cells, dendritic cells, basophils and mast cells, and FcγRIIIb expressed exclusively on neutrophils [17,30,31].
FcγRs are classified as activating Fc receptors, characterized by immunoreceptor tyrosine-based activation motifs (ITAM), or inhibitory Fc receptors, characterized by immunoreceptor tyrosine-based Table 1
Main modifications of the Fc region aiming to improve half-life or mechanisms of action. The modifications are on human IgG1Fc region, unless otherwise stated.
Fc region Receptor Important residues/modifications described Biological outcome References
CH2/CH3 interface FcRn H310, H435, H436, I253, S254, Y436, M252, M428 M252Y/S254T/T256E
M428L/N434S
Half-life modulation [2,13,15,16,18,19,21,22,24,25]
CH2 C1q D270, K322, P329, P331 K326W/E333S
CDC modulation [41,42]
Hinge FcγRI E235La ADCP modulation [43]
CH2 FcγRIIIa S239D, I332E, S239/I332E S239/I332E/A330L
ADCC/ADCP modulation
[35]
CH3 FcγRI E382V/M428I ADCP modulation [39]
CH2 C1q/FcγR N297, glycosylation pattern Stability, ADCC, ADCP and CDC modulation [14,17,53–56,58–62,64–66]
inhibitory motifs (ITIM) [30,32,33]. Cross-linking between IgG and ac-tivating FcγR recruits Src tyrosine kinases that phosphorylate ITAM ty-rosine residues. Downstream signaling components are activated, which eventually results in different cellular responses such as phago-cytosis, granule exophago-cytosis, ADCC, or cytokine synthesis [34]. FcγRIIb is the only inhibitory receptor. Simultaneous engagement of FcγRIIb and the B-cell receptor (BCR) by immune complexes triggers tyrosine phos-phorylation of ITIM, activation of phosphatases, suppression of BCR sig-nals and, consequently, inhibition of intracellular sigsig-nals and B-cell activation [30].
Common to all myeloid cell types is the coexpression of both activat-ing and inhibitory FcγRs on their surface. Therefore, mAbs can bind to activating and inhibitory FcγRs on the same cell and even have a greater affinity for inhibitory FcγRIIIb than for activating FcγRs, inhibiting the immune response, instead of activating it [33]. A balance between acti-vating and inhibitory signals is an important mechanism that the im-mune system uses to regulate the imim-mune response. In cancer immunotherapy, mAbs are designed to trigger a response and not in-hibit it, so if a therapeutic mAb binds to an inin-hibitory receptor, its effi -ciency is decreased [32]. One well-established approach of antibody engineering to generate an anti-cancer antibody with better potency is to increase the affinity of the Fc region for activating Fc receptors such as FcγRI and FcγRIIa and especially FcγRIIIa and decreasing affinity for FcγRIIb inhibitory receptor [31,35,36]. Enhanced ADCC was de-scribed in FcγRIIb knockout mice treated with Trastuzumab and Rituxan [37] and anti-E-cadherin antibodies [38]. Trastuzumab contain-ing E382V and M428I substitutions in the Fc region with selective bind-ing to FcγRI but not to the inhibitory receptor, FcγRIIb, exhibited stronger ADCC than clinical grade Trastuzumab [39]. These studies sug-gest that the ratio of activating FcγR/inhibitory FcγR binding is an im-portant factor in determining the efficacy of antitumor mAbs. However, recruitment of FcγRIIb can be important in the treatment of autoimmune and allergic diseases. The same Fc engineering approaches used to refine affinity for activating receptors have also been adopted for FcγRIIb [32] developing mutated antibodies in the Fc region to gen-erate variants with greater affinity for FcγRIIb with the ability to sup-press effector cell activation [40].
While the Fc region binds to FcRn in the CH2-CH3 junction, FcγRs' interaction sites on Fc are located within the lower hinge-upper CH2 do-main. Crystal structures demonstrate that C1q also binds to Fc between the lower hinge and upper CH2 region [17]. The key Fc residues involved in binding to FcγR/C1q have been characterized (Table 1). Some resi-dues (D270, K322, P329, and P331) were described for C1q binding on human Fc [41]. Moreover, K326W/E333S mutations in an IgG1variant of Rituximab suggested that these amino acids are important for C1q binding [42]. Additionally, a single E235L mutation in the IgG2b-Fc re-gion of mice has suggested that this position is essential to FcγRI bind-ing [43]. Furthermore, amino acid residues at positions 234 and 237 seem to influence the interaction with FcγRII [44].
Currently, IgG1's Fc region and its subunits in the CH2 and CH3 do-main have been engineered to improve effector functions (ADCC, ADCP and CDC). Polymorphisms in FcγR can compromise clinical re-sponses of mAbs [45,46]. The FcγRIIIa receptor contains a polymor-phism at position 158 of its amino acid sequence. While 20% of the white population express a valine residue at this position, the rest of the population has a phenylalanine [14]. Interestingly, FcγRIIIa-V158 has higher affinity for the Fc region than does FcγRIIIa-F158, driving a better ADCC response. Homozygous patients for valine had a better clin-ical response to Rituximab, used for treating Non-Hodgkin Lymphoma, than did patients who are heterozygous or homozygous for phenylala-nine [46]. Based on this perspective, efforts have been made to enhance the affinity of the Fc region for FcγRIIIa. Lazar and colleagues [35] dem-onstrated that single (S239D or I332E), double (S239D/I332E), and tri-ple mutations (S239D/I332E/A330L) in anti-CD52 Alemtuzumab, Rituximab and Cetuximab antibodies improved affinity for the human activating FcγRIIIaV158/F158 allele and enhanced ADCC/ADCP function.
A new approach to improve the effector functions of mAbs is the de-velopment of chimeric CH variants, in which the Fc region of IgG1is shuffled with the Fc region of IgG3, constructing a new Fc that retains the ADCC activity from IgG1and the CDC activity from IgG3[17]. Natsume et al. [47] shuffled constant domains of IgG1and IgG3to create chimeric isotypes of anti-CD20 antibodies producing variants with en-hanced ADCC/CDC activities compared to the wild-type.
4. The nature of carbohydrates on the Fc region: their role in anti-body structure, effector functions and glycoengineering
It is known that the nature of carbohydrates in the CH2 domain plays a prominent role in antibody effector functions. N-glycosylation at as-paragine 297 in the CH2 domain is important for maintaining the Fc conformation and binding to FcγR and C1q, but not FcRn. Therefore, this post-translational modification modulates the affinity of the Fc re-gion for FcγR [17,48]. The glycan moiety is composed of two N-linked biantennary oligosaccharide chains consisting of a heptasaccharide [N -acetylglucosamine (GlcNAc) and mannose (Man)] core even though other residues like terminalN-acetylneuraminic acid, galactose (Gal), bisectingN-acetylglucosamine (GlcNAc), and fucose (Fuc) have also been reported [49–51]. The complexity and heterogeneity of therapeu-tic IgG molecules when expressed in mammalian cells can affect the therapeutic profile of mAbs [48]. The most commonly used cell line for mAb production is Chinese Hamster Ovary (CHO) cells because of their numerous advantages. These cells can reach substantial produc-tion rates, can be adapted to grow in suspension and in different serum-free media, are refractory to infection by human viruses, offering low risks for commercial production, have regulatory approval and pro-duce recombinant glycoproteins with human-like glycans [52].
The nature of the mAb-associated carbohydrates is dependent on which enzymes are expressed by the cell line used for antibody produc-tion. CHO cells contain the enzymeα1,6-fucosyltransferase, encoded by the FUT8 gene, which transfersL-fucose sugar to the glycan core. Many studies have demonstrated that the addition of fucose interferes with IgG binding to FcγRIII, and, consequently, decreases ADCC. An alterna-tive approach focuses on glycoengineering the Fc domain since non-fucosylated antibodies exhibit better ADCC activity because of increased binding to FcγRIIIa [53,54]. FUT8 knockout CHO has been used to obtain non-fucosylated mAbs [55]. Bloemendaal et al. [56] glyco-engineered an anti-TNF Fc region and analyzed its binding to FcγR and its efficacy in mice with colitis. Administration of hypo-fucosylated anti-TNF en-hanced the number of macrophages in the colon compared to the con-trol Adalimumab and showed to be more efficient in reducing colitis. Bruggeman et al. [57] engineered an anti-Rhesus D (RhD) IgG antibody producing four different isotypes (IgG1-4) with or without fucose and evaluated the engagement of all FcγR human types by anti-RhD. All hypo-fucosylated anti-RhD IgG isotypes presented increased binding to FcγRIII isoforms. These antibodies were tested with Fcγ RIIIa-expressing NK cells and enhanced erythrocyte lysis was observed with IgG1and IgG3but not with IgG2and IgG4. Obinutuzumab is a non-fucosylated anti-CD20 mAb that was designed to increase the affinity for FcγRIII and consequently the ADCC in relation to the reference mol-ecule Rituximab [58]. Capuano et al. [59] reported an enhancement in the affinity of FcγRIII by Obinutuzumab compared to Rituximab and higher production of TNF by cells exposed to Obinutuzumab.
mAb expression in mammalian cell systems results in high produc-tion costs. An alternative strategy to address this problem is the devel-opment of aglycosylated antibodies since they can be expressed in bacteria or yeast [39,60,61]. Although the absence of glycans in the Fc region can lead to the loss of effector functions, recent studies have re-ported that aglycosylated antibodies can be genetically engineered to display wild-type-like properties or even further improved functions. Jung et al. [39] described an aglycosylated IgG variant of Trastuzumab, with amino acid substitutions (E382V and M428I) in the Fc region, expressed inE. coli.The antibody displayed highly selective binding to
FcγRI and enhanced killing of tumor cells by dendritic cells (DCs) com-pared to glycosylated Trastuzumab (E382V, M428I) expressed in HEK293 or clinical grade Trastuzumab. Chen and coworkers [61] engineered three aglycosylated IgG variants capable of triggering phagocytosis where FcγRI and FcγRIIIA dimers were the main receptors involved. These aglycosylated variants represent a step toward the pro-duction, in any expression system, of mAbs capable of eliciting immune responses.
Although the removal of IgG Asn297 glycans does not influence IgGs' solubility and in vivo half-life [62], efforts are still needed to improve their thermostability [63] and their binding affinity to C1q [17,64,65]. Fc region glycosylation helps to maintain the conformation andfl exibil-ity of the IgG CH2 domain, thus it has been shown that aglycosylated mAbs do not bind to C1q. The inability of aglycosylated mAbs to activate CDC limits their application, where complement activity plays an im-portant role. However, they can be useful in the treatment of autoim-mune diseases that result from inappropriate or excessive activation of the complement system [66].
5. Conclusion and future perspectives
Modulating antibodies' half-life and effector functions has been a strategy to create novel antibody-based drugs. Since the Fc region mod-ulates its serum half-life and related effector mechanisms (ADCC, CDC, ADCP) via FcRn and FcγRs interactions respectively, Fc engineering has been a promising tool to enhance the therapeutic efficacy of mAbs. In this review, we have explored different Fc engineering ap-proaches used to modulate Fc affinity for FcRn, activating FcγRs (FcγRI, FcγRIIa, and FcγRIIIa) and inhibitory FcγR (FcγRIIb). Strategies in modifying key amino acids or altering mAbs-associated carbohy-drates (glycoengineering) were summarized. A better understanding of the interactions between the Fc region and their receptors can bring to light the next generation of approved mAbs with novel interesting features, minimal side effects, and reduced manufacturing costs.
Contributors
MHGF contributed to the conception, design and drafting of the manuscript; GPF contributed to the preparation and revision of the manuscript; MRLB participated in drafting the article; LQP participated in design and revision of the manuscript; CFCF participated in drafting the article and critically reviewed it for intellectual content.
Declarations of interest
None.
Acknowledgements
This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (459046/2014-04). Marcus Rafael Lobo Bezerra and Larissa Queiroz Pontes, who are gradu-ate students of Programa de Pós Graduação em Biotecnologia de Recursos Naturais da Universidade Federal do Ceará (UFC) were the beneficiaries of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Master's fellowship. The authors thank Amy Nicole Grabner for the English review of the manuscript. There is no conflict of interest statement.
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