Effects of bsAb fragments capable of interacting with NK cells via CD16 have also been investigated. Gleason and coworkers demonstrated the enhanced efficacy of bscFv CD16 × CD19 and trispecific scFv CD16 × CD19 × CD22 constructs in targeting tumor cells by coengaging NK cell effector function (70). These molecules were termed as bispecific and trispecific killer cell engagers (BiKEs and TriKEs) (**Figures 1T,U**), respectively. Such molecules are constructed by linking specific binders together through a short linker derived from human muscle aldolase. Further, a BiKE construct against CD16 and CD33 has been shown to activate CD16+ NK cells to lyse CD33+ HL60 target cells (71). Recently, a TriKE construct with additional specificity against IL-15 induced enhanced NK cell cytotoxicity, degranulation, and cytokine production against CD33+ HL60 cells *via*

increased NK cell proliferation and survival (72). Reusch et al. designed a Tandab-based anti-CD16A × CD30 bispecific tetravalent fragment and found that this format is better than normal monoclonal anti-CD30 IgG, optimized monoclonal anti-CD30 IgG for FcγR binding, and diabody (bispecific and bivalent anti-CD16A and anti-CD30) formats in triggering NK cell lysis of Hodgkin lymphoma cells (CD30 as the antigen) (95, 96). This format of anti-CD16A × CD30 bispecific fragment (known as AFM13) is under phase-2 clinical trial (**Table 2**) for treatment of refractory or relapsed Hodgkin lymphoma patients (97).

# MONOMERIC Ig SCAFFOLDS

Much work in the last 5 years has focused on engineering the IgG constant domains as scaffolds. The IgG-Fc region and isolated CH domains have shown promising virtues in terms of small size, antigen targeting, effector function, and serum half-life.

Soluble monomeric Fc (mFc) was developed by destabilizing the CH3–CH3 domain interface. A phage-displayed library was constructed with random mutations introduced within seven key residues known to be involved in CH3 domain homodimerization and selected against binding of protein G first to enrich well-folded mutants and then against human FcRn (16). The selected clones were diverse in their CH3 domain, and monomer promoting mutations were identified (16). These variants with each containing six to seven mutations had considerably lower melting temperature but exhibited comparable stabilities and pHdependent FcRn binding profiles to that of dimeric Fc (16). The experimental conceptualization was proved by mFc67.3–m36VH fusion (**Figure 1V**) targeting HIV-1 envelope glycoprotein (ENV) and neutralization of viral isolates (16). Based on the identified mFc mutants, the same group (98) optimized mFc to generate mutants with fewer mutations and improved thermostability while maintaining similar pH-dependent FcRn binding as that of wild-type Fc dimer. Interestingly, the new mFc mutants when expressed either in bacteria or mammalian cells all showed selective binding to only FcγRI but not to other receptors including FcγRIIa, FcγRIIb, FcγRIIIa, and C1q (98). This selectivity attractively made the mFc mutant as a potential carrier to treat chronic inflammatory diseases where inflammatory macrophages showed increased expression of FcγRI (98–103). In addition, this selectivity also excluded unwanted cytotoxicities such as ADCC from FcγRIIIa and CDC from C1q (98).

In another strategy to keep Fc in a monomeric form, asparagine-linked glycan structures were engineered into the CH3–CH3 interface of IgG1 Fc lacking the hinge (104). A mutant incorporating N-glycosylation sites (asparagine at positions 364 and 407) yielded a stable, soluble, and mFc scaffold with wild-type-like FcRn binding activity, which suggests that N-glycosylation promotes functional monomeric state and improves the biophysical characteristics (104).

Smaller scaffolds like CH2 domains also possess interesting properties like a properly folded structure, conformational flexibility, lesser propensity to dimerize, and associated FcRn/C1q binding functions, which can have therapeutic benefits (103, 105). A CH2 domain variant (m01) harboring disulfide bond mutations (L12C and K104C) was found to exist solely as a monomer and remained thermally more stable than native counterparts (105). Further, 50% of monomeric CH2 unfolded at a urea concentration of 6.8 M as compared to 4.8 M for the wild-type CH2 (105). The biophysical attributes of the CH2 domain (m01) were further optimized by developing a shorter version where the unstructured random coil at the N-terminus was shortened by seven residues (m01s) (106). This strategy increased the melting temperature of CH2 isolate up to 82.6°C, enhanced stability in serum and mediated stronger binding to soluble FcRn (106). Similarly, a structure-guided design allowed the engineering of an IgG1-CH3 FcRn recognition motif into the CH2 scaffold to impart enhanced pH-dependent FcRn binding, prolongation of serum half-life, and epithelial transcytosis in comparison to parent CH2 isolate (m01s) (107).

Antigen recognition activity was engineered in CH2 scaffolds carrying mutations in the BC and FG loops by selecting against the HIV-1 envelope glycoprotein (gp120)–CD4 complex (15), which yielded CH2 fragments bearing the same BC but different FG loops, suggesting the importance of the latter in such interactions. One such isolate (m1a1) specifically recognized a highly conserved CD4i epitope on the HIV-1 gp120 protein and neutralized HIV-1 isolates in a cell based pseudovirus assay (15). In another approach, a randomly selected VH domain targeting the HIV-1 gp41 protein was grafted into the BC/FG loops of "m01s" without affecting the flanking regions to develop antigen targeting CH2 domains (m2a1) (108). This domain bound to sp62 peptide of HIV-1 envelope membrane-proximal external region, neutralized HIV-1 isolates, and showed pH-dependent FcRn interaction (108).

Initial success with the mFc and CH2 domains prompted the development of a monomeric CH3 (mCH3) scaffold (103, 109). The seven contact residues of the CH3 domain from the previously reported mFc molecules (16) were mutated, and a combination of mutations was found to be required for CH3 monomer to exist (109). These mutations were responsible for stronger intermolecular hydrophobic interactions, which resulted in an intact, folded and thermally stable CH3 monomer (109). The mCH3 scaffold exhibited significant binding to FcRn and protein G (109). Furthermore, mCH3 fused to a VH domain of an HIV-1 targeting antibody (m36.4) showed satisfactory stability, viral neutralization, and pH-dependent FcRn binding (109). A comparative analysis between the dimeric and monomeric scaffolds has demonstrated increased solubility, comparable serum stability, and higher pH-dependent FcRn binding by monomers (103). These properties make antibody scaffolds promising potential therapeutic candidates.

# Fc ANTIGEN-BINDING FRAGMENT

The Fc has also been developed as an antigen-binding domain "Fcab" (**Figure 1W**) by imparting specificity against antigens through yeast surface display (110). The AB and EF loops of IgG1-CH3 domain were randomly mutagenized, cloned into a yeast surface display vector, and selected against HER2/neu extracellular domain (ECD) for two rounds. The selected binders were again randomized in the AB loop and selected on HER2/ neu ECD. One of the isolated Fcab molecules (H10-03-6) showed specificity for binding to HER2, retained binding to FcγRI/protein A, and exhibited an *in vivo* pharmacokinetics profile similar to the native human Fc (110). Post-affinity maturation, Fcab exhibited ~10-fold enhanced binding to the antigen as compared to the parental molecule and elicited NK cell-mediated cytotoxicity *in vitro* against the breast cancer cell line Calu-3, but the potency was ~20-fold weaker than that of Herceptin (110).

Introducing antigen-binding ability into the Fc fragment could compromise stability, and therefore, additional disulfide bonds were engineered within the CH3 domain to provide increased stability (111). Further, these mutations did not affect pH-dependent FcRn binding, which suggests the correct folding of the engineered Fc fragments (111). Similarly, immune modulating activity of Fcab was demonstrated using a HER2 targeting Fcab (HAF3–4), bearing CD16a modulating mutations (111). Both mutants showed binding to the recombinant or cell surface expressed HER2 and had an expected CD16a modulating behavior which correlated with the ADCC potency (111). Later, Woisetschlager and coworkers (112) showed the *in vivo* tumor reduction efficacy of anti-HER2 Fcab (H10-03-6) by simultaneous engagement of the HER2/CD16a and the involvement of ADCC (112).

Since antigen–antibody interaction at an acidic pH can negatively affect drug pharmacokinetics, anti-HER2 Fcab (H10-03-6) variants with weaker binding at pH 6.0 were developed (113). The residues in the AB and EF loops were randomized and selected against HER2-ECD alternately at pH 7.4 and 6.0 using yeast display. The isolated binders exhibited lower affinity at an acidic pH and similarly engaged HER2+ cells in a pH-dependant manner (113). A year later, Leung et al. (114) developed Fcab that could degrade HER2 and induce apoptosis. The AB and EF loop residues of IgG-Fc were randomly mutagenized and selected *via* yeast display for binding to HER2-ECD. After affinity maturation, the candidate Fcab (FS102) exhibited an affinity for HER2-ECD that was equivalent to that of pertuzumab and trastuzumab and an extended serum half-life comparable to the native Fc (114). Further, EC50 values of 1.1 and 3.3 nM were observed against SKBr3 and HCC1954 breast cancer cells, respectively, and a complete tumor regression was shown using HER2+ patient-derived colorectal/gastric cancer xenografts (114). This effect correlated with caspase 3/7 activation in SKBr3 cells in a dose-dependent manner indicating the induction of tumor cell apoptosis (114).

### CHALLENGES IN CLINICAL DEVELOPMENT OF bsAbs AND FRAGMENTS

Smaller antibody fragments (nanobody, human single-domain Ab, scFv, or Fab) and bsAb fragments offer a number of advantages over full-length IgG, including ability to penetrate tissue, cost effective and facile manufacturing methods, and high yields (23, 115). However, their small size leads to a shorter serum halflife, lesser tissue retention, and rapid clearance from the blood through kidneys. This is true for blinatumomab, which has a serum half-life of around 2 h while the serum half-life of fulllength IgG1 is around 2–3 weeks. Thus, patients need a regimen of at least 3-cycle treatment with each cycle consisting of continuous infusion for 4 weeks in a cycle of 6 weeks (116). On the other hand, the fast clearance of small size bsAb fragments may be desirable in imaging and radioimmunotherapy (117, 118).

Specific approaches can be used to increase the longevity of small bsAb fragments in blood and tissue by (1) fusion of the Fc region of IgG molecules or human serum albumin to prolong the serum half-life. These fusions not only increase molecular size of bsAb fragments and therefore protect them from being excreted out of the body but also mediate binding to the FcRn expressed on the endothelial cells to enter IgG serum stabilization pathway (25, 119–121); (2) multimerization of antibody fragments to increase the molecular size for stabilizing concentration in blood and enhancing the valency of antigen binding (120, 122). The multimerization approach, however, runs a risk of imparting heterogeneity to the molecule and can also lead to undesirable effects by crosslinking of the target receptor (120); and (3) linking of a hydrophobic molecule like polyethylene glycol, a clinically proven technology for serum half-life extension (120, 123). Besides, other polymers like polysialic acid, *N*-(2-hydroxypropyl) methacrylamide, and dextran can also provide protection to the small antibody fragments (25, 120).

Currently, there are more than 60 bsAb formats (18). Of note, the success story of blinatumomab in the format of BiTE, but not in other formats including diabody, Tandab, and quadroma indicates that bispecific molecule design has to consider more than one format from the beginning (93). Moreover, expression and purification of these variable formats require tailored procedures based on each design. This may pose a great challenge in developing bispecific molecules. **Table 1** summarizes relevant information from available literatures.

### FINAL REMARKS

Considerable progress has been made in the development of bispecific molecules based on different scaffolds in recent years. Much of this understanding has made it possible to enter an era of bispecific clinical development with two such molecules (catumaxomab and blinatumomab) having been approved for clinical use in humans (18). The field of bsAbs has been evolutionary and revolutionary, which is reflected in our ability to obtain 100% pure heterodimers, the complete evasion of heavy and light chain mispairing, fairly standardized production/purification/ analytical methods and, importantly, clear ideas of their potential applications.

The full-length mAbs have relatively poor tissue penetration ability. In contrast, smaller antibody fragments like BiTE, BiKE, and TriKE can effectively penetrate tumor tissue and efficiently recruit and activate immune effector cells to lyse tumor cells (23, 70, 93, 115). Besides, the recent success in delivering a bsIg across the blood–brain barrier by targeting transferrin receptor and β-secretase to reduce brain amyloid-β in non-human primate model may open the era of specific antibody brain delivery and treatment of neurodegenerative diseases (124). In another possibility, combining non-antibody small fragments such as lambody, affibody, and aptamer with current antibody scaffolds may expand the treatment arsenal of bispecific molecules.

Moreover, monomeric Ig domain scaffold and Fcab are potent new formats to target difficult to reach sites in the body and to add additional antigen specificity along with enhanced stability, effector function, and extended serum half-life. These properties make them suitable for clinical validation.

In the future, bispecific molecules based on various scaffolds will represent an indispensable class of therapeutic options to treat a variety of clinical indications. However, efforts to improve production and purification on an industrial scale must continue to ensure harvesting of the full benefits of these entities.

### REFERENCES

# AUTHOR CONTRIBUTIONS

DW conceived the topic; DW, HL, and AS wrote the manuscript; HL and AS contributed equally in writing the manuscript; DW and SS revised the manuscript.

# FUNDING

This work was supported by National Natural Science Foundation of China (Grant No.: 81572698) to DW.

fungus adherence to vaginal epithelium and protect against experimental vaginal candidiasis. *J Infect Dis* (2007) 195:149–57. doi:10.1086/509891

or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study. *Lancet Oncol* (2015) 16:57–66. doi:10.1016/ S1470-2045(14)71170-2

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Liu, Saxena, Sidhu and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Engineering of Fc Fragments with Optimized Physicochemical Properties Implying Improvement of Clinical Potentials for Fc-Based Therapeutics

*Chunpeng Yang1,2†, Xinyu Gao1,2† and Rui Gong1 \**

*1CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China, 2University of Chinese Academy of Sciences, Beijing, China*

### *Edited by:*

*Lee Mark Wetzler, Boston University, United States*

### *Reviewed by:*

*Mohey Eldin El Shikh, Queen Mary University of London, United Kingdom Johannes S. Gach, University of California, Irvine, United States*

### *\*Correspondence:*

*Rui Gong<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 11 September 2017 Accepted: 07 December 2017 Published: 08 January 2018*

### *Citation:*

*Yang C, Gao X and Gong R (2018) Engineering of Fc Fragments with Optimized Physicochemical Properties Implying Improvement of Clinical Potentials for Fc-Based Therapeutics. Front. Immunol. 8:1860. doi: 10.3389/fimmu.2017.01860*

Therapeutic monoclonal antibodies and Fc-fusion proteins are successfully used in treatment of various diseases mainly including cancer, immune disease, and viral infection, which belong to the Fc-based therapeutics. In recent years, engineered Fc-derived antibody domains have also shown potential for Fc-based therapeutics. To increase the druggability of Fc-based therapeutic candidates, many efforts have been made in optimizing physicochemical properties and functions mediated by Fc fragment. The desired result is that we can simultaneously obtain Fc variants with increased physicochemical properties *in vitro* and capacity of mediating appropriate functions *in vivo*. However, changes of physicochemical properties of Fc may result in alternation of Fc-mediated functions and *vice versa*, which leads to undesired outcomes for further development of Fc-based therapeutics. Therefore, whether modified Fc fragments are suitable for achievement of expected clinical results or not needs to be seriously considered. Now, this question comes to be noticed and should be figured out to make better translation from the results of laboratory into clinical applications. In this review, we summarize different strategies on engineering physicochemical properties of Fc, and preliminarily elucidate the relationships between modified Fc *in vitro* and the subsequent therapeutic influence *in vivo*.

Keywords: monoclonal antibody, Fc-fusion protein, Fc-based therapeutics, optimization, physicochemical property, stability, aggregation

### INTRODUCTION

Since the hybridoma technology for the production of monoclonal antibodies (mAbs) was invented more than 40 years ago, mAbs are widely used as diagnostics and therapeutics. The first commercial therapeutic mAb, muromonab-CD3 (trade name Orthoclone OKT3), was approved by the U.S. Food and Drug Administration (FDA) for prevention of kidney transplant rejection in 1986 (1). Because it is a murine antibody, administration of this antibody in human might lead to the production of human anti-mouse antibody (HAMA) responses. To reduce the immunogenicity, the development of therapeutic mAbs undergoes four generations: murine mAbs, chimeric mAbs, humanized mAbs, and fully human mAbs (2). Nine therapeutic mAbs have been approved by the U.S. FDA

**47**

since the start of 20171 (3) while the total sales of 70 mAbs for clinical treatment will exceed 100 billion U.S. dollars this year. Among those mAbs, Adalimumab (trade name Humira) the first approved fully human mAb derived from phage display, led the list of top-selling pharmaceutical products with global sales of 16 billion U.S. dollars in 2016 (4).

Fc-fusion proteins are composed of an immunoglobulin (Ig) Fc domain that is directly linked to another peptide, protein, or protein domain. For therapeutic propose, the first description of CD4-Fc fusion protein showed the inhibitory activity against the formation of syncytia during HIV-1 infection in 1989, which showed the proof-of-concept of use of therapeutic Fc-fusion proteins for treatment of HIV-1 infection (5). Subsequently, many modified CD4-Fc fusion proteins were constructed including PRO 542, which was still clinically evaluated (6, 7). Etanercept (trade name Enbrel), a recombinant human tumor necrosis factor (TNF) receptor-Fc fusion protein, was the first TNF-α antagonist approved in the USA for the treatment of rheumatoid arthritis in 1998 (8, 9). The total sale of etanercept is about six billion U.S. dollars in 2016 reported by Amgen (Chairman and CEO Letter and Amgen Inc. 2016 Annual Report2 ), which demonstrates huge market for therapeutic Fc-fusion proteins.

Fc-derived antibody domains are emerging candidates as Fc-based therapeutics. Since they are part of Fc fragments, they may have complete or partial Fc-mediated functions. Therefore, they could be used as scaffold for selection of functional binders, or as carrier for generation of novel fusion proteins for potential clinical use (10). Several candidates have been selected based on Fc-derived scaffolds while Fc-derived domain-fusion protein were also constructed, which were promisingly functional in the aspect of potential therapeutic significance. However, the gap between current progress and clinical use is still huge.

Although great success has been achieved, there are still many limitations during the development of Fc-based therapeutics from bench to bedside (1, 11). The poor physicochemical properties are considerable factors that lead to failure of potential candidates in clinical trials. For marketed drugs, such drawbacks could increase their adverse effects. Therefore, a new-developed candidate should be well characterized in its physicochemical properties which may need to be optimized for better therapeutic outcomes, while increase of physicochemical properties could also be one strategy to make current biodrugs better (biobetter). Although a lot of effort has been put into modification of physicochemical properties on Fc fragment, whether modified physiochemical properties can lead to desired efficacy *in vivo* has not be well understood yet. Here, we summarize the methodology in engineering of physiochemical properties of Fc and try to discuss how the improvement of physicochemical properties influences the efficacy for administration *in vivo*.

# Fc-BASED THERAPEUTICS

As described above, Fc-based therapeutics are now widely used in therapy and prevention of various diseases. Fc is the crystallizable fragment derived from Ig which has five classes including IgG, IgA, IgD, IgM, and IgE in human (12). An Ig molecule is composed of antigen-binding fragments (Fab) Fc. Fc plays multiple roles in dimerization for formation of Y-shaped structure of Ig and maintenance of the structure, and Fc-mediated effector functions and extension of serum half-life. There are two domains: second constant domain (CH2) and third constant domain (CH3) in monomeric Fc of IgG. CH2 domain has a single N-linked glycan at N297 (all the antibody residues here are numbered according to EU numbering (13) unless specified). Two CH3 domains interact strongly with each other to form homodimer resulting in dimerization of Fc. These elements contribute to the physicochemical characteristics of Fc. Through the binding of Fc in IgG to its receptor Fc-gamma receptors (FcγRs), immune leukocytes presenting FcγRs on their surface membrane are recruited and activated, which subsequently triggers antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis to kill and clear target cells (e.g., tumor cells) (14, 15). In addition, Fc can bind to the serum complement molecule (C1q) to initiate the assembly of membrane attack complex formed by complement cascade proteins to destroy target cells, which is termed complement-dependent cytotoxicity (CDC) (16, 17). These effector functions are important for the pharmaceutical efficacies of Fc-based therapeutics. Besides mediation of effector functions, Fc can also bind to neonatal Fc receptor (FcRn) in a pH-dependent manner, which leads to the extension of the serum half-life of IgG (18). In addition, binding of Fc to immune-related molecules such as Fc receptors can regulate immune response *in vivo* (19). Taking together, Fc part in an Fc-based therapeutic protein plays important roles in biological and pharmacological properties including (i) increased stability and aggregation resistance; (ii) acquired multivalent binding to the target; (iii) enhanced Fc-mediated effector functions; (iv) extended serum half-life; and (v) modulated immunogenicity.

### Full-Size mAbs

The therapeutic mAbs have been successfully used for the treatment of various diseases including cancer, immune disease, and infection (20–22). They are highly specific and efficient. Currently, most of the U.S. FDA-approved therapeutic mAbs are full-size IgG molecules with a molecular weight of approximately 150 kDa. Within the IgG isotype, there are four subtypes (IgG1–IgG4) with differing properties distinguished by different hinge regions and Fc fragments. Most of the currently marketed therapeutic IgGs are of the IgG1 subtype while no mAb of IgG3 format is approved yet. A typical IgG1 molecule contains two copies of Fab fragments and one Fc fragment. Each Fab fragment contains a heavy chain variable domain (VH), a light chain variable domain (VL), a first heavy chain constant domain (CH1), and a light chain constant domain (CL). The Fc fragment is dimeric which contains two CH2 domains and two CH3 domains. Fab and Fc are connected by flexible hinge region. In total, a full-size mAb has 12 Ig-fold domains, which means the formation of corrected folding is a complicated process in the production of functional IgG1 molecule. Therefore, how to

1http://www.antibodysociety.org/tag/approved-antibodies/.

2http://investors.amgen.com/phoenix.zhtml?c=61656&p=irol-reportsannual.

prevent them from unfolding and aggregating is still a big challenge in modern biopharmaceutical industry (23).

### Fc-Fusion Proteins

Lots of endogenous proteins in our body and other foreign proteins may have potential to treat various diseases due to the antagonistic or agonistic effects. However, these proteins may have many limitations (e.g., instability and rapid clearance rate from the circulation), which constrains their further application in clinic. To solve this problem, fusion of them to the antibody Fc fragment is an effective strategy. Fc-fusion proteins are molecules in which the Fc fragments are fused to proteins of interests, such as extracellular domains of receptors, soluble cytokines, ligands, enzymes, engineered domains, or peptides (24–27). Therefore, Fc-fusion proteins inherit some antibody-like properties such as relatively good physicochemical characteristics for easy expression, purification, formulation, storage and transportation, bi- or multivalent, long serum half-life, enhanced function, and adjustable immunogenicity, which increases the possibilities for clinic use. However, the similar problems as mentioned in the above paragraph should also be considered during clinical development of Fc-fusion proteins.

### Engineered Domains from Fc Fragment as Potential Therapeutics

A major problem for full-size mAbs is their poor penetration into tissues (e.g., solid tumors) and weak or absent binding to sterically restricted regions on the surface of some molecules (e.g., on the viral envelope glycoproteins), which are fully accessible only by molecules with small size (28). Reduction of molecular weight of full-size mAbs such as identification of the minimum binding domain is one of attractive directions to overcome the drawbacks. Therefore, lots of derivates from intact Igs appeared during last two decades. These derivates include Fab, single chain variable fragment (scFv), heavy chain variable domain (VH) and light chain variable domain (VL) (29). The functional VH from camelidae, specially termed as nanobody, has been clinically developed by Ablynx. However, due to the lack of Fc part, the serum half-lives of these variants are relatively short, which is one of the major obstacles for *in vivo* administration (29). Other Ig domains including CH2 and CH3 derived from Fc and Fc itself have been proposed as new scaffolds for development of novel Fc-based therapeutics (10, 28, 30, 31). They are also smaller than full-size mAbs. Compared with those variants from Fab-based backbone, these scaffolds from Fc-based backbone might offer additional Fc-mediated advantages such as high stability, potential effector functions, and long half-life due to incorporation of full length or truncated Fc fragment (10, 28). Optimization of the Fc-based scaffolds on their folding and aggregation resistance could confer better potentially therapeutic outcomes.

The full-size mAbs, Fc-fusion proteins, and engineered domains from Fc fragment share Fc fragment or portion of Fc fragment as common region. Therefore, engineering of Fc could be benefit for all of them. Here, we focus on the current progress in increase of physicochemical properties of Fc including stability and aggregation resistance toward better clinic consequences.

# INCREASE OF PHYSICOCHEMICAL PROPERTIES OF Fc FRAGMENT

In general, the stability and aggregation resistance of a protein are two major physicochemical properties we most concern. The stability is the ability of a protein which retains its correctly folded conformation under harsh conditions such as high temperature, chemical denaturant, protease, and others, while the aggregationresistant property is to reduce the formation of soluble oligomers and insoluble precipitates during expression, concentration, storage, and others.

One important restrictive factor in development of Fc-based therapeutics is that these proteins may tend to unfold and aggregate upon exposure to various stresses (32), including agitation (33, 34), high temperature (35, 36), low pH (37, 38), high protein concentration for subcutaneous therapeutic delivery (39), freeze– thaw cycle (40, 41), and transportation and long-term storage (42, 43), which means loss of function and increase of immunogenic risk. For example, aggregation may lead to not only loss of activity but also immune response, and negatively impact on many production processes including expression, purification, and formulation (44). A stable protein typically could be correctly expressed at high level, easily purified with no requirement of specific conditions such as low temperature and additional protease inhibitor, which makes the manufacture process much easier and cheaper. It remains active during long-term storage and after administration *in vivo*, which achieves less frequent usage at a lower dosage. An aggregation-resistant protein could be concentrated to high concentration without formation of inactive oligomer, which reduces the injection volume and makes patient more comfortable. Although stability and aggregation propensity are in different descriptions, the relationships between them are close (45). In many cases, increased stability can also lead to less aggregation propensity. Hence, during the development of recombinant therapeutic proteins, prevention of unfolding and aggregation is essential for ensuring efficacy and safety. The structure of a protein is maintained by covalent and non-covalent interactions (46). The covalent interactions are typically caused by the formation of disulfide bonds between two cysteine residues. The non-covalent interactions include hydrogen bonds, Van der Waals forces, hydrophobic interactions, and salt bridges (ionic bonds). These interactions guide the correct folding of a protein to form secondary structure such as α-helix and β-sheet, then form advanced tertiary structure and quaternary structure. Therefore, optimizing the covalent and non-covalent interactions is major direction to increase the physiochemical properties of Fc fragment.

# Role of CH2 and CH3 in Maintaining Fc Structure

The antibody heavy chain constant domain is generally defined as CH1–CH2–CH3 in IgG, IgA, and IgD, with an additional domain (CH4) for IgM and IgE (12). The crystal structure of a fucosylated human IgG1 Fc is used here for presenting (PDB 3AVE) (47) (**Figure 1**).

Structural comparison of fucosylated CH2 in IgG, IgA, and IgD is equal to CH3 in IgM and IgE. The primary sequence of IgG

CH2 has a glycosylation motif N297–S298–T299 that results in N-linked glycosylation (48). The oligosaccharides are important for the stability, aggregation propensity, and effector functions of Fc fragment (49). For example, removal of the oligosaccharides results in reduction of aggregation resistance in IgG1 under acidic conditions (50).

The secondary structure of CH2 consists of two β-sheets forming a barrel as solved crystal structure of glycosylated IgG1 CH2 in an intact IgG1 molecule (51), a fucosylated human IgG1 Fc (47), or isolated aglycosylated CH2 (52) (**Figure 1**). There are seven β-strands from A to G connected by three loops (loops BC, DE, and FG) and two helices (helix 1 and 2) in CH2 (**Figure 1**). The native disulfide bond between strand B (C261) and F (C321), buried in the hydrophobic core of the molecule, should be important for the structural stability of CH2 although the direct evidence is still lacking (**Figure 1**). CH2 is relatively unstable compared with other Ig domains such as CH3 (53, 54). For example, during thermo-induced unfolding, the melting temperature (Tm) of mouse IgG1 CH2 is only 41°C. The Tm of human IgG1 CH2 is 54°C, which is also low but higher than that of mouse IgG1 CH2 (55). In serial comprehensive studies, it was found that (i) different IgG subclasses have different stability and aggregation propensities due to the sequence variation of their CH2 domains, and IgG1 is the best one among all the four subclasses in general (56–59); (ii) Fc aggregation induced by low pH is firstly triggered by CH2 unfolding associated with the protonation of specific acidic residues (54, 60). Therefore, it is one of the major determinants for the unfolding of Fc-based molecules (61). Engineering of CH2 to increase its stability and aggregation resistance is expected to improve the physicochemical properties of Fc, which could be used to modify Fc-based therapeutics.

CH3 is the Ig domain following CH2. The structure of CH3 is very similar to that of CH2 (**Figure 1**). It also contains seven β-strands from A to G connected by three loops (loops BC, DE, and FG) and two helices (helix 1 and 2) with a native disulfide bond between strand B (C367) and F (C425) as CH2 although strand D seems to be shorter than that in CH2 according to the crystal structure (**Figure 1**) (47, 51). Distinguishing from two CH2 domain with in Fc, two CH3 domains can interact with each other very strongly, which leads to the formation of dimeric Fc structure. Probably due to the homo-interaction, dimeric CH3 has much higher stability than monomeric CH2 (62). In the interface of two IgG1 CH3 domains, there are at least six residues (T366, L368, P395, F405, Y407, and K409) involved in the interactions (**Figure 1**) (63). In detail, residues T366 and Y407 form a hydrogen bond and represent the principal intermolecular and intramolecular contact with each other, while residue K409 forms a hydrogen bond with residue D399 on the partner CH3 domain. Residues L368 and F405 form intermolecular and intramolecular contacts through van der Waals interactions only. These five residues locate on the two internal antiparallel β-strands and form a patch at the center of the interface. P395 contributes to the flexibility of the proline-containing loop constituting the domain–domain interface. Furthermore, the direct evidence showed that the native disulfide bond is not only involved in the folding of single CH3 domain, but also related to the dimerization process of two CH3 domains (64, 65), which is helpful for prevention of aggregation (66). Combination of display technology and high-throughput sequencing discloses a stability landscape of the CH3 domain (67). For example, it has been found that hotspots locate at C- and F-strand (positions 378–383 and 423–428, respectively) which are tolerant and intolerant to substitution due to their different orientations in a β-sheet. The side chains of hydrophobic residues V379, W381, F423, and V427, as well as the disulfide bond forming residue C425, are directed to the hydrophobic core of the CH3 domain and interact with residues of the inner β-sheet. They are highly intolerant to mutation. By contrast, the solvent-exposed side chains of A378, E380, E382, S424, S426, and M428 are more tolerant to mutation. This strongly suggests that the intolerance to mutation of a particular residue is not primarily caused by its localization in a secondary structural element, but by side-chain interactions with other parts of the molecule. The factors are more complicated for those residues in the inner β-sheet because they interact with other residues either in other chains in the same domain or in the same chains in the symmetrical partner CH3 domain.

Interestingly, although the glycan chain does not contact the CH3 domain, the CH3 domain in glycosylated Fc reveals a higher stability than that in aglycosylated Fc, implying an indirect contribution of the glycan chain to maintain the CH3 structure domain possible through stabilization of CH2 domain (68). Although the CH2 domain firstly unfolds during acid-induced unfolding, the colloidal stability of the CH3 homodimer in the fully unfolded state is lower than that of CH2 monomer and aglycosylated Fc, Yang et al. Engineering Fc for Clinical Potentials

and the unfolded CH3 homodimer forms much larger aggregates (68, 69). Therefore, it was concluded that (1) the unfolding process of CH2 and CH3 domains is independently from each other in the aglycosylated Fc region; (2) the colloidal stabilities of the CH2 and CH3 domains affect the aggregation process of the unfolded aglycosylated Fc region in a compensatory manner; and (3) the CH3 domain plays the most critical role among different Ig domains in driving intact antibody aggregation under acidic conditions (68, 69).

Although different antibody subclasses and subtypes have different Fc sequences, Fc is the common part of all the full-size antibodies and Fc-fusion proteins. To increase the stability and aggregation resistance of Fc, most previous work has focused on introducing mutations to form covalent interactions such as disulfide bonds or enhance the non-covalent interactions. In addition, change of the glycosylation form may also alter the folding of the Fc-based molecules.

### Introduction of Covalent Bond in Fc

Disulfide bonds are main kind of covalent interactions in intra- or inter-Ig domains, formed by the oxidation of two thiol groups within cysteine residues, which fix and stabilize the structure of proteins in an oxidative environment. There are 12 intra-domain disulfide bonds (each domain has one intra-domain disulfide bond locating in hydrophobic core) that exist in IgG, while 4, 6, 13, and 4 inter-chain disulfide bonds exist in IgG1, IgG2, IgG3, and IgG4, respectively, due to different length of hinge regions (70). Disruption of the intra-domain disulfide bonds in CL, VH, VL or CH3 results in a significant reduction of the structural stability of those domains, especially the thermodynamic stability (66, 71–77). Similarly, inter-domain disulfide bonds also have an effect on structural stability of IgG (68, 78). The existence of one disulfide bond in hydrophobic core of IgG domain could stabilize the structure. Hence, additional disulfide bond may improve the stability of Ig domain. The IgG1 Fc fragment consisting of two CH2 domains and two CH3 domains has four inherent intradomain disulfide bonds (one disulfide bond in each domain). Additional disulfide bonds could be engineered in single domain or between two domains.

### Engineering of Disulfide Bond in CH2

There is one intrinsic disulfide bond in hydrophobic core of CH2 domain between β-strand B and F (C261 in strand B and C321 in strand F) as mentioned earlier (**Figure 1**). In our previous research, based on possible distance of forming disulfide bond between two cysteines, five pairs of amino acids were substituted to cysteines among which two of them could be well expressed in bacterial expression system (55). Those two mutants termed m01 and m02, respectively, in which an additional disulfide bond between β-strand A and G was engineered by mutations on L242C and K334C in m01 (**Figure 2A**) as well as V240C and L334C in m02 (**Figure 2B**). The Tm of m01 is 73.8°C, which is almost 20°C higher than that of wide-type CH2 (wtCH2). Both of them are also much more stable against urea unfolding compared with wtCH2. Meanwhile, the secondary structure of m01 is not affected by this additional disulfide bond measured by circular dichroism (CD) and nuclear magnetic resonance. The stability of

different mutations for introduction of additional disulfide bonds presented by PyMOL. (A) CH2 mutant L242C/K334C. (B) CH2 mutant V240C/K334C. (C) CH2 mutant A287C/L306C. (D) CH2 mutant R292C/V302C. (E) CH3 mutant S375C/P396C. (F) CH3 mutant P343C/A431C. (G) CH3 mutant P343C/A431C/K447C. The native disulfide bond is colored by yellow, whereas the mutated residues for additional disulfide bonds are colored by red. All the marked distance is the measured between two α-carbon atoms in related two cysteines after mutagenesis by using PyMOL.

m02 is also much better than that of wtCH2, but the aggregationresistant property is not as good as m01. It has been shown that the Tm of CH2 with mutations in L242C and L334C in IgG1 is 8.7°C higher than that in wtIgG1 (79). In another research (80), to improve the stability of an IgG1 variant with mutation of N297G (mAbW.IgG1, an effector function silenced IgG1), an additional disulfide bond according to the design of m01 was introduced between position L242 and K334 (81). As expected, the stability of this IgG1 variant (mAbW.SEFL2.0) with mutations on L242C/ N297G/K334C is improved particular in the thermal stability, but it shows faster clearance in the rat in pharmacokinetics study. Another four IgG1 variants with different additional disulfide bond formation were designed, constructed, and expressed in CHO expression system, among which two variants mAbW. SEFL2.1 with mutations of A287C/N297G/L306C in CH2 domain (**Figure 2C**) and mAbW.SEFL2.2 with mutations of R292C/ N297G/V302C in CH2 domain (**Figure 2D**) with improved stability, decreased rate of clearance, and longer half-lives in both the rat and cynomolgus monkey models compared with mAbW. IgG1. Importantly, the Tm values of mAbW.SEFL2.1 and mAbW. SEFL2.2 are about 8°C higher than that of mAbW.IgG1, while mAbW.SEFL2.0 is about 2°C higher than that of mAbW.IgG1, which indicates introduction of disulfide bonds in different positions results in different outcomes.

### Addition of Disulfide Bond in CH3

The same as CH2 domain, CH3 domain also has one intrinsic disulfide bond in hydrophobic core (C367 and C425). To investigate the influence of this disulfide bond in CH3, a variant of CH3 reducing disulfide bond was engineered (66). And in the following studies it has been shown that the thermal stability of the reduced form is lower than that of the oxidized form measured by CD and differential scanning calorimetry (DSC) results. In addition, the reversibility after unfolding is also significantly lower. And although most of reduced form retained a stable dimeric structure, aggregation is improved. Similarly to CH2, introduction of additional disulfide bond inside the domain could also improve the stability of CH3. In another study, an additional disulfide bond was engineered into isolated monomeric CH3 by mutation of P343 and A431 to two cysteines, resulting in improved protein expression (up to fivefold) and elevated Tm (from 40.6 to 76.0°C), without affecting FcRn binding ability (82). In a more previous study, two additional disulfide bonds were engineered into CH3 domain, respectively. Hence the Tm value of Fc variant with dual mutations of P343C/A431C and S375C/ P396 in CH3 domain (**Figures 2E,F**, respectively) was obviously improved measured by CD and DSC experiment compared with wtFc (83). Since two CH3 domains interact non-covalently with each other, in addition to introduction of intra-domain disulfide bonds, there are some works on engineering of additional interdomain disulfide bonds between two CH3 domains. The last three amino acids (P445, G446, and K447) at C-terminal of CH3 were replaced by G, E, and C, respectively, derived from CL domain (**Figure 2G**), which could introduce an additional inter-domain disulfide bond between two CH3 domains in Fc or dimeric CH3 (68, 84). And the conformational stability of both the CH2 and CH3 domains could be improved in an Fc variant with this kind TABLE 1 | Introduction of disulfide bonds and engineering of non-covalent interactions in CH2 and CH3 domains.

*All the domains are from human IgG1 if not specified.*

*bAll the islolated domains are expressed in Escherichia coli, others are expressed in eukaryotic cells.*

of inter-domain disulfide bond (68). In another research, an Fc variant (P343C, A431C P445G, G446E, and K447C) with not only additional intra-domain but also inter-domain disulfide bond could increase the thermostability of CH2 and CH3 (84). The Tm of CH3 in this variant was improved by 18.1°C compared with the wtFc. Furthermore, the properties of pH and aggregation resistance of this variant were also increased when compared with the wtFc. The mutations in CH2 and CH3 domains for introducing disulfide bonds are listed in **Table 1**.

### Optimization on Non-Covalent Interaction in Fc

Although introduction of disulfide bonds can significantly increase the stability of a protein, the risk of increasing aggregation propensity caused by incorrectly paired cysteines might not be neglected. Therefore, optimization of non-covalent interactions is another efficient strategy to improve the stability or aggregation resistance of Fc fragments. The optimization could be performed by site-directed mutations (without forming covalent disulfide bond) under the aid of sequence and structural information, and computation, which could change the local residue-to-residue interactions and influence the whole Fc-based molecule.

*a*

### Introduction of Intramolecular Non-Covalent Interactions in CH2

The exposure of hydrophobic residues may increase the aggregation propensity due to non-specific hydrophobic interactions, especially when the large hydrophobic clusters form. Therefore, rational disruption of those continuous hydrophobic residues without affecting the molecular structure may be helpful for decreasing the formation of aggregation. A computational technology termed spatial aggregation propensity (SAP) was developed to measure the dynamic exposure of hydrophobic patches and identify the location and size of these aggregation-prone regions based on the atomistic molecular dynamics simulations, which can guide the performance of target mutations for engineering of protein stability and aggregation resistance (45, 85, 86). By this technology, several mutations with different combinations were introduced to generate several IgG variants (45). One variant with combinational mutations of L235K and L309K in the CH2 domain (**Figure 3A**) showed not only increased thermostability stability but also improved aggregation resistance. Being a hydrophilic amino acid, lysine can discontinue the hydrophobic patch and reduce the non-specific interactions between hydrophobic patches. Furthermore, as mentioned earlier, CH2 domain is typically the least stable domain in the Fc portion, which influences the whole molecular stability and aggregation propensity. Therefore, the physicochemical properties of the antibody molecule could be optimized after rational introduction of proper hydrophilic amino acids for disruption of the large hydrophobic aggregation-prone clusters. In addition to SAP (87–89),

mutated residues for additional disulfide bonds are colored by red.

several online programs such as TANGO3 (90–92), PASTA4 (93), AGGRESCAN5 (94), and Aggrescan3D6 (95) are also widely used to predict aggregation-prone regions within proteins. Therefore, it is desired that new Ig variants could be identified in the future.

In many instances, it might be desirable for a therapeutic mAb and Fc-fusion protein to lack/reduce effector functions. According to previous studies, two sets of mutations in CH2 domain including "TM" (triple mutations of L234F/L235E/ P331S) and "YTE" (M252Y/S254T/T256E) are effective in abolishing the binding of Fc to FcγRs (96) and C1q and enhancing the pH-dependent binding to FcRn (97, 98), respectively, which can generate an IgG variant (TM–YTE) with extended half-life but without Fc-mediated effector functions *in vivo*. However, these mutations adversely influence the stability and aggregation propensity and result in many difficulties for further applications (99). For example, it has been shown that replacement of the original amino acids M252, S254, and T256 to Y, T, and E significantly reduced the stability and increased the aggregation tendency due to the increased local flexibility of the 244–254 segment in CH2 (100, 101). To compensate the decreased physicochemical properties, several mutants were designed with different combinations of mutations with the strategies including substitution of charged residues (e.g., E) by uncharged residues (e.g., Q). According to a serial of experiments by characterization of designed mutants, one novel mutant (FQQ–YTE) with combinational mutations of L234F/L235Q/K322Q/M252Y/S254T/T256E (**Figure 3B**) was identified, which had significantly improved conformational stability while retaining the same biological activities as TM–YTE mutant (99).

### Introduction of Intra- and Intermolecular Non-Covalent Interactions in CH3

Sequence analysis among different Ig classes and subtypes can provide useful clues for Fc optimization. One good example is the mutation on bovine IgG1 CH3 for increased stability deduced from the sequence and structural information (102). According to sequence alignment and frequency analysis among 36 unique IgG Fc sequences originating from 19 different mammalian species, bovine IgG1 demonstrated the closest resemblance to the consensus sequence other than primate IgG sequences, while a few crucial positions could be mutated to make the molecule more stable. Therefore, the residues S174, Y179, G197, S207, and T246 in bovine IgG1 CH3 numbered according to the reference (102) which are partnered with G371, D376, K392, G402, and L441 in human IgG1 CH3 were selected to be substituted by G, D, K/A, G, and L, respectively. Four of these 5 mutant positions are at residue sites of reasonable heterogeneity (i.e., the measured positional entropies are in the top 70% for all residue positions), while mutation at S207 (G402) was the only position that highly conserved because only 4 of the 36 sequences have another residue other than G. In spatial structure, each residue is far away from the four others. After measurement of

5http://bioinf.uab.es/aap/.

3http://tango.crg.es/.

4http://protein.bio.unipd.it/pasta2/index.html.

6http://biocomp.chem.uw.edu.pl/A3D/.

heat-induced unfolding, one combination with G197K/S207G/ T246L (**Figure 3C**) showed the highest Tm, which increased about 10°C compared with that of wide-type bovine IgG1 CH3. The experimental results could be explained as follows. First, among these mutations, replacement of G197 to K392 inserts a lysine at the interface between the CH3 dimer locating at the hydrophobic portion of the side-chain that packs against a hydrophobic patch created by F210 (F405) and V202 (V397) of the opposite dimer subunit. Although adding a hydrophobic moiety like alanine to this cavity is also better than original G, introduction of positive charged lysine at this position is shown to be best due to the adjacency to D204 (D399), a negative charged amino acid can possibly enhance the favorability of lysine at this position. Second, substitution of S207 by G402 places G in a tight turn between β-strands, which likely releases strain induced by the tight turn since the backbone dihedral angles of this residue in the crystal structure are outside the generally allowed ranges for α-substituted amino acids (all amino acids other than G). Finally, the mutation of T246 to L441 completely buries the isobutyl side chain into the hydrophobic interior of the β-sandwich. Although burial of three groups are expected for more favorable contributions, the actual effect is not as obvious as desired. The reason is that the side chain of T can compensate somewhat for the loss of buried hydrophobic surface area due to steric placement or hydrogen bonding.

This finding indicates that cross-species analysis provides useful information about the relationship between residues and physicochemical properties, and further guides the rational design for a better Fc with more stable, more aggregation-resistant and more soluble characteristics.

### Engineering of Loop and Other Regions in CH2 and CH3

Both CH2 and CH3 domains have several flexible loop regions, which could also be the targets for optimization of the physicochemical properties. For example, to stabilize the CH2 domain, an enhanced aromatic sequon (EAS) (Q295F/Y296A) (**Figure 4A**) was engineered into the top of *N*-glycosylated DE loop, which led to a 4.8°C increase of the Tm of the purified IgG1 Fc fragment (103). This strategy could be used in optimizing a full-length IgG1 molecule for enhancement of its resistance to unfolding and aggregation. The crystal structure of the EAS-stabilized IgG1 Fc fragment reveals the importance of the GlcNAc1·F295 interaction, as well as the participation of the core fucose (Fuc) attached to GlcNAc1 in an interaction with F295. As mentioned earlier, the yeast display technology is also used in high-throughput screening of improved CH3 mutants with replacement of its original loops for optimization on Fc and development of antigen-specific Fc binders [Fc-antigen binding (Fcab)] (104, 105).

It has been shown that natural β-sheet proteins use negative design to avoid edge-to-edge aggregation, which indicates that the residues at N- and C-termini may be involved in aggregation formation (106). A shortened CH2 (CH2s) (**Figure 4B**) was constructed with truncation of seven unstructured N-terminal residues according to a crystal structure of an intact IgG1 (PDB 1HZH) (51), which showed significantly increased aggregation

resistance and potential Fc-mediated functions (107). Engineering of C-terminal residues in CH2 might also have the same influence as described earlier. The optimizations on non-covalent interactions in CH2 and CH3 domains are summarized in **Table 1**.

### Glycoengineering in Fc

There are only two symmetrical N-glycosylation sites in the Fc fragment, which locate at amino acid position N297 in IgG1 CH2 domain. It has been widely accepted that N-glycan has a critical impact on the structure and effector functions of Fc-based therapeutics (108–111). Absence of the N-glycan can cause dramatic conformational change and decreased stability of the Fc (112–117). Without N-glycan, the binding of Fc to various receptors and their associated biological functions is either reduced or completely lost (112, 116, 118–120). The factors influencing the oligosaccharide profiles of Fc-based therapeutics are very many, such as the use of different cell lines, cell culture conditions, scales, and other factors (121, 122). Therefore, variation of glycoform is one of the main factors that cause heterogeneity of therapeutic antibodies and Fc-fusion proteins. Although aglycosylated Fc-based therapeutics have been explored for clinical use, the majority of these therapeutic proteins are still glycosylated. In most conditions, the function of N-glycan is irreplaceable for the treatment of some diseases.

According to the difference of oligosaccharides on the outer arms, N-glycan can be classified into three categories including high mannose (Man), complex, and hybrid (49). All of these classes share an invariable core structure containing two copies of primary *N*-acetylglucosamine (GlcNAc), one primary Man and the two secondary Man residues. High Man oligosaccharides are composed of Man only in the outer arms. Complex oligosaccharides are composed of GlcNAc and galactose and potentially sialic acid in the outer arms. Meanwhile, complex oligosaccharides can exist with or without the core Fuc. Hybrid oligosaccharides are made of one arm with complex and the other arm with high Man residues (**Figure 5**) (49). Therefore, different configurations and compositions of N-glycan can lead to >400 different variants concerning the two CH2 domains of IgG Fc (123).

The position of glycosylation on the Fc determines that it has no impact on antigen–antibody binding and FcRn binding, but oligosaccharides are critical for the binding of FcγRs and C1q, which trigger different immune responses (FcγRs for ADCC and C1q for CDC). On the other hand, changes occurring on the composition of N-glycan can influence the conformation of the whole antibody or Fc-fusion protein molecule, causing alteration in the binding affinity for various FcγRs (124). That is one of the reasons for the glycosylation playing such effective role in engineering Fc-based therapeutics.

The process of glycosylation only occurs in eukaryotes, so *Escherichia coli* and Chinese Hamster Ovary cell (CHO) derived IgG1 Fc were usually used to illustrate the impact of N-glycosylation on the stability of Fc, representing aglycosylated and glycosylated Fc proteins, respectively (50). Both of them behave similarly during heat and low pH induced unfolding. First, the tertiary structure and CH2 domain are unfolded, then the secondary structure and CH3 domain are changed. Due to the interaction of oligosaccharides, the glycosylated Fc protein is more compact (smaller hydrodynamic radius) than the aglycosylated Fc protein at neutral pH. In the aspects of thermostability and pH resistance, the Tm of glycosylated CH2 domain is 4–5°C higher than that of aglycosylated domain and the acid resistance of glycosylated Fc is ~0.5 pH lower than aglycosylated Fc (50).

Previous studies have proved that truncation of N-glycosylation is a major method to understand the relationship between the functions, structures and Fc glycoforms. Fc glycoform variants with partial or complete removal of glycan carbohydrates were compared with wtFc by using long molecular dynamics simulations (125). The results indicated that glycan truncation or removal can cause quaternary structural deformation of the Fc due to the disruption or loss of a lot of inter-glycan contacts. Because the existence of the weak binding through two oligosaccharides, glycan truncation/removal can also cause the tertiary structural deformation of CH2 domains, which results in destabilization of individual CH2 domains. During elevating Tms, glycan truncation is differentially affecting structural deformation in locations of helices 1 and 2 in CH2 that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the binding surface to FcRn, could affect the binding to FcRn if these regions are unable to refold after Tm normalization. During elevated Tm simulations of the deglycosylated variant, CH2 domains collapse onto CH3 domains. All these studies show that glycosylation plays an important role in the stability of Fc. Besides that, different formations of glycosylation in N297 site were compared, containing three Fc proteins produced from the yeast *Pichia pastoris* with three kinds of glycosylation sites (di-, mono-, and nonglycosylated) and another three different forms of nonglycosylated Fc (mutating N297 to two different amino acids and enzymatic digestion of the Fc glycoforms), to examine the differences of structural stability. Under different pH conditions, the di- and monoglycosylated forms of Fc showed the highest and lowest levels of stability, respectively, while the stability of nonglycosylated form was in the middle and depended on the solution pH (126, 127). Furthermore, hemi-glycosylated (same to monoglycosylation) Fc shows that the binding affinities toward all FcγRs were significantly decreased and a moderate decrease (~20%) in C1q binding, representing change of effector functions (128).

Lots of previous studies have proved that glycosylation also deeply affects the aggregation of antibodies and Fc-fusion proteins (126, 129–131). By comparing the differences between glycosylated and deglycosylated antibodies, it has been found that deglycosylated antibodies had not only less thermostability and resistance to GdnHCl-induced unfolding, but also higher aggregation rates at a accelerated stability study (129). The possible reason for these findings is that disruption of protein–carbohydrate interactions leads to the exposure of aggregation-prone motifs and further results in the aggregation of the whole deglycosylated antibodies (130). The interactions between two monomeric Fc regions of an antibody include protein–protein, carbohydrate– carbohydrate, and protein–carbohydrate, all these forces have contributions to the stability and aggregation resistance. So any change of oligosaccharides (e.g., truncation and variation on glycoforms) might have serious impact on the physicochemical properties of Fc-based therapeutics.

Although the importance of N-glycosylation on Fc is clear, the production of Fc-based proteins with selected glycoforms is still very difficult. For now, the enzymatic modification of recombinant IgGs *in vitro* or engineering the host expression system can be used to modify the glycoforms of Fc-based therapeutics (108).

Due to the complexity and limit of technology for engineering the N-glycoform, lots of work still focuses on the generation of IgG variants with aglycosylation, which only needs to introduce a mutation into N297, for decrease of the effector functions to treat some chronic diseases. However, as described earlier, these mutants may influence the physicochemical properties of Fc-based proteins deeply and cause the loss of efficacy, which will be further discussed in the following paragraph.

# RELATIONSHIPS BETWEEN PHYSICOCHEMICAL PROPERTIES AND CLINICAL POTENTIAL

The development of therapeutic antibodies, Fc-fusion proteins, and Fc-based antibody domains faces many challenges from bench to bedside. For example, an mAb may have excellent activities *in vitro* but lose the entire or major function *in vivo* and *vice versa*. Hence, to make a successful Fc-based therapeutics, it may need many modifications before clinical use.

Historically, either IgG2 or IgG4 isotypes have been selected for applications where cytotoxic effector functions are not required due to their limited cytotoxic effector functions (132). However, IgG1 is still the first choice due to its comprehensive advantages in many cases. Therefore, to eliminate undesired effector functions, removal of the glycosylation is an efficient way. A major concern is that deglycosylation will lead to instability and aggregation. Hence, the IgG1 scaffold with stable effector functions missing (81) needs to be further optimized. Based on the computational modeling, two variants mAbW.SEFL2.1 and mAbW.SEFL2.2 with tri-mutations on A287C/N297G/L306C and R292C/N297G/V302C, respectively, as mentioned earlier showed improved stability, decreased clearance rate, and longer half-life (80), which could be used for development of therapeutic mAbs without effector functions.

As mentioned, Etanercept is one of the most successful recombinant Fc-fusion proteins in the market. When administered *in vivo*, the dose of each injection is large (e.g., 25 mg/twice or 50 mg/once per week), which requires high concentration of Etanercept in the formulation. The risk of aggregate formation in this condition is increasing, which may lead to loss of functions and increase of side effects. To improve the solubility and reduce the aggregation, two mutations D239E and L241M were introduced into the Fc portion of Etanercept, which may increase the stability and aggregation resistance of whole recombinant protein (133). Actually, the formation of aggregation should be seriously considered when developing a biosimilar of Etanercept (134).

As a domain from Fc, CH2 has been proposed as a scaffold for development of C-based single domain antibody (C-sdAb), which has been extensively reviewed (10, 28). CH2 could have or partially have Fc effector functions and affinity to FcRn. Therefore, it could have relatively longer serum half-life after engineering due to the containment of FcRn binding sites inherited from Fc (135, 136) compared with other Ig domains (e.g., VH) as binders (29). Based on this scaffold, a panel of C-sdAbs has been selected against different targets (137, 138). However, in general, their activities are modest. Since it has been shown that CH2-based

### REFERENCES

binders tend to aggregate, a mission for development of clinically potential C-sdAbs is to further modify the scaffold to increase its stability and decrease its aggregation propensity (55, 107, 135, 139, 140), as well as for development of other engineered domains such as Fcab (104, 105, 141), monomeric Fc (142) and monomeric CH3 (82) derived from Fc fragment. Although preliminary results show the proof-of-concept, there is still very long way to achieve the final aim.

### SUMMARY AND PROSPECTIVE

The problems caused by instability and aggregation propensity are major restrictions in Fc-based biopharmaceutical industry. Many other reviews have already been focused on these issues (32, 46, 143). In this review, we summarized and discussed current status in the related field in Fc engineering. Although lots of work has been done to increase the stability and aggregation resistance of Fc, it seems that the *in vivo* outcomes are not very clear after optimization. On the other hand, plenty of work has also been done to enhance the Fc-mediated effector functions and extend the half-life. However, the value for clinical application is not well verified. At present the fact is that only a few candidates among many finally enter the market due to various reasons. Therefore, more attentions should be paid toward the relationship between physicochemical optimizations and potential clinical applications, which may accelerate the development of Fc-based therapeutics.

# AUTHOR CONTRIBUTIONS

RG designed the review article topic and text structure; CY, XG, and RG wrote the manuscript.

# ACKNOWLEDGMENTS

The authors are thankful to The Core Facility and Technical Support, Wuhan Institute of Virology, Wuhan Institute of Biotechnology as well as Wuhan Key Laboratory on Emerging Infectious Diseases and Biosafety for the supports. This work was funded by the "Personalized Medicines—Molecular Signaturebased Drug Discovery and Development", Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA12020346), the Key Program of Chinese Academy of Sciences (Grant No. ZDRW-ZS-2016-4), the National Key Research and Development Program of China (Grant No. 2016YFC1202902), and the "One-Three-Five" Strategic Programs of Wuhan Institute of Virology, Chinese Academy of Sciences (Grant No. Y605221SA1).

aggregation propensity. *J Pharm Sci* (2012) 101(1):102–15. doi:10.1002/ jps.22758

its stability and aggregation resistance. *Mol Pharm* (2013) 10(7):2642–52. doi:10.1021/mp400075f

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Yang, Gao and Gong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Ji-Hee Ha1 , Jung-Eun Kim1 and Yong-Sung Kim1,2\**

*1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon, Korea*

### *Edited by:*

*Tianlei Ying, Fudan University, China*

### *Reviewed by:*

*Sang Taek Jung, Kookmin University, South Korea Roland E. Kontermann, University of Stuttgart, Germany*

> *\*Correspondence: Yong-Sung Kim<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Immunotherapies and Vaccines, a section of the journal Frontiers in Immunology*

*Received: 14 July 2016 Accepted: 16 September 2016 Published: 06 October 2016*

### *Citation:*

*Ha J-H, Kim J-E and Kim Y-S (2016) Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins. Front. Immunol. 7:394. doi: 10.3389/fimmu.2016.00394*

The monospecific and bivalent characteristics of naturally occurring immunoglobulin G (IgG) antibodies depend on homodimerization of the fragment crystallizable (Fc) regions of two identical heavy chains (HCs) and the subsequent assembly of two identical light chains (LCs) via disulfide linkages between each HC and LC. Immunoglobulin Fc heterodimers have been engineered through modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form heterodimers rather than homodimers. Many research groups have adopted different strategies to generate Fc heterodimers, with the goal of high heterodimerization yield, while retaining biophysical and biological properties of the wild-type Fc. Based on their ability to enforce heterodimerization between the two different HCs, the established Fc heterodimers have been extensively exploited as a scaffold to generate bispecific antibodies (bsAbs) in full-length IgG and IgG-like formats. These have many of the favorable properties of natural IgG antibodies, such as high stability, long serum half-life, low immunogenicity, and immune effector functions. As of July 2016, more than seven heterodimeric Fc-based IgGformat bsAbs are being evaluated in clinical trials. In addition to bsAbs, heterodimeric Fc technology is very promising for the generation of Fc-fused proteins and peptides, as well as cytokines (immunocytokines), which can present the fusion partners in the natural monomeric or heterodimeric form rather than the artificial homodimeric form with wild-type Fc. Here, we present relevant concepts and strategies for the generation of heterodimeric Fc proteins, and their application in the development of bsAbs in diverse formats for optimal biological activity. In addition, we describe wild-type Fc-fused monomeric and heterodimeric proteins, along with the difficulties associated with their preparations, and discuss the use of heterodimeric Fc as an alternative scaffold of wild-type Fc for naturally monomeric or heterodimeric proteins, to create Fc-fusion proteins with novel therapeutic modality.

Keywords: bispecific antibody, Fc engineering, heterodimeric Fc, Fc-fusion proteins, immunocytokines, antibody engineering

### INTRODUCTION

Bispecific antibodies (bsAbs) simultaneously bind two different antigens or two distinct epitopes on the same antigen as a single molecule; they differ from naturally occurring immunoglobulin G (IgG) monospecific antibodies (mAbs) (1, 2). Owing to their additional targeting ability, bsAbs often offer improved clinical benefits for the treatment of complicated diseases, such as cancers and immune disorders, wherein multiple cell-surface receptors or ligands are engaged (1–3). Numerous efforts have been made to engineer mAbs into bsAbs, which has resulted in the generation of more than 60 different bsAb formats (3–5). Many bsAbs have been engineered by linking antibody fragments, such as single-chain variable fragments (scFv), antigen-binding fragments (Fab), and heavy (VH) and light chain (VL) variable domains, as well as their appendages to IgG-format mAbs (3–6). However, these novel formats, deviating from the conventional IgG structure, often suffer from poor physicochemical properties, such as low solubility and aggregation, difficulties in large-scale manufacturing, poor pharmacokinetics, and potential immunogenicity (3–6). To improve the developability, bsAbs in the formats of intact IgG or IgG-like (containing an Fc) architectures have been extensively developed (5–7).

Conventional IgG antibodies are bivalent and monospecific, the assembly of which depends upon *in vivo* homodimerization of two identical heavy chains (HCs), which is mediated by homodimeric associations between CH3 domains, and subsequently disulfide linkages between each HC and each light chain (LC), in B cells (8–11). Thus, the development of bsAbs, using intact IgG formats with wild-type HCs and LCs, faces HC–HC and HCVH-CH1–LC mispairing problems (5, 7). Some approaches for IgG-based bsAbs, based on wild-type homodimeric Fc regions, have been utilized, such as a dual-action Fab (DAF, two-in-one antibody) (12), a κλ-body (13), and a rat/mouse chimeric antibody (14). However, DAF and κλ-body technologies require extensive antibody engineering and screening and are not easy to generate with previously established mAbs. The rat/mouse chimeric antibody requires multiple purification stages, suffers from low purification yields, and faces potential immunogenicity.

To address the HC mispairing problem, heterodimeric Fc technology, which enables two different HCs to be preferentially assembled together, rather than with the same HCs, has been developed (5, 7, 15). Heterodimeric, Fc-based, intact IgG-format bsAbs have been developed in combination with a common LC approach (16) or with two distinct LCs, using the CrossMab technology (17) and ortho-Fab IgG technology (18). Additional heterodimeric Fc scaffolds have been extensively exploited for the generation IgG-like bsAbs by appending antigen-binding antibody fragments, such as VH, VL, scFv, Fab, and single-chain Fab (scFab). Depending on the designed architectures, the resulting bsAbs differ in antigen specificity (from monospecific to tetraspecific) and antigenbinding valency (from monovalent to tetravalent). In addition to the heterodimeric IgG scaffolds for bsAbs, heterodimeric Fc fragments are now emerging as excellent scaffolds to create Fc-fused monomeric or heterodimeric proteins or cytokines, which are challengeable formats to be achieved by wild-type homodimeric Fc.

In this review, we first focus on the design and generation of heterodimeric Fc and their application in the development of therapeutic bsAbs in diverse formats. We then describe the current status of homodimeric Fc-fused monomeric proteins and peptides and propose that heterodimeric Fc fragments, which can present the fusion partner as native-like monomeric or heterodimeric forms, represent a promising scaffold for the next generation of Fc-fused proteins and cytokines.

### HETERODIMERIC Fc ENGINEERING

Wild-type Fc homodimerization is initially mediated by a large, tightly packed interface (~2469 Å2 buried surface area), between the two identical CH3 domains with sub-nanomolar affinity, and subsequently by disulfide linkages in the hinge region (**Figure 1A**) (8, 9). For this reason, heterodimeric Fc variants have been mainly engineered through the replacement of homodimer-favoring interactions at the CH3 domain interface with heterodimer-favoring interactions. This is achieved by introducing asymmetric mutations in each CH3 domain, which promotes the assembly of HCs from two different antibodies (**Figure 1B**). This heterodimeric Fc engineering, using CH3 variant pairs, has been approached using two strategies: (1) structure-based rational design and (2) directed evolution.

X-ray crystal structures of human IgG1 Fc show that Fc homodimerization is driven by both hydrophobic interactions at the center of the CH3 interface core and symmetric electrostatic interactions surrounding the rim of the hydrophobic core (19, 20), as schematically shown in **Figure 2A**. Thus, structure-based rational design of heterodimeric Fc fragments has been utilized to generate heterodimeric CH3 variant pairs (CH3A:CH3B) with different mutations in each chain at the CH3 interface core such that the variant pair thermodynamically favors the formation of heterodimers over the homodimers. The structure-based rational design of such heterodimeric CH3 variant pairs can be classified into four strategies: (i) symmetric-to-asymmetric steric complementarity design (e.g., KiH, HA-TF, and ZW1) (21–24), (ii) charge-to-charge swap (e.g., DD-KK) (25), (iii) charge-to-steric complementarity swap plus additional longrange electrostatic interactions (e.g., EW-RVT) (26), and (iv) isotype strand swap [e.g., strand-exchange engineered domain (SEED)] (7, 24), as summarized in **Table 1**. Critical parameters in heterodimeric Fc engineering include the yield of heterodimeric Fc formation over unwanted homodimeric Fc contaminants, minimal loss in stability relative to the natural Fc, and maintaining natural Fc-like properties, such as serum half-lives and effector functions. Heterodimeric Fc-directed bsAb formation usually yields greater than 90% of the desired product by co-expressing two heterodimer Fc-based antibodies. This makes it feasible for large-scale production and quality control to meet clinical needs. In contrast to heterodimeric Fc engineering, an approach to generate monomeric Fc fragments was explored by introducing four mutations at the CH3 interface (27).

# Symmetric-to-Asymmetric Steric Complementarity Design

In a pioneering approach, Carter and colleagues from Genentech conceived the heterodimeric Fc variant and invented the so-called "Knobs-into-Holes (KiH)" Fc technology (21, 22). The concept was to introduce a "knob" in one CH3 domain (CH3A) by substitution of a small residue with a bulky one (i.e., T366WCH3A in EU numbering). To accommodate the "knob," a complementary "hole" surface was created on the other CH3 domain (CH3B) by replacing the closest neighboring residues to the knob with smaller ones (i.e., T366S/L368A/Y407VCH3B) (**Figure 2B**) (15). The "hole" mutation was optimized by structured-guided phage library screening (21). X-ray crystal structures of KiH Fc variants (28, 33) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the inter-CH3 domain core interface, whereas the knob–knob and the hole–hole interfaces do not favor homodimerization owing to steric hindrance and disruption of the favorable interactions, respectively. The KiH Fc was further engineered to improve purity and stability by introducing an additional inter-CH3 domain disulfide bond pair, S354CCH3A– Y349CCH3B, generating the KiHS-S Fc variant. This exhibited a high yield of heterodimerization (~95%) and improved thermal melting (*T*m) of the CH3 domain by 6 to ~78°C (16). Most KiH Fc-based bsAbs currently being evaluated in clinical trials have adopted the KiHS-S Fc as a scaffold (**Table 2**).

Extensive strategies combining structure-based computational design of many variants and subsequent experimental validation to assess the heterodimerization yield have been adopted to generate other heterodimeric Fc variants with sterically complementary mutations. These include HA-TF Fc from Xencor (23) and ZW1 Fc from Zymeworks (24). Particularly, ZW1 was generated by a two-stage approach, combining negative design to first destabilize the natural Fc homodimer-favoring interactions at the CH3–CH3 interfaces and then positive design of the previously designed variant to increase biophysical stability (24, 45). Negative design, maximizing unfavorable interactions in potential homodimers while promoting specificity between heterodimeric species, generated an Fc variant with F405A/ Y407VCH3A–T366L/T394WCH3B, and resulting in a heterodimerization yield of ~95% (24). However, the *T*m of the CH3 domain was ~72°C, similar to that of KiH Fc (22), but ~10°C lower than that of the wild-type CH3 domain (30). The subsequent positive design, which added T350V/L351YCH3A–T350V/K392LCH3B mutations to the first variant, yielded ZW1 (T350V/L351Y/F405A/ Y407VCH3A–T350V/T366L/K392L/T394WCH3B), which retained the high Fc heterodimerization yield of ~95% and exhibited Fc stability similar to that of the wild-type CH3 domain with a *T*m value of 81.5°C (24). The high heterodimer purity of ZW1, validated by stable expression in Chinese hamster ovary (CHO) cells, and the favorable biophysical properties could improve the manufacturability of ZW1-based bsAbs (45).

More recently, Leaver-Fay et al. (29) computationally produced heterodimeric Fc variants, using an explicit multistate design (sequence optimization) combined with negative design to destabilize and eliminate homodimer-favoring inter-CH3 interactions. One of the best clones, 7.8.60 Fc, was first designed by introducing mutations of D399MCH3A–K409VCH3B to eliminate the conserved electrostatic interactions of D399-K392/ K409 in the wild-type CH3 interface. In the next-round, computational optimization identified the following substitutions sequentially: Y407ACH3A–T366VCH3B to minimize CH3B

homodimerization and to improve the heterodimer formation, K360DCH3A − Q347RCH3B to eliminate CH3B homodimers, and finally E345RCH3B to strengthen the K360DCH3A−Q347RCH3B interaction (**Table 1**). The crystal structure determined clearly resolved the interface and showed that the models were very accurate. The 7.8.60 heterodimeric Fc combined with orthogonal Fab interface mutation technology (18) produced intact IgG-format bsAbs with ~93% purity (29).

### Charge-to-Charge Swap Design

As an alternative approach, Gunasekaran et al. from Amgen (25) sought to design a heterodimeric Fc by reversing symmetric charge complementarity at the CH3 domain interface, while retaining hydrophobic core integrity (**Figure 2C**). Owing to twofold symmetry at the inter-CH3 interface, the targeted electrostatic interaction pairs were symmetrically duplicated on both peripheral sides of the hydrophobic core (**Figure 2A**). They converted the symmetric charge-pair residues into asymmetric charge polarity by first designing a K409DCH3A–D399KCH3B pair variant and then further screening for additional charge-pair mutations through experimentally assessing heterodimerization yield. This resulted in DD-KK with K409D/K392DCH3A–D399K/E356KCH3B mutation pairs. The asymmetric charge mutation pairs drove heterodimerization, whereas "positively charged" and "negatively charged" homodimers were suppressed by unfavorable repulsive charge interactions. Particularly, E356KCH3B was introduced in an attempt to suppress the FcCH3B–FcCH3B homodimerization to increase the purity of FcCH3A–FcCH3B heterodimer. The DD-KK design resulted in greater than 90% heterodimerization depending on the transfection ratio of the two chains.

## Charge-to-Steric Complementarity Swap and Long-Range Electrostatic Interaction Design

The concept of KiH and DD-KK Fc design can be summarized as the replacement of the symmetric hydrophobic and electrostatic

### Table 2 | Heterodimeric Fc-based antibody formats, generated in previous studies, and now being evaluated in clinical trials.

interactions conserved at the CH3 interface with the same, but asymmetric, interactions. In contrast to the sterics-based KiH design and charge-swap DD-KK design, Choi et al. (26) replaced the conserved, symmetric electrostatic interactions at the buried interface of the CH3 domains with asymmetric hydrophobic interactions. This resulted in the generation of a W-VT Fc variant with K409WCH3A–D399V/F405TCH3B mutations, favoring Fc heterodimer formation (~77% purity) due to complementary hydrophobic interactions, while disfavoring homodimers owing to the loss of the K409–D399 electrostatic interactions and the steric collision of K409WCH3A–F405CH3A. To increase the heterodimerization yield of W-VT, they replaced a K360–Q347 pair with negligible interactions, owing to the long distance (4.61 Å) at the rim of the CH3 interface (19) with the K360ECH3A–Q347RCH3B asymmetric mutation pair, which favors the heterodimer because of the long-range electrostatic interaction at a distance of 3.45 Å (30). The combined heterodimeric Fc, called EW-RVT (**Figure 2D**), showed a heterodimer yield of ~91% and a CH3 domain *T*m value of ~77.5°C, which were comparable to those of KiH and DD-KK Fc variants (26). The X-ray crystal structure of the EW-RVT Fc heterodimer led to the addition of an inter-CH3 disulfide bond with a Y349CCH3A–S354CCH3B pair, yielding EW-RVTS-S Fc. The asymmetric disulfide bond was confirmed by resolving the crystal structure (30). EW-RVTS-S Fc showed improved heterodimer yield (by ~3%) and higher thermodynamic stability of the CH3 domain (by ~2.8°C) compared to those parameters of the parent EW-RVT Fc (30). The crystal structures of EW-RVT and EW-RVTS-S Fc fragments, obtained to determine the molecular details of the CH3A/CH3B interface, revealed that the mutations did not cause any significant changes in the Fc structure (30). In agreement with the crystal structure, the EW-RVT Fc heterodimer displayed the native IgG1-like, pHdependent, neonatal Fc receptor (FcRn) binding pattern, and Fcγ receptor (FcγR) interaction, which were equivalent to those of the native IgG1 (previously reported) (46, 47). This suggests that the EW-RVT-based antibody will have similar serum half-lives and effector functions to those of the conventional human IgG1.

### Isotype Strand Swap Design

Davis et al. from Merck Serono (31) designed a heterodimeric Fc by mixing human IgG and IgA CH3 domain segments to create a complementary CH3 heterodimer, which was referred to as SEED Fc. Native CH3 domains of human IgA and IgG are structurally similar (10), but cannot dimerize because of lower homology within interface residues. They analyzed the structural dimerization motif in the CH3 domain of each isotype and devised SEED through the exchange of some β-strand segments in the CH3 domain of each isotype to drive heterodimer formation through steric complementary contact surfaces. The SEEDbased antibodies (e.g., SEEDbody) were purified with a protein A resin and exhibited long serum half-life and effector functions comparable to those of wild-type human IgG1 Fc-based antibodies (48). However, owing to the artificial sequence of the SEED CH3 heterodimer, the potential immunogenicity has not been determined.

### Directed Evolution of Heterodimeric Fc

Aforementioned Fc heterodimers have been generated through structure-based rational design or computational modeling. Recently, Choi et al. (32) generated heterodimeric Fc variants through directed evolution combined with yeast surface display and high-throughput screening (49, 50). A combinatorial heterodimeric Fc library display system was developed by mating two haploid yeast cell lines; one haploid cell line displayed an Fc chain library (displayed FcCH3A) with mutations in one CH3 domain on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B) with mutations in the other CH3 domain. In the mated cells, secreted FcCH3B was displayed on the cell surface through heterodimerization with the displayed FcCH3A. Fluorescence-based detection of this interaction enabled screening of the library for heterodimeric Fc variants by flow cytometry. For the proof-of-concept, they constructed combinatorial heterodimeric Fc libraries with simultaneous mutations on targeted residues of both CH3A and CH3B based on the template of the W-VT Fc variant, and screened the libraries to isolate numerous heterodimeric Fc variants with heterodimerization yields of ~90%. This directed evolution approach identified unexpected heterodimer-favoring mutation pairs at the CH3 interface, such as hydrogen bonding or cation–π interactions, as well as homodimer-disfavoring pairs, which have not been tested by structure-guided rational design. The best clone, A107, exhibited a heterodimerization yield of ~93%, which was much higher than that (~77%) of the parent W-VT Fc variant.

# HETERODIMERIC Fc-BASED ANTIBODIES IN DIVERSE FORMATS

Heterodimeric Fc are compatible with independent fusion of two different antigen-binding units to the N- and/or C-terminus of each Fc chain. This facilitates the creation of diverse heterodimeric Fc-based antibodies with differences in specificity, binding valency, geometry of target-binding sites, among others, as summarized in **Figure 3** and **Table 2**.

### Monospecific and Monovalent Antibodies

Heterodimeric Fc-based monospecific antibodies (mAbs) with monovalent antigen binding have been generated by fusion of the Fab to the N-terminus of only one Fc chain (Fc/Fab-Fc), the scFv to the N-terminus of only one Fc chain (Fc/scFv-Fc), the VH-CH1 (HCVH-CH1) and VL-CL to the respective N-termini of each heterodimeric Fc chain (HCVH-CH1-Fc/LC-Fc), or the VH and VL to the respective N-termini of each heterodimeric Fc chain (VH-Fc/VL-Fc) (**Figure 3**).

The Y-shape of typical IgG antibodies often limits their utility against some targets, whereby bivalent target-binding dimerizes and agonizes, rather than antagonizes, the intended targets. For example, targeting the c-Met receptor tyrosine kinase with bivalent antibodies can mimic the ligand hepatocyte growth factor (HGF)-mediated agonism via receptor dimerization (51). To address this issue, Genentech developed a KiH Fc-based, onearmed Fc/Fab-Fc format monovalent antibody, onartuzumab (MetMab), through fusion of a humanized anti-c-Met 5D5 Fab to the N-terminus of only the "hole" of the Fc chain (34). Whereas the bivalent anti-c-Met 5D5 IgG antibody acted as an agonist causing c-Met-activated tumor cell growth, rather than growth inhibition, the monovalent MetMab bound the receptor in a one-to-one fashion to inhibit HGF binding and block receptor activation, thus acting as an antagonist. Co-expression of three chains, specifically the Fc, HCVH-CH1-Fc, and LC, from three respective cistrons on a single plasmid, and targeting these chains to the periplasmic space of *Escherichia coli*, efficiently resulted in the production of MetMab. Purification by sizeexclusion chromatography yielded ~95% purity (34). A clinical trial of MetMab combined with the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib to block metastasis in non-small cell lung cancer was recently halted in Phase 3, but other clinical trials with MetMab are expected. The EW-RVT Fc variant was also applied to make a MetMAb-like monovalent antibody in the VH-Fc/VL-Fc format, called msMet, wherein the VH and VL of MetMab were fused to the N-terminus of each heterodimeric Fc chain (26). As a single agent, the msMet antibody suppressed the *in vivo* tumor growth of human gastric cancer xenografts in mice.

DD-KK Fc was exploited to generate a monovalent 14D2 antibody, in the HCVH-CH1-Fc/LC-Fc format, to target tumor necrosis factor receptor 1 (TNFR1) by linking the VH-CH1 and VL-CL of the anti-TNFR1 14D2 antibody to the N-terminus of each heterodimeric Fc chain (25). The parent bivalent 14D2 antibody showed undesirable activity, specifically activation of TNFR1 at low doses via cross-linking of the receptor, similar to that observed for the TNF ligand. However, the monovalent antibody was devoid of agonistic activity, and blocked TNF-mediated chemokine induction in mice. Importantly, the monovalent 14D2 antibody exhibited similar pharmacokinetic profiles as the fulllength IgG when injected in mice.

# IgG-Like Formats with Appendages of scFv and scFab

Heterodimeric Fc technology can resolve the HC mispairing problem, but still faces the challenge of a LC-pairing problem, specifically, cognate HCVH-CH1-LC pairing in the Fab region, to generate bsAbs in intact IgG formats (5, 7). To address the HCVH-CH1-LC-pairing problem, single-chain antigen-binding units, such as scFv and scFab, have been, respectively, linked to each heterodimeric Fc chain or tandemly linked to one heterodimeric Fc chain, generating IgG-like bsAbs in formats of scFv-Fc/scFv-Fc, scFab-Fc/scFab-Fc, Fc/scFv2-Fc, Fab-Fc-VH/ Fab-Fc-VL, Fab-Fc/scFv-Fc, Fab-Fc/scFab-Fc, and scFab-Fc- (scFv)/scFab-Fc-scFv (**Figure 3**).

The EW-RVT Fc was exploited to generate a bsAb, dubbed bsVeMet, simultaneously targeting both c-Met and VEGF receptor 2 (VEGFR-2), with two respective antigen-specific scFvs fused to the N-termini of each heterodimeric Fc in the format of scFv-Fc/scFv-Fc (26). The bsVeMet more potently inhibited HGF and VEGF-stimulated cell proliferation, downstream signaling, and *in vivo* angiogenesis than the two parent mAbs.

Furthermore, bsAbs have been extensively designed to recruit immune effector cells, such as T cells, to tumor cells (52, 53). Bispecific T-cell Engager (BiTE) was constructed by tandem

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connection of two scFvs, one of which was specific to CD3 on T cells and the other was specific to a tumor-associated antigen on tumor cell surfaces. The goal of this strategy was to engage cytotoxic T cells for lysis of the targeted antigen-expressing cancer cells, and was first developed by Mack and colleagues (54). Later, the CD19 × CD3 BiTE blinatumomab was developed by Micromet and clinically approved in 2014 for the treatment of relapsed/refractory B-cell acute lymphocytic leukemia (55). However, the serum half-life of tandem scFv-based BiTEs in mice was only 5–6 h (56), which was much shorter than that of Fc-containing antibodies. The fast clearance of blinatumomab requires infusions or repeated injections to maintain a therapeutically effective dose over a prolonged period, specifically, daily infusions for 8 weeks in clinical trials (57). To improve pharmacokinetics of such scFv-based BiTEs, a DD-KK Fc-based bsAb was generated in the scFv-Fc/scFv-Fc format by fusing two scFvs with different binding specificities, one against human CD3 and the other against a tumor-associated receptor tyrosine kinase (TARTK), to each Fc heterodimer chain (25). Due to the presence of the Fc region, the scFv-Fc/scFv-Fc antibody exhibited a much longer *in vivo* half-life in mice and could be administered more infrequently compared to other types of CD3 bispecific scFv fragments (25). The CD3 × TARTK bsAb completely suppressed the growth of the TARTK-positive glioma in a xenograft mouse model.

Xu et al. from Sutro Biopharma (35) produced a KiH Fc-based BiTE bsAbs using the *E. coli*-based cell-free expression system. They designed epithelial cell adhesion molecule (EpCAM) × CD3 bsAbs in the two-armed scFv-Fc/scFv-Fc format or the onearmed Fc/scFv2-Fc (i.e., Fc/BiTE-Fc) format and further reversed the two formats by exchanging the "knob" Fc and "hole" Fc fusion partner. They found that the expression yield and biological activities, such as dual targeting and tumor cell killing by engaging T cells, was highly variable and depended on the formats. This suggested that a more sophisticated optimization process is necessary to determine the optimal ratio of the two expression plasmids for enhanced cell-free expression of the Fc fusion partner for the two scFvs or the tandem scFv BiTE. Intriguingly, the KiH Fc-based bsAb (100 kDa) showed significantly longer serum half-life (*T*1/2 ≈ 4.9–5.2 days) than the BiTE control (50 kDa) (T1/2 ≈ 0.2 days), but were less stable than trastuzumab IgG (150 kDa) (T1/2 ≈ 25 days) and scFv-Fc (100 kDa) (T1/2 ≈ 9.1 days) in mice. This might be attributed to destabilizing mutations in the CH3 domains of the KiH Fc (35).

Using an HA-TF Fc scaffold, Xencor generated Her2 × CD3 or Her2 × CD16 bsAbs in Fab-Fc-VH/Fab-Fc-VL (dubbed mAb-Fv) format through the fusion of VH and VL domains of CD3 or CD16 antibodies to the C-termini of two distinct HCs (23). The advantage of the mAb-Fv format is improved tumor targeting due to bivalent antigen engagement through two intact arms of the IgG mAb, compared to the monovalent target-binding formats, such as scFv-Fc/scFv-Fc and scFab-Fc/scFab-Fc. However, mAb-Fv showed non-native oligomeric species and the C-terminal Fv exhibited inferior affinity to the CD16 or CD3 antigen compared to the parent Fab. This was most likely due to steric hindrance and suggested the need for C-terminal-fused Fv optimization. For these reasons, Xencor adopted an HA-TF Fc-based Fab-Fc/

scFv-Fc bsAb format (dubbed "plug-and-play" platform), which showed native mAb-like stability and non-compromised antigen-binding activity (36). For example, the Fab-Fc/scFv-Fc formatted bsAbs, such as CD123 × CD3 (XmAb14045) and CD20 × CD3 (XmAb13676), are expected to begin clinical Phase 1 trials for acute myeloid leukemia (NCT02730312) and B-cell malignancies, respectively, in 2016.1 Glenmark Pharm is also developing bsAbs in a heterodimeric Fc-based Fab-Fc/scFv-Fc format (dubbed "BEAT" by Glenmark), in which the heterodimeric Fc was designed by introducing mutations, mimicking the natural association of the heterodimeric T-cell receptor (TCR) α and β chains, into the interface between the two CH3 domains of IgG (58).

Schanzer et al. from Roche (37, 59) generated a KiH Fc-based bsAb XGFR in the format of Fab-Fc/scFab-Fc for targeting EGFR with the Fab format in one arm and the insulin-like growth factor receptor type I (IGF-1R) with scFab format in the other arm. The format of XGFR bsAb, the so-called one arm scFab IgG (OAscFab-IgG) was devised to prevent incorrect pairing of LCs, while maintaining the closest possible format to intact IgG. XGFR was produced with high expression yields comparable to those of the respective parent IgG mAbs. However, other tested bsAb formats of IgG-scFv (IGF-1R IgG with C-terminal attachment of disulfidestabilized EGFR scFv) and dual-variable domain (DVD)-IgG (IGF-1R IgG with the N-terminal attachment of EGFR VH and VL in the HC and LC, respectively) showed ~2-fold and ~16-fold lower expression yields than that of the OAscFab-IgG format (37, 59). To further increase immune effector functions triggered by XGFR bsAb, the heterodimeric Fc portion was glycoengineered to remove the fucosylation, leading to strong antibody-dependent cell-mediated cytotoxicity. The resulting XGFR demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models (37, 59). In addition, Castoldi et al. from Roche (38) constructed a KiHS-S Fc-based trispecific antibody (dubbed TriMAb) in the scFab-Fc-(scFv)/scFab-Fc-scFv format, in which anti-EGFR and anti-IGF-1R scFabs are on the respective arm of IgG with anti-Her3 scFv to fused to the C-terminus of both chains or only one chain. The EGFR × IGF-1R × HER3 TriMAb showed growth inhibitory effects comparable to that of a combination of each single parental antibody, in a model cell proliferation assay.

### Intact IgG Formats with Correct LC Association

IgG-like bsAbs based on appending scFv and scFab to heterodimeric Fc fragments have overcome HCVH-CH1-LC mispairing issues within the Fab region, but they are limited in their use of linkers, the length and composition of which often needs to be optimized. A sub-optimal linker can cause undesirable problems, such as loss of antigen binding, due to binding site hindrance, poor expression levels, immunogenicity, poor pharmacokinetics, and *in vivo* cleavage. Thus, many attempts have been made to develop bsAbs that deviate minimally from natural IgG antibodies without the use of artificial linkers. Since antigen specificity usually resides in the Fv chain composed of the VH and VL, correct

1http://xencor.com/

pairing between the cognate HC and LC during co-expression and assembly of bsAbs is essential to ensure antigen-binding specificity and affinity. LC mispairing occurs because the Fab heterodimerization interfaces of VH–VL and CH1–CL between two distinct antibodies are almost identical, though the complementarity determining regions (CDRs) of VH and VL are quite different. Thus, to generate bsAbs using a full-length IgG format, cognate HCVH-CH1-LC pairing should be ensured, in addition to correct HC pairing by heterodimeric Fc technology. The LC mispairing problem has been addressed by using a common LC (15, 16), a domain crossover (CrossMab) (17), and a designed orthogonal Fab interface (18) (**Figure 3**).

The use of a common LC, compatible with the two distinct HCVH-CH1s, is a straightforward way to construct bsAbs with intact IgG formats (15). Two mAbs can be first isolated from a human scFv phage library sharing identical VLs and then assembled into a single IgG format using KiH Fc-based HC heterodimerization (16, 60). However, this approach constrains antibody discovery for two different targets with a single LC or requires novel antibody libraries, and is difficult to adopt when using two different previously established antibodies. A notable example of an IgG-format bsAb using a common LC in combination with a KiH Fc is emicizumab (ACE910) developed by Chugai, which targets factor IXa and factor X (FIXa × FX) (61, 62). They first generated various FIXa- and FX-specific mAbs by immunizing mice with human FIXa and FX antigens and then generated a lead chimeric bsAb based on the retention of biological activity and the feasibility of using only one LC (62). FIXa and FX form a complex with cofactor FVIII but the complex formation is deficient in hemophilia A patients. Standard treatment for this disease includes frequent injection of recombinant or plasma-derived factor VIII (62). Emicizumab mimics FVIII functions by allowing FIXa and FX to be close for the formation of the complex. This is because the distance between the FIXa- and FX-binding sites of FVIIIa is similar to that between the two antigen-binding site arms of the Y-shaped IgG. Emicizumab overcomes the short half-life (T1/2 ≈ 0.5 days) of recombinant FVIII and is expected the lower immunogenicity. Emicizumab is now in a Phase 3 clinical study in patients with hemophilia A (NCT02622321) (42).

To facilitate antibody generation with a common LC, Merus developed a transgenic mouse (MeMo), engineered to generate antibodies with a single human common LC and diverse human HCs (63). The MeMo technology combined with their own CH3 domain engineered heterodimeric Fc technology allows them to generate intact IgG-format bsAb (dubbed Biclonics by Merus).2 A Biclonics-based bsAb MCLA-128 targeting Her2 × Her3 is now being evaluated in Phase 1/2 clinical trials for patients with solid tumors (64).

CrossMab technology from Roche (17) is another strategy to solve the HCVH-CH1–LC association problem in combination with KiH Fc technology. This technology keeps one Fab untouched, whereas the VH or CH1 domain of the other Fab is switched with partner VL or CL domains. In the early proof-of-concept stage of CrossMabs, they were evaluated in three formats according to the crossover region: (1) CrossMabFab, crossover of the complete VH-CH1 and VL-CL domains, (2) CrossMabVH-VL, crossover of only the VH and VL domains, and (3) CrossMabCH1-CL, crossover of only the CH1 and CL domains. Although the three CrossMab variants showed comparable thermal stability to that of the parental IgG mAbs, the CrossMabCH1-CL format showed only minimal unwanted side products; however, the other two formats produced some portion of side products (17). For this reason, the CrossMabCH1-CL format was predominantly used over the other two formats. CrossMab-based bsAbs might be easily derived from pre-existing antibody pairs using domain crossover without the need for identification of common LC and the Fab interface mutations, required for correct LC association (39). Many CrossMAbs have been generated and evaluated and currently four different CrossMab-based bsAbs are now in active Phase 1/2 clinical trials (39) (**Table 1**). For example, two versions of a VEGF × Ang2 CrossMabCH1-CL are currently being evaluated in clinical trials: (1) RG7221 (Vanucizumab), one VEGF × Ang2 CrossMabCH1-CL, in Phase 1 in patients with cancers (NCT01688206) and (2) RG7716, the other tailor-made VEGF × Ang2 CrossMabCH1-CL, in Phase 2 trials for patients with macular degeneration (NCT02484690).

The CrossMabCH1-CL format was further extended by fusion of a Fab to the N-terminus to generate the Fab-CrossMabCH1-CL format for trivalent bsAbs (**Figure 3**). Using this format, IgG-based T-cell bsAbs (TCBs) with bivalent binding to carcinoembryonic antigen (CEA), dubbed CEA-TCB, was developed (40). Currently, a Phase 1 clinical trial of CEA-TCB is ongoing (NCT02324257) for the treatment of CEA-expressing solid tumors (39). Furthermore, the genetic fusion of the two different DAFs (dual-action Fabs, i.e., two-in-one antibody), one EGFR × Her3 DAF and another Her2 × VEGF DAF, to the N-terminus of each arm of a KiH-based four-in-one CrossMabCH1-CL format generated the EGFR × Her3 × Her2 × VEGF targeting tetraspecific IgG-format antibody (41) (**Figure 3**). The same group also generated an EGFR × Her3 × Her2 × VEGF targeting tetraspecific, tetravalent antibody by combining the CrossMabCH1-CL and DVD-IgG technologies (65), resulting in the Fv-/Fv-CrossMabCH1-CL IgG format (41) (**Figure 3**).

An alternative approach for enforcing correct HCVH-CH1–LC association includes introduction of a set of mutations at the heterodimeric VL–CL and VH–CH1 interface (18, 66, 67), similar to modification of the CH3 interface for the heterodimeric Fc design. In an ortho-Fab IgG approach (18), structure-based regional design introduced complementary mutations at the LC and HCVH-CH1 interface in only one Fab, without any changes being made to the other Fab (**Figure 3**). Based on the ortho-Fab IgG format combined with heterodimeric Fc technology, the EGFR × c-Met bsAb (LY3164530) from Eli Lilly was generated, which is currently being evaluated in a Phase 1 clinical study of patients with advanced or metastatic cancer (NCT02221882). Zymeworks is also currently developing intact IgG-format bsAbs generated by the combination of ortho-Fab IgG and ZW1 Fc technologies.3

Frontiers in Immunology | www.frontiersin.org October 2016 | Volume 7 | Article 394

2http://www.merus.nl/

3http://www.zymeworks.com/

### *In Vitro* Assembly of Intact IgG Formats

Heterodimeric Fc technology alone, without the combination of HCVH-CH1–LC association technology, can generate intact IgG-format bsAbs by separate expression of two parental half-antibodies [HC–LC (HL) pairs] and subsequent *in vitro* recombination into desirable bsAbs, the assembly of which is driven by heterodimer-favoring Fc interactions under controlled biochemical conditions (60). This approach allows the correct HCVH-CH1–LC association during the expression stage of the parental half-antibody and maintains the cognate HCVH-CH1–LC pairing in the next *in vitro* assembly step. Spiess et al. from Genentech (68) further combined the two separate processes into one step by co-culture of two transformed *E. coli* cell lines, each expressing a half-antibody. They demonstrated the robustness of the technology by producing 28 unique IgG bsAbs. However, this *E. coli* expression system was limited to production of non-glycosylated antibodies. Shatz et al. (69) applied this technology to a mammalian expression system to produce glycosylated antibodies. A notable example of a bsAb prepared using the above manufacturing system is the TfR × BACE1 bsAb that targets TfR (transferrin receptor) and BACE1 (β-site amyloid precursor protein-cleaving enzyme) (43, 44). In a human TfR knock-in mouse, the bsAb could cross the blood–brain barrier and accumulated in the mouse brain where it inhibited BACE1 to reduce brain Aβ production (44). This bsAb was validated in preclinical models, and is now on the path to clinical trials.

Unlike the human IgG1 isotype, IgG4 has been shown to undergo Fab arm exchange (FAE) both *in vivo* and *in vitro*, in which one half-antibody (HL pair) recombines with other half-antibodies from other IgG4 molecules (10, 70, 71). The structural basis of IgG4 for FAE resides in the IgG4 core hinge region (226Cys-Pro-Ser-Cys-Pro230), and especially the Ser228 residue and the CH3 domains (critically, the R409 residue) (70, 72). To confer FAE ability to IgG1, two groups engineered FAE-capable IgG1 by introducing the FAE-associated IgG4 specific mutation pairs at the core hinge region and inter-CH3 interface, for example, D221E/P228E/L368ECH3A–D221R/P228R/ K409RCH3B (dubbed EEE-RRR) from Rinat-Pfizer (73), or only at the inter-CH3 interface, such as K409RCH3A–K405LCH3B (dubbed Duobody) from Genmab (72). The retention of the stable wild-type IgG1 hinge in the Duobody technology might be beneficial for the generation of bsAbs, which are resistant to reduction under physiological conditions *in vivo* and are much more stable during storage after FAE-mediated manufacturing (74). It should be noted that the above two mutation pairs at the CH3 interface are not enough to predominantly produce heterodimeric Fc fragments when the two HCs are coexpressed, unlike the KiH, DD-KK, and EW-RVT heterodimeric Fc technology. Instead, two mAbs based on the FAE-capable IgG1 are separately expressed and purified and then mixed together under mild redox conditions, resulting in a stable IgG1 bsAb with high yields (greater than 90%) (72–74). The EGFR × c-Met bsAb (JNJ-61186372) generated by Duobody technology was effective against EGFR inhibitor-resistant lung tumors in mice (75).

# Fc-FUSED PROTEINS WITH THE NATURAL MONOMERIC OR HETERODIMERIC FORM

The genetic fusion of proteins, such as ligand-binding soluble receptors, cytokines, growth factors, enzymes, and peptides, to the Fc domain of human IgG1 is a well-established strategy to extend the serum half-life and often enhance biological activity through dimerization of the fusion partner (76–78). Conjugation to the Fc may also enhance tissue penetration of the fusion partner by FcRn-mediated transcytosis (79, 80). In addition, the Fc moiety in many cases improves the biophysical properties of its fusion partner, such as the solubility and stability of the protein (81). At the end of 2015, about 11 Fc-fusion proteins had been clinically approved (82). All of the marketed Fc-fusion proteins and many others in clinical trials are based on the wild-type human IgG1 Fc, which presents the fusion partner as a homodimer due to the homodimeric Fc structure. However, some cytokines and hormones naturally occurring in monomeric or heterodimeric forms exhibit markedly improved pharmacokinetics and even biological activities when they are fused to the Fc in natural forms (83). Thus, Fc-fusion, while keeping the original configuration of the fusion partners, is desirable for those proteins. However, as described below, generation of such Fc-fused monomeric or heterodimeric proteins based on the wild-type Fc is not straightforward (**Figures 4A–C**) due to the homodimeric structure. A promising solution to this limitation is the use of a heterodimeric Fc as an alternative scaffold to wild-type Fc (**Figures 4D,E**).

### Wild-Type Fc-Fused Protein Monomers

Naturally occurring monomeric cytokines and hormones, such as erythropoietin (Epo), interferon α/β (IFNα/β), and Factor IX (FIX), Fc-fused proteins in the native monomer format, dubbed Fc-fused monomers, showed improved pharmacokinetics and even biological activities, compared to Fc-fused dimers (83). Epo is a glycoprotein hormone drug of ~34 kDa that exerts its effects by binding to the Epo receptor to stimulate red blood cell production (84). Bitonti et al. (85, 86) demonstrated that human IgG1 Fc-fused Epo in monomeric format (Epo-Fc monomer), consisting of a single molecule of Epo conjugated to a wild-type Fc dimer (**Figure 4A**), exhibited enhanced pharmacokinetic and pharmacodynamic properties, compared to the Epo-Fc dimer, in both cynomolgus monkeys and humans. The inferior activity of the Epo-Fc dimer might be attributed to steric hindrance introduced by two molecules of Epo linked to a dimeric Fc; this could adversely affect the binding to respective receptors (83). To prepare an Epo-Fc monomer, two plasmids encoding FLAG-tagged Epo-Fc at the C-terminus (Epo-Fc-FLAG) and untagged Epo-Fc were first co-transfected into CHO cells. Three protein products were produced by the cells: the Epo-Fc/Epo-Fc homodimer (Epo-Fc dimer), the EpoFc/Epo-Fc-FLAG heterodimer (Epo-Fc monomer), and the Epo-Fc-FLAG/Epo-Fc-FLAG homodimer. The Epo-Fc monomer was purified using a combination of protein A chromatography, size-exclusion chromatography, and subsequently FLAG-affinity chromatography (85, 87).

Similar to the Epo-Fc monomer, Fc-fused monomer formats of IFNβ, IFNα, and FIX exhibited much better biological activity in the monkey models compared to corresponding Fc-fused dimers (83). Intriguingly, in the case of IFNα, the IFNα-Fc monomer showed very similar pharmacokinetics to that of the IFNα-Fc dimer; however, the IFNα-Fc monomer displayed much better *in vivo* efficacy compared to the IFNα-Fc dimer (83). This enhanced efficacy of IFNα-Fc monomer was also dependent on the length of the linker between the IFNα and Fc regions. Longer linker length resulted in increased specific activity of the IFNα-Fc monomer, indicating that there might have been some steric hindrance between IFNα and its receptor. The Fc-fused monomer was then prepared as described previously for the Epo-Fc monomer. Thus, the desired Fc-fused monomer could theoretically be prepared up to only 33% of the total expression yield. Despite many desirable properties of Fc-fused monomers, the development of wild-type Fc-based monomers faces difficulties associated with isolating desired Fc-fused monomers from other format mixtures using robust purification steps, and is also limited by the inherent poor yield of a maximum of 33%.

For Fc-fused monomeric peptides, Kang et al. from Hanmi Pharm (88) developed a glucagon-like peptide-1 (GLP-1)/glucagon (GCG) dual agonist for the potential treatment of obesity and diabetes by activating both GLP-1 and GCG receptors based on LAPScovery™ (Long-Acting Protein/Peptide Discovery) technology. They chemically conjugated GLP-1/GCG peptides to only one arm of human IgG1 aglycosylated Fc fragments, produced from *E. coli* via a flexible polyethylene glycol (PEG) linker (**Figure 4B**). The GLP-1/GCG-Fc monomer exhibited markedly improved pharmacokinetics and greater *in vivo* biological activity compared to the GLP-1/GCG-Fc dimer (89). However, the preparation of the GLP-1/GCG-Fc monomer was complicated, requiring separate Fc preparation, *in vitro* chemical conjugation of the GLP-1/GCG peptide to the Fc via a PEG linker, and, finally, multi-step purification of the desired product from the reaction mixture (89). Based on LAPScovery technology, aglycosylated Fc-fused monomers for insulin, human growth hormone, and granulocyte-colony stimulating factor were developed, and are now in clinical trials in Europe.4

### Wild-Type Fc-Fused Protein Heterodimers

For heterodimeric proteins composed of two different subunits, the Fc-fusion format, achieved by fusion of each subunit independently to separate Fc fragments might be desirable to maintain biological activity, in comparison to the homodimeric form by tandem subunit linkage to wild-type Fc. Follicle-stimulating hormone (FSH) is a non-covalently linked heterodimeric protein consisting of α and β subunits, and its recombinant protein is commonly used for therapy in the treatment of both male and female infertility (90). However, it requires daily injections for several days for up to months due to its short half-life (35 h in humans) (90). To prepare long-acting FSH, Low et al. (91) designed a human IgG1 Fc-fused FSH in two different formats. One is a single chain-linked FSH-Fc (Fc-FSH tandem homodimer) with tandemly linked α-β subunits fused to the N-terminus of the Fc fragment. The other is a heterodimeric FSH-Fc (Fc-FSH heterodimer) with α and β subunits fused separately to the N-termini of each arm of the Fc fragment (**Figure 4C**). They found that the Fc-FSH heterodimer was significantly more active than the Fc-FSH tandem homodimer, whereas the two Fc-fusion proteins had similar terminal serum half-lives in both rodents and monkeys. It is likely that the architecture of the Fc-FSH heterodimer might present the α and β subunits in a more favorable conformation for bioactivity than that of the Fc-FSH tandem homodimer. To purify Fc-FSH heterodimers, they inserted a 6 × His-tag into the C-terminus of the β subunit-fused Fc fragment only. As a result, whereas the Fc-FSH tandem homodimer was ~90% pure following a single protein A chromatography step, the Fc-FSH heterodimer required additional purification by nickel affinity chromatography to achieve similar purity. However, in addition to multi-step purifications, such preparations of Fc-FSH heterodimers gave lower purification yields (33%) compared to the total expression yield.

# Antibody-Fused Cytokines, Immunocytokines

Cytokines are key players in controlling immune responses during physiological and pathophysiological processes (92). Various cytokines have been clinically approved for diverse immunerelated diseases, including interleukin-2 (IL2) and tumor necrosis factor (TNF) as well as INFα for malignant cancer therapy (92, 93). However, the therapeutic efficacy of cytokines is often hampered by poor pharmacokinetics and significant toxicity associated with their systemic administration. To overcome such limitations, antibody–cytokine fusion proteins, the so-called immunocytokines, have been devised in an attempt to enhance antibody-mediated tumor tissue targeting of cytokines, and to increase serum half-lives (93, 94). However, a recent study in mice using an immunocytokine of IL2-fused to tumor antigenspecific IgG in format of IgG-IL2, showed that the biodistribution and pharmacokinetics are entirely governed by the cytokine IL2 moiety, rather than the expected antibody-targeting antigen specificity (95). The IgG-IL2 predominantly remained systemic and was associated with innate immune cells expressing IL2 receptors regardless of antigen specificity or FcγR interactions, whereas the parent IgG antibody was preferentially accumulated in antigen-targeted tumors (95). Rather than the IgG-IL2 immunocytokine, a combination of an anti-tumor antigen IgG antibody and an untargeted Fc-IL2 monomer was approached for synergistic anti-tumor immunotherapy (96).

Furthermore, the biodistribution of immunocytokines was considerably influenced by the ratio of antibody to cytokine. IL12 is a heterodimer (~70 kDa) consisting of heavy (p40) and light (p35) chain subunits, which are covalently linked by disulfide bonds (97). In an attempt to develop IL12-based immunocytokines combined with an L19 scFv that targets the EDB domain of fibronectin, Gafner et al. (98) constructed three different forms of immunocytokines differing in the ratio of antibody to cytokine. They found that the format with 2:1 antibody to cytokine ratio of p40-L19 scFv/L19 scFv-p35, a heterodimeric fusion protein in which the disulfide-linked p35 and p40 subunits of IL12 are fused to L19 scFv, displayed the highest tumor-targeting performance in biodistribution studies. Thus, enhanced therapeutic activity was observed when compared to the other two formats having 1:1 or 2:2 ratio of antibody to cytokine. Similar results were also obtained with IL12 immunocytokines generated with a different F8 scFv antibody (99). With a monomeric cytokine of IL7 (~20 kDa), the same group evaluated the biodistribution of three different formats of IL7-fused F8 scFv immunocytokines differing in antibody to cytokine ratios (100). They found that, as in the case of IL12, the format with a 2:1 antibody to cytokine ratio showed the highest tumor localization compared to the other two formats with 1:1 and 2:2 ratios.

### Heterodimeric Fc-Fused Immunocytokines

The above results suggest that increasing the antibody to cytokine ratio results in improved tumor tissue localization via decreasing cytokine-mediated targeting, which can potentially reduce the dose-related toxicity. IgG-based immunocytokines have been typically generated by fusion of cytokines to the C-terminus of homodimeric Fc fragments of HCs, which present the cytokine as a homodimeric form (31). However, such IgG-based immunocytokines might display poor pharmacokinetics and distribution, as shown with IgG-IL2 formats (95, 101). To generate an IgG-based cytokine monomer format for monomeric (e.g., IL2, IL4, IL7, IL9, IL15, and IL21) and heterodimeric cytokines (e.g., IL12, IL23, IL27, and IL35), a heterodimeric Fc is an attractive scaffold to present cytokines with native structures. Roche (102, 103) generated CEA-targeted IgG-fused IL2 monomeric immunocytokines (CEA-IL2v) by fusion of an engineered IL2 variant (IL2v) to the C-terminus of an antibody with the KiH heterodimeric Fc to obtain a 2:1 antigen-binding valence to cytokine ratio (**Figure 4D**). Similarly, they also developed the fibroblast activation protein (FAP)-directed IgG-IL2v monomeric immunocytokine (FAP-IL2v) (103, 104). Both CEA-IL2v (NCT02004106, NCT02350673) and FAP-IL2v (NCT02627274) are now being evaluated in Phase 1 clinical trials.

4http://www.hanmipharm.com/

# CONCLUSION AND PROSPECTS

In summary, immunoglobulin Fc heterodimer technology is very useful in generation of bsAbs with various antigen specificities and binding valencies in full-length IgG and IgG-like formats, while retaining many favorable properties of natural IgG antibodies. As of July 2016, more than seven heterodimeric Fc-based IgGformat bsAbs are now being evaluated in clinical trials (**Table 2**). Nonetheless, there are some issues to be considered for heterodimeric Fc-based bsAbs. All of the heterodimeric Fc-based bsAbs require assembly with three or four separate chains, which often hampers cell line development for high yield expression. Since each heterodimeric Fc chain has different mutations in each CH3 domain, the expression level and stability are somewhat different from each other, often requiring optimizing processes to identify the best pair between heterodimeric Fc chain and fusion partner (35). Furthermore, since the so far developed Fc heterodimers do not reach a 100% heterodimerization yield, the downstream process to ensure high bsAb purity, to meet clinical needs, should be considered from the initial stage of the heterodimeric Fc-based bsAb design.

In addition to bsAbs, heterodimeric Fc technology is now emerging as a promising scaffold for the generation of Fc-fused proteins and cytokines in monomeric or heterodimeric formats. The generation of Fc-fused monomeric proteins with wild-type Fc fragments is not any more desirable because it requires additional artificial sequences, such as extra tags for selective affinity chromatography and also multiple purification steps with limited purification yields (maximum 33%). Fusion to heterodimeric Fc fragments can present the fusion partner in its natural monomeric form for monomeric proteins and peptides as

REFERENCES

well as natural heterodimeric forms for heterodimeric proteins, rather than the artificial homodimeric form for wild-type Fc fragments (**Figure 4E**). This heterodimeric Fc capability will facilitate the development of the next generation of Fc-fused proteins that maintain full function of the fusion partner, while retaining the Fc-mediated extended half-life and immune effector functions. The reduced binding valency might further help to reduce target receptor-mediated clearance. Particularly, in tumor-targeting IgG-based immunocytokines, the heterodimeric Fc-fused cytokines, in their natural forms, improve tumor tissue accumulation by reducing associations with immune cells, and thereby minimizing systemic toxicity. This facilitates their development as therapeutic agents. Some heterodimeric Fc-based IgG-fused immunocytokines are now in clinical trials and many more are expected to be developed.

### AUTHOR CONTRIBUTIONS

JHH, JEK, and YSK designed the review article topic, surveyed relevant literatures and patents, and analyzed the data; YSK designed and supervised the review article; and JHH and YSK wrote the manuscript with input from all the other authors.

### FUNDING

This work was supported by grants from the Pioneer Research Center Program (2014M3C1A3051470) and the Global Frontier Project (2013M3A6A4043874) from the National Research Foundation (NRF), and the Korea Health Technology R&D Project (HI16C0992) through the Korea Health Industry Development Institute (KHIDI), funded by the Republic of Korea.

activation for the treatment of pancreatic cancer. *MAbs* (2016) 8(4):811–27. doi:10.1080/19420862.2016.1160989

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Ha, Kim and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Corrigendum: immunoglobulin Fc Heterodimer Platform technology: From Design to Applications in therapeutic Antibodies and Proteins

*Ji-Hee Ha1 , Jung-Eun Kim1 and Yong-Sung Kim1,2\**

*1Department of Molecular Science and Technology, Ajou University, Suwon, South Korea, 2Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon, South Korea*

Keywords: bispecific antibody, Fc engineering, heterodimeric Fc, Fc-fusion proteins, immunocytokines, antibody engineering

### **A corrigendum on**

### *Edited and Reviewed by:*

*Tianlei Ying, Fudan University, China*

> *\*Correspondence: Yong-Sung Kim<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 19 October 2017 Accepted: 02 November 2017 Published: 13 November 2017*

### *Citation:*

*Ha J-H, Kim J-E and Kim Y-S (2017) Corrigendum: Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins. Front. Immunol. 8:1582. doi: 10.3389/fimmu.2017.01582*

**Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins**

*by Ha J-H, Kim J-E, Kim Y-S. Front Immunol (2016) 7:394. doi: 10.3389/fimmu.2016.00394*