Modulating Igg Effector Function by FC Engineering and Glycoengineering Andy Racher † & Olga Obrezanova ‡

Modulating IgG effector function by FC engineering and glycoengineering Andy Racher † & Olga Obrezanova ‡

Lonza Biologics, 228 Bath Road, Slough, Berkshire, SL1 4DX, UK

† Corresponding author: [email protected] ‡ Current address: AstraZeneca, Cambridge, UK

Summary several Fc engineered and glycoengineered antibodies in late stage clinical trials. Several proprietary technologies were Antibodies are the fastest growing group of biotherapeutics. In developed for glycoengineering of monoclonal antibodies early 2020 more than 100 antibody-based molecules, including including Potelligent® technology (KHK / BioWa), GlycoMAb® biosimilars, had been approved globally. Oncology and (GlycArt / Roche), EMABLing® (LFB), GlycoExpress™ (Glycotope) inflammatory disorders remain the major therapeutic areas for and GlymaxX® (ProBioGen). antibody-based molecules but there is a growing interest in using mAbs for treating infectious disease. During the last 10 Encouraging clinical results and prospects in multiple disease years antibody engineering focussed on development of “fit- areas make it likely that more Fc engineered and for-purpose” antibodies with modulated effector functions glycoengineered antibodies will enter clinical trials and such as increased or muted antibody-dependent cell-mediated subsequently be approved. According to market research cytotoxicity (ADCC), antibody-dependent cellular phagocytosis studies, the glycoengineered antibodies are likely to emerge as (ADCP), complement dependent cytotoxicity (CDC), and to the forerunner in the short term (84% of the market share by increase half-life. This paper summarises approaches for 2021) and, subsequently, the Fc protein engineered antibodies modulating antibody effector functions and pharmacokinetics, are likely to gain a higher proportion (55% by 2026). and provides examples of antibodies in clinical studies Glycoengineering strategies focussing on enhancing ADCC employing such approaches. effector function could potentially encounter competition from Tuning of effector functions can be achieved by either antibody Fc-engineering (via amino acid mutation) strategies. As more Fc engineering or by glycoengineering. Fc engineering involves Fc-engineered antibodies become approved for market use, the amino acid modifications in the Fc with the aim of enhancing or Fc protein engineering may become a preferred approach to reducing / eliminating effector functions. One class of modulate ADCC effector function. For the other effector antibodies with reduced effector functions is aglycosylated functions such as ADCP and CDC, there exists a breadth of antibodies, which is antibodies without glycans attached to the knowledge on how to enhance/reduce ADCP and CDC by Fc Fc. Aglycosylated antibodies can be generated by using mutations. There are no glycoengineering strategies yet to expression platforms incapable of glycosylation as well as by manipulate these effector functions consistently. amino acid modifications. There are currently at least four approved antibodies with engineered Fc: eculizumab (an IgG2/IgG4 hybrid Fc), durvalumab and atezolizumab (an aglycosylated antibody) and ocrelizumab. The first three are engineered to reduce effector functions and the fourth is engineered to enhance ADCC and reduce CDC. Glycoengineering strategies involve low or no fucosylation for enhanced ADCC function, the key mechanism of cell killing in oncology applications, or higher levels of sialylation for dampened immune responses (a less common strategy). There are currently three approved glycoengineered antibodies on the market: mogamulizumab and benralizumab are non- fucosylated antibodies generated by Potelligent® technology; while obinutuzumab is manufactured using the GlycoMab® to produce an antibody with low fucose content. There are also

Introduction

Monoclonal antibodies (mAbs) have high specificity and low toxicity, which when combined with their versatility makes them very attractive for disease treatment. For many years’ use of recombinant mAbs was focussed on cancer and inflammatory disorders. The increasing frequency of infections caused by multidrug resistant bacteria or viruses, and the recent viral disease epidemics, feeds the growing need for new prophylactic- or therapeutic-approaches that include anti- infective mAbs. As of February 2020, 100 antibody therapeutics, excluding biosimilars, have been approved globally 1.

Cancer cells are targeted by mAbs through specific recognition of tumour-associated antigens. Binding of the mAb to its target triggers effector functions upon engagement of receptors present on a range of immune cells including natural killer (NK) cells, T cells, dendritic cells, and the complement pathway components (Figure 1). This results in death of the target cell through a number of mechanisms such as complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). For treatment of inflammatory disorders, mAb modes of action (MOAs) include ligand and receptor blockade, receptor downregulation and signalling induction in 2 addition to the cell killing MOAs . Pathogens are targeted Figure 1: Antibody modes of action: (A) modes of action in oncology and (B) for directly and indirectly by mAbs (Figure 1). Antibodies bind to infectious disease. Cell lysis through activation of complement dependant the surface of pathogens and the Fc region interacts with cytotoxicity (CDC); interaction with Fc receptors on effector cells to engage immune systems such as phagocytes (opsonisation). antibody dependent cellular cytotoxicity (ADCC); signalling for ingestion of a pathogen or target cell by a phagocyte. Agglutination of the pathogen results from mAbs binding to the pathogens’ surface. Neutralisation occurs when mAbs stick to As of February 2020, all approved full-length antibodies are of pathogens and blocks their binding sites. Infected cells are the IgG isotype. An IgG antibody is made of two antigen killed by ADCC whilst pathogens can be killed directly following binding fragments (Fab) and the crystallisable fragment (Fc), complement activation (CDC). In addition, mAbs also block (Figure 2). The Fab fragment is responsible for antigen binding. toxin secretion and neutralise toxins. For these MOAs The Fc fragment is responsible for binding to the immune cell enhancing effector functions can improve mAbs’ efficacy and receptors like Fcγ receptors (FcγRs), the complement protein safety in both cancer and inflammatory disorders along with C1q and the neonatal receptor (FcRn). Interactions with FcγRs infectious diseases. and C1q lead to recruitment of effector functions such as ADCC, ADCP and CDC. Binding to FcRn prolongs the half-life of In some cases, it is desirable to reduce or eliminate effector antibodies. functions to prevent target cell death, unwanted cytokine secretion or off-target cytotoxicity. An example of such Recently, considerable efforts have been put into enhancing/ undesirable effector function is unwanted cell killing through reducing antibody binding to Fc receptors. One route is Fc ADCC, ADCP or CDC when a mAb’s function is blocking engineering. Fc amino acid residues involved in interactions receptor-ligand interactions and target cell death is not with FcγRs, C1q and FcRn interactions were mutated. The required. Another example is for antibody-drug conjugates second route is glycoengineering the Fc N-glycan. This paper where off-target interaction with receptors present on immune summarises the approaches used to modulate antibody cells is not wanted so as to prevent off-target cytotoxicity. If effector functions, and provides examples of antibodies in molecules are designed to bind immune system components, clinical studies where such approaches have been employed. then effector functions may not be desirable so as to achieve greater safety.

reduced binding of C1q to IgG2 and IgG4 subclasses, but also to differences in downstream events of the complement cascade. IgG3 has the most potent CDC activity 7, 8.

The amino acid residues involved in C1q binding are located in the lower hinge-upper CH2 region of Fc (Figure 2). The interaction is dependent on N-glycosylation at a conserved site (Asparagine 297, N297) in the CH2 region. Without N-linked oligosaccharides, there is no binding of Fc to C1q and there is no associated CDC function. C1q interacts with at least two antibody Fc regions in close proximity 3, with hexameric conformation of antibody complexes demonstrated to be highly efficient 9. Three recent reviews provide details of interaction residues for IgG1 antibodies and highlight structural differences for the other IgG subclasses 6, 7, 10.

Figure 2: Schematic representation of IgG overall structure and its binding regions with FcγRs, C1q and FcRn. The heavy and light chains linked by inter- Several approved antibodies such as rituximab and 11 chain disulphide bonds are shown. The site of interaction of FcγRs/ C1q with Fc ofatumumab show potent in vitro CDC activity . is shown in brown; the site of interaction of FcRn with Fc is shown in red. ADCC and ADCP effector functions The cellular immune response occurs mostly due to the Overview of effector functions interactions between the antibody and FcγRs. There are 5 activating FcγRs and one inhibitory receptor FcγRIIb (Table 1). The FcγRs are heterogeneous in terms of their cellular Efficacy of anti-cancer mAbs is achieved through both its expression and Fc binding affinities 10. antigen binding mechanism and its Fc-dependent mechanism. The Fab region can directly bind tumour cells, facilitating tumour clearance, inhibition of tumour growth and tumour cell Table 1: Fcγ receptors, their binding affinity and cellular expression death. The Fc region can further improve efficacy of mAbs by mediating effector functions such as CDC, ADCC and ADCP. Fcγ receptors Affinity strength Expressing cells

The effector mechanism that is most important for the efficacy FcγRI (activating) High affinity, Mononuclear phagocytes, -8 -9 of direct-targeting mAbs is widely debated. The choice depends KD ~ 10 –10 M dendritic cells and upon the antibody target, the nature of the epitope bound and neutrophils effector cells. Widely different activities can be elicited from FcγRIIa (activating) Lower affinity, Neutrophils, macrophages -7 different mAbs to the same target, including differences in FcγRIIc (activating) KD ~ 10 M effector mechanisms engaged. For example, anti-CD20 antibody ofatumumab has greater CDC activity than rituximab, FcγRIIb (inhibitory) Lower affinity, B-cell lymphocytes -7 also an anti-CD20 antibody. Trastuzumab is reported to initiate KD ~ 10 M 3 ADCC better than pertuzumab, both are anti-Her2 antibodies . FcγRIIIa (activating) Exists in Lowest affinity, NK cells, macrophages and -5 two alleles: V158 - moderate KD ~ 10 M T-cell subsets Complement-based effector function binding; F158 - low binding When an IgG molecule binds target surfaces, it can simultaneously engage the humoral immune system. The FcγRIIIb (activating) Lowest affinity, KD Neutrophils -5 humoral immune response is initiated by interactions with C1q, ~ 10 M a protein of the complement system, and a consequent activation of the complement pathway, resulting in CDC activity 4, 5. Intracellular signalling through the activating receptors leads to effector functions such as ADCC and ADCP, and inflammation The four IgG subclasses differ in their efficiency of triggering via the induction of cytokine secretion. In contrast, intracellular complement-based effector function. IgG1 and IgG3 can signalling through the inhibitory FcγRIIb counter-balances efficiently trigger the classical route of complement 6 but IgG2 activating signalling pathways. FcγRIIa and inhibitory FcγRIIb are and IgG4 do so much less efficiently. This is due, mostly, to the critical for immune regulation. The available evidence suggests that FcγRIIa is the dominant player in the induction of ADCP by

macrophages. FcγRIIIa is the key receptor for NK- and T cell- Fc engineering to modulate antibody effector mediated ADCC response. Cells expressing the higher affinity FcγRIIIa-V158 allele mediate ADCC more effectively 12. FcγRIIIb functions and half-life lacks signal generation capacity 10. Reviews by Wang et al. and Saxena & Wu focus on recent There are differences between the four IgG subclasses in their advances in Fc engineering approaches to modulate effector 7, 10 ability to bind FcγRs and trigger effector function. IgG1 and functions and serum half-life . Table 2 is a non-exhaustive list IgG3 interact efficiently with most FcγRs and are potent triggers of Fc engineered mAbs that are approved for use or are in of effector functions. IgG2 and IgG4 show reduced affinity to a clinical trial. number of FcγRs and will induce more subtle responses but only in certain cases. Monomeric IgG3 binds more efficiently Various strategies have been used to develop more effective than monomeric IgG1 to FcγRIIa, FcγRIIIa and FcγRIIIb. antibodies with desired ADCC, ADCP, and CDC activities. These Vidarsson et al. provide details of IgG subclasses binding include site-directed mutagenesis, alanine scanning, structure- profiles to each FcγR 6. based computational design, directed evolution technologies, yeast display, and asymmetric engineering. The results have FcγRs bind to a region partially overlapping the C1q binding yielded numerous mutations that modify Fc-FcγR interactions site, in the hinge and proximal CH2 region of Fc (Figure 2). The and the resultant effector functions. Details of these mutations interaction is dependent on the glycan at the N297 site in the and references to corresponding studies are detailed elsewhere 7, 10 CH2 region. . The following modulations of effector functions were identified: Without N-glycans, the binding of Fc to FcγRs and the associated effector functions is reduced or eliminated. Re- • Enhance ADCC: by increased FcγRIIIa binding, on its own or engineering of Fc region can reinstate or enhance FcγR binding together with decreased FcγRIIb binding. and associated effector functions even in the absence of • Enhance ADCP: by increased FcγRIIa and FcγRIIIa binding. glycosylation 13. We refer to recent reviews for the details of • Enhance CDC: achieved by increased C1q binding or by interaction residues and differences between IgG subclasses 6, 7, hexamerization. 10. • Reduce effector functions: by reduced FcγR and C1q binding or by aglycosylation. The efficacy of several anti-cancer mAbs (e.g. rituximab and • Increased half-life, achieved by increased FcRn binding at trastuzumab) is believed to rely, in part, on recruitment of pH 6.0 FcγR-based effector functions. These include activation of NK • Increased coengagement of antigen and FcγRs: by increased cells via FcγRIIIa and resultant ADCC and inflammatory cytokine FcγRIIb binding, or by decreased FcγRIIIa binding and release, macrophage-mediated ADCP through interactions with increased FcγRIIa binding. multiple FcγRs, and recruitment and activation of other immune cells (e.g., neutrophils). Studies on rituximab and The majority of studies considered IgG1 subclass antibodies, trastuzumab have suggested that ADCC is the key mechanism of but there are several examples where mutations were made in action to eliminate cancer cells 12. IgG2, IgG3 and IgG4 antibodies. In most of the studies, 1-3 amino acid mutations were made in the Fc, but in some FcRn receptor and antibody half-life antibodies the Fc was engineered using five or more mutations. The neonatal Fc receptor (FcRn) prolongs the half-life of mAbs through a recycling mechanism in a pH-dependent interaction Enhanced ADCC. Stavenhagen et al. utilised yeast surface with the Fc region. display to identify the variant with mutations F243L/R292P/Y300L/ V305I/P396L that has increased FcγRIIIa The pharmacokinetic profiles of mAbs vary among subclasses. binding, and showed 100-fold increased ADCC activity 15. It is known that the serum half-life of subclasses IgG1, IgG2 and Margetuximab, an anti-HER2 IgG1 mAb with these 5 mutations IgG4 is ~23 days as compared to 2-6 days for IgG3 10. except that V305I was replaced with L235V to reduce FcγRIIb binding, showed enhanced ADCC activity over the wild-type The binding site enabling interactions with FcRn is located in equivalent 12, 16, 17. Margetuximab is now in phase III clinical trial the interface of CH2-CH3 regions (Figure 2). The absence of for patients with metastatic breast cancer glycans in CH2 region of Fc does not significantly affect FcRn binding and antibody half-life 13, 14. The IgG residues involved in interactions with FcRn are detailed in two recent reviews 7, 10.

Table 2: Examples of Fc-engineered antibody therapeutics approved or under clinical evaluation

Antibody Target Fc modification Intended function Disease Clinical Company development

Eculizumab C5 Cross-subclass reduced effector function PNH and atypical hemolytic Approved Alexion (Soliris) IgG2/IgG4 uremic syndrome Pharmaceuticals Durvalumab PD-L1 L234F/L235E/P331S reduced effector function Bladder cancer Approved AstraZeneca/ (Imfinzi) MedImmune Atezolizumab PD-L1 N298A, aglycosylated reduced effector function Bladder cancer Approved Genentech/ (Tecentriq) Roche Ocrelizumab CD20 NA enhanced ADCC, reduced CDC Multiple sclerosis Approved Genentech/ (Ocrevus) Roche BI836826 CD37 NA enhanced ADCC CLL Phase-1 Boehringer

JNJ56022473 CD123 NA enhanced ADCC AML Phase 2 Janssen R&D

XmAb2513 CD30 NA enhanced ADCC Hodgkin/large cell Phase 1 Xencor, Inc. lymphoma XmAb5871 CD19 S267E/L328F increased inhibitory FcγRIIb binding, SLE Phase 1 Xencor, Inc. immune inhibition XmAb7195 IgE S267E/L328F increased inhibitory FcγRIIb binding, Allergic diseases Phase 1 Xencor, Inc. immune inhibition XmAb5774 CD19 S239D/I332E enhanced ADCC/ADCP CLL Phase 1 Xencor, Inc.

Margetuximab HER-2 L235V/F243L/R292P/ enhanced ADCC Breast cancer Phase 3 MacroGenics Y300L/P396L TRX4 CD3 N297A - aglycosylated reduced effector function Type-1 diabetes mellitus Phase 3 GSK/Tolerx (autoimmune) Onartuzumab MET N297A - aglycosylated reduced effector function NSCLC/gastroesophageal Phase 3 Roche cancer ALD518 IL-6 N297A - aglycosylated reduced effector function RA/NSCLC/oral mucositis Phase 2 Alder TRX518 GITR N297A - aglycosylated reduced effector function Malignant melanoma Phase 1 Tolerx

PNH, paroxysmal nocturnal hemoglobinuria; CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; SLE, systemic lupus erythematosus; NSCLC, non- small cell lung cancer; RA, rheumatoid arthritis; Adapted from Saxena & Wu 10 and from Kaplon & Reichert 18

Enhanced CDC. It is known that IgG1 is the most potent ADCC Reduced effector functions. IgG2 and IgG4 have very limited activator, while IgG3 has highest potency to recruit ability to elicit ADCC and are recognized as being able to recruit complement system 19. Therefore, IgG1 and IgG3 Fc regions can complement less effectively than IgG1 and IgG3 7. A cross- complement one another to maximize the immune effector subclass approach was used to reduce effector function. The response. The variants with chimeric CH regions showed a 25– approved anti-C5 therapeutic, eculizumab, has the IgG2 amino 60% increase in ADCC and CDC activity compared to wild type acids 118 to 260 and the IgG4 amino acids 261 to 447 and has IgG1 and IgG3 molecules 19. The CDC activity of a humanized been shown to have limited or undetectable effector function 7, anti-CD20 IgG1 (ocrelizumab) was increased by around 23-fold 21. As such, eculizumab represents the first proof-of-concept while retaining normal IgG1 ADCC by combining a triple mutant that reduced effector function mAbs can be safely utilised in an (S267E/H268F/S324T) with earlier reported G236A/I332E in the approved mAb-based therapeutic. CH2 domain 10, 20.

Half-life extension. Attempts have been made to engineer Fc conformational flexibility of the CH2-CH3 domain interface, as regions to improve antibody pharmacokinetics 7, 10. Enhanced compared to the glycosylated counterpart 28. Fc-FcRn interaction at acidic pH can extend IgG’s serum half- life, which may benefit patients by greater therapeutic efficacy, The inherent glycan heterogeneity of mAbs when expressed in less frequent dosing and lower cost burden. mammalian cell systems can cause high production costs and variations from batch to batch. Bypassing glycosylation provides A Fc variant with mutations M252Y/S254T/ T256E - termed YTE bioprocessing advantages by avoiding the heterogeneity. - identified by screening a phage display Fc variant library Additionally the aglycosylated antibodies can be produced in showed enhanced binding to human FcRn at pH 6.0 22. A later prokaryotic and lower eukaryotic hosts, leading to much faster study demonstrated that the YTE variant had 10-fold increased development timelines. binding to human and cynomolgus FcRn at pH 6.0, which translated into a 4-fold increase in half-life in a cynomolgus PK It is worth noting that removal of appended glycans lowers an study 7, 23. Motavizumab-YTE, a humanized antibody targeting IgG’s thermostability, affects its resistance to proteolysis and respiratory syncytial virus, is the first antibody engineered for increases its aggregation propensity in vitro 13, 29, 30. The initial FcRn mediated half-life extension tested in human subjects 7, 24. concerns that the absence of the N297 glycan in the Fc may The results from the phase I clinical trial in healthy adults result in immunogenicity, poor stability during formulation and demonstrated a 2- to 4-fold increase in half-life relative to storage, and unfavourable in vivo pharmacokinetics have been motavizumab IgG1 depending on the dose, which provided largely dismissed based on the results from clinical trials of proof-of-concept that FcRn low pH enhancing modifications aglycosylated antibodies. The aglycosylated antibodies display translated into increased half-life in humans. This mutation has pharmacokinetics that are at least comparable to that of been incorporated into a few early stage candidates. ‘normal’ IgG therapeutics. No adverse immunogenicity effects have yet been reported. Other half-life extension mutations of the Fc have been made. These include Xencor’s Xtend technology, which has been Currently, there is at least 1 approved antibody (atezolizumab) incorporated into at least two early stage clinical candidates, and 4 aglycosylated antibodies in clinical trials in phases I, II and Alexion’s anti-C5 mAb, ALXN5500, and National Institutes of III: TRX-4, onartuzumab, ALD518 and TRX 518. Glycans were Health’s anti-CD4 mAb, VRC01LS 25, 26. removed by introducing a Fc modifying mutation at N297A in all four mAbs 10, 13. One anti-CD4 antibody, TLX-1 (from Tolerx) Aglycosylated antibodies was discontinued after phase I trials. Onartuzumab is the first Aglycosylated antibodies have no glycans attached to the therapeutic full-length antibody that is manufactured in E. coli glycosylation site in Fc CH2 domain, which is achieved by 10, 13, 31. This is significant because E. coli has a very limited and mutating the amino acid sequence at glycosylation site N297 specific intrinsic glycosylation machinery 32. Consequently, E. with mutations such as N297A, N297Q, N297G (see 7 and coli can produce antibodies without any N-linked or O-linked papers listed therein). glycans (i.e. fully aglycosylated) while leaving residue N297 unmodified. Aglycosylated antibodies in clinical trials have been Removal of the N297 glycan reduces the binding affinity to produced in E. coli, yeast and CHO cells. Production in algae has FcγRI and abolishes binding to the weaker affinity FcγRII and been described although no clinical trials have been reported FcγRIII. The binding to C1q is also reduced 10-fold. The reduced 13, 33. binding abolishes ADCC and CDC effector functions. This may be advantageous in the cases where ADCC/CDC action is not Recent studies have shown that engineering aglycosylated required or is considered a liability, as is the case of antibodies can reintroduce binding to FcγRs, and thus elicit neutralising, agonistic (receptor activating) or antagonistic ADCC. Additionally, engineering of aglycosylated antibodies can (receptor inactivating) mAbs. In such cases, the antibody MOA be used to elicit unique FcγR selectivities resulting in novel is to bind cell-surface receptors with the purpose of blocking or effector functions and mechanisms of action that do not activating receptor-ligand interactions and target cell death is appear possible with their glycosylated counterparts 13, 34. The not required (see 2, 13 and papers cited therein). aglycosylated antibodies, due to greater conformational flexibility of Fcs, provide possibilities to manipulate the ratio of Based on available crystal structure data, the presence or affinities to activating receptors over the inhibitory FcγR absence of the glycan at N297 should not influence FcRn receptors that could allow greater control over activation of binding and the pharmacokinetics of aglycosylated antibodies certain cell types in therapeutic setting 13, 27, 35, 36. 10, 13, 27. Several studies in mice and clinical trials demonstrated that this is generally the case 13, 14. The three-dimensional The bioprocessing advantages of production in prokaryotic and structure of human aglycosylated antibody suggests a greater lower eukaryotic hosts, and therapeutic advantages of little

glycan heterogeneity, together with promising clinical trials Interactions between Fc glycan structures with Fcγ and C1q results make aglycosylated antibodies an attractive therapeutic receptors involved in binding are described in the review 29. format.

Trends and opportunities in Fc engineering Over the last few years, the focus of antibody engineering was upon the generation and application of “fit for purpose” antibodies with tuned/increased ADCC, ADCP and CDC or reduced effector functions. There are several Fc antibodies engineered to modulate effector functions that are approved for market use, including eculizumab (Fc hybrid to reduce effector functions), durvalumab (Fc mutations to reduce effector functions), atezolizumab (aglycosylated) and ocrelizumab (Fc mutations to increase effector functions). In addition, several Fc-engineered antibodies with modulated effector functions are in late stage clinical trials. Fc mutations to extend antibody half-life have been incorporated in several early stage clinical candidates. Clinical success of such Figure 3. The complex N-linked glycan found at position 297 of a full-length mAb antibodies shows that that altering the effector functions by Fc heavy chain polypeptide. The glycan can exist as a core structure (in background engineering is likely to be widely used in the development of box) or with either the addition of fucose, bisecting N-acetylglucosamine new therapeutics. (GlcNAc), one or two galactoses, and one or two sialic acid residues.

Xencor’s proprietary XmAb antibody engineering technology Sialic acid. Several studies listed in Liu et al. found that the creates modifications to the mAb’s Fc domain enhancing presence of sialic acid reduced binding to Fcγ receptors and the binding to FcγRs, producing Fc domains capable of potent associated biological activity. In contrast, several listed studies immune inhibition (400-fold increased affinity for FcγRIIb) or demonstrated that removal of sialic acid has no impact on FcγRI potent cytotoxicity towards NK cells and macrophages (40-fold binding, ADCC and CDC. In general, sialic acid has no impact on greater affinity for FcγRIIIa). Xencor’s Xtend technology for half- half-life 29. It is a major factor that contributes to the formation life extension enables increased circulating half-life of an of antibody acidic species. The presence of terminal α2,6-linked antibody up to 3-fold. XmAb Fc domains are plug-and-play and sialic acid residues in the IgG Fc has been implicated in anti- can be substituted into nearly any antibody. Other companies inflammatory activity 6, 37-39. have their own Fc mutations, some of them possibly protected by patents. Bisecting GlcNAc. Bisecting GlcNAc is present at very low levels and only present in mAbs expressed in murine cell lines, e.g. Impact of Fc oligosaccharides on antibody NS0. CHO cell lines have been engineered to express the enzymes needed to add a bisecting GlcNAc residue 40. effector functions Increased levels of bisecting GlcNAc correlated with increased ADCC, with minimal impact on CDC. However, this A recent review by Liu et al. focusses on the impact of IgG Fc enhancement of ADCC is apparent only when a relatively high oligosaccharides on recombinant antibody structure, stability, 29 level of bisecting GlcNAC was present in antibodies with mainly safety and efficacy . Three main categories of N-linked core-fucosylated oligosaccharides 29. oligosaccharides, complex, high mannose and hybrid, are described as well as structures of commonly observed 29 Galactose. Galactosylation has no impact on in vivo clearance. oligosaccharides in monoclonal antibodies . The complex N- However, its impact on ADCC and complement activation may linked glycan is shown in Error! Reference source not found.. need to be evaluated case-by-case, depending on the High mannose glycans are composed of mannose only in the mechanisms of action. Some studies suggest an increase in outer arms. Hybrid oligosaccharides are made of one arm with galactose leads to increased ADCC 41-43 and others to decreased complex and the other arm with high mannose residues. ADCC 41, 42. The current understanding is that the terminal galactose has a minimal impact on ADCC, but a more Fc glycans have no impact on binding to FcRn, as the binding substantial impact on CDC 29, 30. Pereira et al. reported that Fc sites are located in the CH2-CH3 domain interface. In contrast, galactosylation leads to increased FcγRIIIa binding although to a oligosaccharides are critical for the binding of Fcγs and C1q. lesser extent compared to a removal of core fucose 12. General decrease in galactosylation has been found in several

autoimmune diseases 6, 44, suggesting galactosylated IgG has an Various approaches have been utilised to produce non- anti-inflammatory activity. fucosylated proteins and antibodies, involving manipulation of host biosynthetic pathways by gene knockdown/knockout or Fucose. The majority of the oligosaccharides in recombinant overexpression of glycoprocessing enzymes, and/or in vitro mAbs are the complex type with core fucose. A small chemoenzymatic glycosylation remodelling 12, 37. Several percentage of complex oligosaccharides are detected as lacking companies have developed proprietary technologies for core-fucose. High mannose types of oligosaccharides do not antibody Fc glycoengineering. These are summarised in Table 3. have core-fucose. Table 4 is a non-exhaustive list of Fc glycoengineered mAbs that are either approved for use or are currently in clinical trial. The overall structure of antibodies with or without the core- fucose is very similar. The lack of fucose has no impact on Table 3: Commercial glycoengineering technologies binding of antibodies to FcγRI, C1q, FcRn, and the His- polymorphic FcγRIIa, but it has slightly increased binding to Technology Description Company Arg-polymorphic FcγRIIa, and FcγRIIb. The most striking discovery is that antibodies without core fucose showed Potelligent® FUT8 gene knockout; afucosylated BioWa significantly improved binding to FcγIIIa, leading to higher ADCC. The pivotal study by Shields et al. demonstrated that GlycoMAb®/ Overexpression of glycoprocessing Roche IgG1s deficient in fucose had an up to 50-fold increase in GlycArt enzymes (GnT-III); low fucose content FcγRIIIa binding relative to IgG1 as well as enhanced ADCC 45. Shinkawa et al. later demonstrated that fucose deficient EMABLing® YB2/0 cell lines; low fucose content LFB technologies antibodies had improved ADCC function compared to 46 antibodies containing bisecting GlcNAc . The enhanced GlycoExpress™ Low fucose content Glycotope affinity to FcγRIIIa is the result of a unique carbohydrate- carbohydrate interaction between the N-glycan of the IgG and GlymaxX® Disruption of the de novo GDP fucose ProBioGen the N-glycan of the FcγRIIIa at N162 12, 29. pathway; manipulation of fucose levels

In one study, afucosylated antibody demonstrated faster GlycoDelete Manipulation of the Golgi N- The VIB and clearance in mice, while a different study did not reveal a half- glycosylation pathway; simplification Ghent University life difference 29. and shortening of glycans

High mannose glycans. The level of high mannose is very low in recombinant mAbs, but there are cases in which up to 10% has FUT8 gene knockout. One approach is based on knockdown or been observed. Several studies demonstrated that the knockout of the FUT8 gene, encoding the enzyme that presence of high mannose reduced or gave comparable binding catalyses the transfer of fucose from GDP-fucose to the to FcγRI and reduced binding to FcγRII. The presence of high innermost GlcNAc residue of the tri-mannosyl core structure. mannose showed enhanced binding affinity to FcγRIIIa and The FUT8 gene in the CHO DG44 cell line was targeted using 47 enhanced ADCC, mainly due to the lack of the core-fucose. sequential homologous recombination . This technology, Antibodies with high mannose show substantial deficiency in developed and patented by BioWa, was used by Kyowa Hakko C1q binding and the classical complement activation (although Kirin to create mogamulizumab (POTELIGEO®), a marketed a superior activity through the alternative complement antibody, expressed in FUT8-knockdown CHO cells to achieve pathway). There are studies demonstrating no difference in complete afucosylation. Another marketed antibody produced half-life in mice, but additional studies demonstrated shorter in by the BioWa technology is benralizumab (MEDI-563, vivo half-life in humans and animal models 29, 30. Fasenra™). The BioWa FUT8 technology is used by Lonza in its Potelligent® CHOK1SV platform.

Glycoengineering to modulate effector The FUT8 gene has also been targeted for inactivation using the functions zinc finger nuclease platform 12, 48 and by small interfering RNA 12. Strategies to produce afucosylated antibodies It is now widely recognised that removal of the core fucose from the Fc N-glycans represents the most effective approach for enhancing ADCC activity 12, 45, 46.

Table 4: Examples of glycoengineered antibody therapeutics approved or under clinical evaluation

Antibody Target Glycoengineering Technology Disease Clinical Company development Obinutuzumab Chronic lymphocytic leukemia ; (Gazyva) CD20 low fucose content GlycoMAb® Non-Hodgkin’s lymphoma Approved Roche Mogamulizumab (POTELIGEO) CCR4 afucosylated Potelligent® Cutaneous T cell lymphoma Approved Kyowa Hakko Kirin Benralizumab (Fasenra) IL-5Rα afucosylated Potelligent® Asthma Approved AstraZeneca Neuromyelitis optica and neuromyelitis optica spectrum Inebilizumab CD19 afucosylated Potelligent® disorders Phase 2/3 Medimmune Chronic lymphocytic leukemia, non-Hodgkin lymphoma, Ublituximab CD20 low fucose content EMABLing® multiple sclerosis Phase 3 TG Therapeutics Bemarituzumab Gastric and gastroesophageal Five Prime (FPA144) FGFR2b afucosylated Potelligent® cancer Phase 3 Therapeutics Cusatuzumab T cell lymphoma, acute myeloid (ARGX-110) CD70 afucosylated Potelligent® leukemia Phase 2 Argenx

Phase 1 ARGX-111 c-MET afucosylated Potelligent® Advanced cancer complete Argenx Lumretuzumab HER3 reduced fucosylated GlycoMAb® Breast cancer Phase 1 Roche TrasGEX/ GT- MAB7.3-GEX HER2 reduced fucosylated GlycoExpress™ Solid tumours Phase 1 Glycotope Roledumab Rh D low fucose content EMABLing® Rhesus disease Phase 2/3 LFB Technologies IL-3Rα / Acute myeloid leukemia, Kyowa Hakko Kirin KHK2823 CD123 afucosylated Potelligent® myelodysplastic syndrome Phase 1 Pharma CHO cells. Use of modified sugars in culture media to SEA-CD40 CD40 afucosylated inhibit fucosylation Cancer and carcinomas Phase 1 Seattle Genetics

Adapted from Pereira et al. 12 and Garber 49

YB2/0 cell lines. Rat hybridoma YB2/0 cells have significantly and completely afucosylated antibodies 12, 51. Double-knock lower levels of the FUT8 mRNA than CHO cells. YB2/0 produced down of FUT8 and GMD also effectively allows stable antibodies have a low level of core fucose and, therefore, an expression of fully afucosylated antibodies with enhanced enhanced ADCC 12, 46. EMABLing® technology (LFB group) uses ADCC 37, 52. YB2/0 cells. Ublituximab, a chimeric IgG1 antibody with low fucose produced using EMABLing® technology, was approved CHO Lec13 cells are naturally defective in GDP-fucose for clinical trials and is currently in phase 1, 2 and 3 trials for formation due to a deficiency in endogenous GDP and were various indications 12. The level of fucosylation is not robust due used for the production of afucosylated antibodies. However, to high sensitivity to process conditions. studies have shown that Lec13 clones display a range of fucosylation levels. In addition, the Lec13 cell line is not Enzymes of GDP-fucose biosynthesis. Another approach is to sufficiently robust to be utilised as a host cell line for knockout the GMD gene, which encodes an enzyme involved in recombinant protein production cell line as expression levels the biosynthesis of GDP-fucose formation 37, 50. A FX-knockout are lower than in other CHO hosts 12, 45. CHO cell line was reported to express antibodies with the desired ratio of primarily-fucosylated to afucosylated glycans,

The de novo GDP-fucose biosynthesis pathway of the host CHO benthamiana 59, 60. The transfer of a glycosylation pathway cell line (antibody expressing) was selectively targeted by from Campylobacter jejuni into E. coli created considerable overexpression of a bacterial reductase enzyme (GTS from interest in use of prokaryotes for glycoprotein production. Actinobacillus actinomycetemcomitans). The GTS enzyme However, development of suitable prokaryotic hosts requires converted the GDP-4-keto-6-deoxymannose of the de novo more than insertion of mammalian glycosyltransferases. Issues pathway to GDP-6-deoxy-D-talose. Diversion of the key pathway such as lack of non-native glycan precursors have to be intermediate disrupted the de novo GDP fucose pathway and addressed 61. generated >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity Biochemical inhibitors of fucosylation. In one study small (11-fold). This glycoengineering strategy allows the subsequent molecules (2-fluorofucose and 5-alkynylfucose) were used to utilization of the alternative GDP-fucose synthesis salvage inhibit antibody fucosylation. The mechanism of action of these pathway through L-fucose supplementation, to modulate the inhibitors is likely due to the depletion of intracellular GDP- IgG N-glycan fucosylation levels 53. Sandig and colleagues fucose with a subsequent block of the de novo pathway or the expressed the prokaryotic enzyme GDP-6-deoxy-D-lyxo-4- inhibition of FUT8 12, 62. hexulose reductase in CHO cells 54. Cytosolic expression of the enzyme deflects GDP-4-keto-6-deoxymannose into a dead-end Other glycoengineering strategies product, GDP-D-rhamnose, again resulting in the production of Besides manipulation of fucosylation, similar strategies can be mAbs lacking core fucose. used to redirect and control other aspects of glycosylation profiles. Since the presence of terminal α2,6- linked sialic acid GDP-fucose transporter. It has been shown that loss-of-function residues in IgG Fc has been implicated for anti-inflammatory mutations in the Golgi GDP-fucose transporter gene SLC35CL activity 37-39, several groups pursued overexpression of α2,6- was able to eliminate fucosylation reactions that occur in the SiaT in the mammalian host cell to enhance terminal sialylation Golgi 12. The SLC35CL gene was inactivated in CHO cells 37, 63, 64. In addition to overexpression of certain glycoprocessing generating CHO-gmt3 cells. An IgG antibody expressed in the enzymes, the use of glycoprocessing inhibitors can manipulate CHO-gmt3 cells was shown to be completely afucosylated 12, 55. glycosylation 37, 65, 66, by preventing production of certain glycan structures. Overexpression of glycoprocessing enzymes. Overexpression of certain glycoprocessing enzymes in mammalian host cell lines Chemoenzymatic remodelling. In vitro chemoenzymatic can enhance glycoforms. GnT-III is not normally expressed in glycoengineering is another attractive method to prepare CHO cells. Overexpression of GnT-III catalyses the formation of homogeneous glycoforms of mAbs or afucosylated mAbs (see 37 bisecting GlcNAc residue, which in turn inhibits the fucosylation and papers cited there). First, the majority of N-glycans are reaction and results in antibodies with reduced core fucose removed from an antibody by treatment with an ENGase content. Co-expression of GnT-III and αManII resulted in (endo-β-N-acetylglucosamidase), such as EndoS highest level of bisecting and afucosylated glycans on IgG (endoglycosidase S), followed by treatment with a fucosidase to antibodies 12, 37, 56. This method is the basis of Roche’s GlycArt remove the core fucose. Selective oxazoline derivatives of (GlycoMAb®) technology which was employed to create complex N-linked glycan structures are prepared by chemical obinutuzumab (Gazyva®), a humanised IgG1 with reduced methods using isolated or synthetic oligosaccharides. The fucose content and an increased ADCC activity. GlcNac or fucosylated GlcNac is then further extended by transglycosylation, in which the preformed glycan oxazoline Glycoengineering in non-mammalian cells. The early steps of N- donor allows a specific N-glycan to be added to the acceptor to glycosylation are conserved in yeast, plant and mammalian achieve a defined, homogeneous antibody glycoform 12, 37. The cells, the processing pathways branch off at later points 37. method is not considered to be cost effective for producing Strategies to humanise glycoproteins produced in yeast and afucosylated therapeutic antibodies 12. The chemo-enzymatic plant cells mainly focus on elimination of the yeast-specific (by approach has been applied to the glycoengineering of human knockout of och or alg3) genes or plant-specific (by knockout of IgG-Fc fragments, as well as various therapeutic mAbs such as β1,2-XylT or α1,3-FucT genes) hypermannosylated glycoforms, rituximab, trastuzumab and the antiviral antibody FI6 (see 37 and subsequent transfer of the mammalian glycan processing and papers cited there). enzymes 12, 37. The examples of such glycoengineering systems applied to therapeutic antibodies include the non-commercial GlycoDelete technology. This technology shortens the Golgi N- production of the rituximab in Pichia pastoris 37, 57, the glycosylation pathway in mammalian cells and simplifies N- afucosylated anti-CD30 mAb in the glycoengineered aquatic glycosylation resulting in proteins with small, sialylated plant Lemna minor 58, and the HIV neutralising mAb 2G12 and trisaccharide N-glycans and reduced complexity compared to the anti-tumour heteromultimeric IgM PAT-SM6 in Nicotiana native mammalian cell glycoproteins 37, 67. A pre-existing GnT I-

deficient human embryonic kidney cell line HEK293S is used as CDC by Fc mutations. There are no glycoengineering strategies a starting point, generating Man5GlcNAc2 glycans. Golgi- yet to manipulate these effector functions. targeted expression of an ENGase, endoT, in HEK293S cells allows the Man5GlcNAc2 to be converted to mono-GlcNAc The presence of sialic acid residues in Fc glycans has been glycoproteins. Subsequent processing by Golgi-resident GalT implicated for anti-inflammatory activity and several groups and SiaT leaves glycoproteins with the galactosylated pursued glycoengineering to enhance sialylation, although this disaccharides (Gal-GlcNAc) or the sialylated trisaccharides did not result in commercial technology or clinical antibody (Neu5Ac-Gal-GlcNAc), and some of the monosaccharide candidates. intermediate mono-GlcNAc. The platform was tested on a therapeutic glycoprotein and an anti-CD20 mAb. The normally Therapeutic antibody landscape glycosylated mAb and the GlycoDelete version share similar stability and antigenicity profiles. Antibody produced with the A number of antibodies with engineered Fc modifications are GlycoDelete technology has less initial clearance from serum in being investigated in clinical trials and several (at least 4) are mice, which might allow a reduced dosage frequency. However, approved for various indications, some examples are provided GlycoDelete antibody has lower binding affinity to human Fcγ in Table 3. The molecules fall into three categories: antibodies receptors, the feature which may be desirable or not with enhanced effector functions for treatment of cancer and depending on therapeutic applications. infectious diseases, molecules inhibiting immune activation for treatment of inflammatory diseases and a new class of Supplementation strategies. Glycan heterogeneity can be aglycosylated mAbs with either inert or active-immune function influenced by several process conditions including 10. This paper focussed on antibodies, but there are two perturbations to temperature, pH and dissolved oxygen; a topic approved fusion proteins with modified Fc functionality to reviewed by Hossler 68. The other strategy is a supplementation reduce effector functions. These are the CTLA4-Fc proteins, of nucleotide-sugar precursors and associated components, abatacept (Orencia®) and belatacept (Nujolix®) 25. such as sugars and amine-sugars, including nucleosides like uridine and cytidine, and ionic forms of the metal manganese At present, at least 35 glycoengineered antibodies, with their 69. Blondeel & Aucoin 69 reviewed supplementation strategies fucose partially or completely removed, have been investigated focussing on several factors, such as differences in in animal models. Twenty-six have been studied in clinical trials enzyme/transporter expression levels across cell platforms, and 3 have been approved for use in clinical practice 12. differences between produced recombinant proteins with Examples of approved antibodies and those in clinical trials are respect to the accessibility of their glycan sites by shown in Table 4. glycosyltransferases, the bioreactor process and sampling timelines and glutamine levels. We refer to this review for the There are currently 70 phase III and 575 phase I/II antibody- details of the supplementation regimes and resulting effects on based drugs in clinical trials 7, 25, and the need to differentiate glycosylation. This approach has been successfully used by 70, 71 antibodies is becoming imperative. The market report “Fc groups working with Lonza’s GS-CHO platform . Protein and Glycoengineered Antibodies Market (2nd Edition), 2016 - 2026” 72 identified that Fc engineered and Trends and opportunities in Fc glycoengineering glycoengineered antibodies account for nearly 70 products in Currently, three glycoengineered antibodies have been marketed, clinical and preclinical stages of development; approved for market use and at least 35 antibodies have been accounting for nearly 60% of the pipeline for molecules in studied in clinical trials and animal models. The clinical development. The approval of the effector modulated glycoengineering strategies employed for all of these mAbs eculizumab, obinutuzumab, mogamulizumab, candidates were focussing on low fucose levels or absence of atezolizumab and ocrelizumab has demonstrated that altering fucose (afucosylation) to enhance ADCC function. the effector function can be a viable way to successfully develop new therapeutics and bring them into clinic. Glycoengineering strategies focussing on enhancing ADCC effector function could encounter competition from Fc- The market research report estimates that the glycoengineered engineering (via amino acid mutation) strategies. As more Fc- antibodies are likely to emerge as the forerunner in the short engineered antibodies demonstrate efficacy and safety in term (84% of the market share by 2021) and, subsequently, the clinical trials and become approved for market use, Fc protein Fc protein engineered antibodies are likely to gain a higher engineering may become a preferred approach to modulate proportion (55% by 2026); specifically, atezolizumab (by Roche) ADCC effector function due to its greater versatility. For the and durvalumab (by AstraZeneca/MedImmune) are expected other effector functions such as ADCP and CDC, there exists a to be blockbusters . breadth of knowledge on how to enhance/reduce ADCP and

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