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LitCovid_Glycan-Motif-Structure

{"project":"LitCovid_Glycan-Motif-Structure","denotations":[{"id":"T1","span":{"begin":1391,"end":1397},"obj":"https://glytoucan.org/Structures/Glycans/G82576YO"},{"id":"T2","span":{"begin":12340,"end":12346},"obj":"https://glytoucan.org/Structures/Glycans/G82576YO"},{"id":"T3","span":{"begin":12376,"end":12382},"obj":"https://glytoucan.org/Structures/Glycans/G82576YO"},{"id":"T4","span":{"begin":13499,"end":13505},"obj":"https://glytoucan.org/Structures/Glycans/G82576YO"}],"text":"IgG Subclasses: Structure and Properties\n\nThe molecular basis of IgG and FcγR interactions\nThe extracellular regions of the FcγR are structurally similar. Each low‐affinity FcγR has two ectodomains, whereas the high‐affinity FcγRI has a third domain but this is not directly involved in IgG binding.62\nThe interaction between the IgG subclasses and the FcγR is most comprehensively defined for human IgG1 by both X‐ray crystallographic7, 62, 63 and mutagenesis structure/function analyses.64, 65, 66 These studies defined key regions of the IgG sequence required for interaction with their FcγRs.\nCrystallographic analyses of the human IgG1‐Fc complexed with FcγRI, FcγRII or FcγRIII show that these interactions are similar in topology, and asymmetric in nature. The second extracellular domain of the FcγR inserts between the two heavy chains. Here it makes contacts with the lower hinge of both H chains and with residues of the adjacent BC loop of one CH2 domain and the FG loop of the other. The N‐linked glycan at asparagine 297 (N297) of the heavy chain is essential for the structural integrity of the IgG‐Fc by affecting the spacing and conformation of the CH2 domains. Indeed, its removal ablates FcγR binding.67 Of particular relevance to therapeutic mAb development is that the normal low‐affinity IgG interaction with FcγRIIIa is profoundly increased by the removal of the core fucose from the N297 Fc oligosaccharide.68\nNo crystallographic data are available for IgG2 or IgG4 Fc in complex with FcγR, but mutagenesis studies of the Fc and the FcγR revealed general similarity, but with critical differences, in the interaction of these subclasses with their cognate FcγR.\n\nUnique features of the IgG2 and IgG4 subclasses\nIn IgG1, the stable interaction of the two heavy chains results from the combined effects of stable covalent inter H‐chain disulfide bonds and strong noncovalent interaction of the two CH3 domains (Table 3). In stark contrast, in IgG2 and IgG4 the interaction of the CH3 domains of each H‐chain is weak. Residues 392, 397 and 409 (Eu numbering) profoundly affect the stability of these interactions. The difference at position 409 (R409 in IgG4 and K409 in IgG1) confers a 100‐fold decrease in stability of the interface between the two CH3 domains of IgG4 compared with that of IgG1 (Table 3).69\nFurthermore, the core hinge of IgG4 differs from IgG1 at position 228 (P228 in IgG1 and S228 in IgG4), resulting in unstable inter‐heavy‐chain disulfide bonds. This, together with the destabilizing amino acids in the CH3, confers the unique property of half‐antibody (Fab arm) exchange between different IgG4 antibodies,69 thereby creating monovalent, bispecific IgG4 antibodies in vivo.69, 70 The similarly unstable interactions between the CH3 domains in IgG2 are conferred by the interface residue M397; however, the stable inter‐H‐chain disulfide bonds of the core and upper hinge prevent half‐molecule exchange (Table 3).69\nIn addition, IgG2 uniquely has three disulfide bond conformers (Table 3). The distinct conformers are formed when (1) each light chain is attached to the Cys131 residue of CH1 in the heavy chain (IgG2‐A conformer), (2) both light chains attach to the upper hinge (IgG2‐B) or (3) one light chain is attached to the CH1 Cys131 and one to the upper hinge of the other heavy chain (IgG2‐AB).71 This results in distinct positioning of the Fabs relative to the Fc portions in the different conformers, which has implications for the interaction with antigen and the capacity of IgG2 to cross‐link target molecules in the absence of FcγR binding, for example, in an agonistic mAb setting.72\nIt should also be noted that IgG3 has not been used in therapeutic mAbs despite its unique biology. The main impediment to its use are its physicochemical properties such as susceptibility to proteolysis and propensity to aggregate that present challenges to industry‐scale production and stability but protein engineering is attempting to overcome these hurdles.73\n\nTherapeutic antibody design: improving mAb potency\nMany factors affecting FcγR‐dependent responses in vivo come into play during mAb therapy. The experience of three decades of clinical use of mAbs taken together with our extensive, albeit incomplete, knowledge of IgG and FcγR structure and immunobiology provides a war chest for the innovative development of new and highly potent mAbs through the manipulation of their interaction with the FcγR.\nTherapeutic mAb engineering strategies are directed by many factors including the biology of the target, the nature of the antigen, the desired MOA and possibly the anatomical location of the therapeutic effect,21 and thus to optimize potency for a desired response, the context of use is critical.\n\nThe nature of the IgG isotype\nDifferent capabilities for the recruitment and activation of the different immune effector functions are naturally found in the Fc regions of the human IgG subclasses. Thus, to achieve a desired MOA, the different IgG subclasses are important starting points for the selection and engineering of the optimal mAb Fc. IgG1 is, in many ways, a proinflammatory or “effector‐active” subclass, as it can initiate the complement cascade and is a “universal” FcγR ligand.74 Notwithstanding it is also a ligand for the inhibitory FcγRIIb, IgG1 elicits proinflammatory responses through all activating‐type FcγRs, including ADCC, ADCP and cytokine release.\nBecause of their more restricted FcγR‐binding profile, IgG2 and IgG4 have offered some choice in potentially avoiding FcR effector function without the need for Fc engineering. They have been used as the backbone for therapeutic mAbs either because recruitment of patients’ effector functions was unlikely to be necessary for the primary MOA of the mAb or is possibly detrimental to the desired therapeutic effect.75 However, the use of these unmodified “inert” subclasses is not without consequences and underscores the need for Fc engineering to modify FcγR interactions—See the “Attenuating and ablating FcγR related functions of IgG” section.\nThus, the choice of IgG subclass for therapeutic mAb engineering is an important first step for engineering of novel mAbs of improved specificity, potency and safety.\n\nFc engineering for enhanced anticancer therapeutics\nIgG1 is the predominant subclass used in the development of cytotoxic mAbs where induction of an activation‐type response, ADCC or phagocytosis, is considered desirable.45, 76, 77\nCytotoxic mAb cancer therapeutics can control disease progression by one or more mechanisms. Their MOAs include direct induction of apoptotic cell death of the cancer cell (anti‐CD20, anti‐CD52) or blocking receptor signaling (anti‐HER2, anti‐EGFR). They may also harness FcγR effector functions, including ADCC in the tumor microenvironment.78 The approved mAbs, rituximab (anti‐CD20), trastuzumab (anti‐HER2) and cetuximab (anti‐EGFR), are formatted on a human IgG1 backbone and all require activating‐type FcγR engagement for optimal therapeutic activity.79, 80 This presents an example where context of therapeutic use is critical for therapeutic mAb design. IgG1 antibodies bind both the activating FcγR (e.g. FcγRIIIa) and the inhibitory FcγRIIb. In some environments effector cells will coexpress FcγRIIb together with FcγRI, FcγRIIa and FcγIIIa, as may occur on a tumor‐infiltrating macrophage. Therapy with an IgG1 anti‐cancer cell mAb may then be compromised by the inhibitory action of FcγRIIb upon the ITAM signaling of the activating FcγR as both types of receptor would be coengaged on such an effector cell by the mAb bound to the target cell. This leads to reduced therapeutic mAb potency. Thus, the relative contributions of the activating (A) and inhibitory (I) FcγR to the response by an effector cell, the A‐to‐I ratio, may be an important determinant in clinical outcome of therapeutic mAb activity,76, 81, 82 that is, the higher the A‐to‐I ratio, the greater the proinflammatory response induced by the therapeutic mAb or conversely the lower the A‐to‐I ratio, the greater the inhibition or dampening of the proinflammatory response.\nThus, the challenge for the development of more potent FcγR effector mAbs is to overcome three major obstacles. First, improving activation potency by selectively enhancing interaction with activating‐type FcγR, particularly FcγRIIIa owing to its predominant role in ADCC‐mediated killing of tumor cells. Second, reducing binding interactions with the inhibitory FcγRIIb. These two approaches improve the FcγR A‐to‐I ratio of cytotoxic IgG1 mAbs. Third, overcoming the significant affinity difference in the interaction with the main FcγRIII allelic forms of FcγRIIIa‐V158 and FcγRIIIa‐F158 76, 83, 84 which appears to be an important source of patient variability in responses to therapeutic mAb treatment of cancer.\nAt the time of writing, some mAbs with improved potency are coming into clinical use. Their improved action has been achieved by modifying the N‐linked glycan or the amino acid sequence of the heavy‐chain Fc (Table 4).\nTable 4 Fc or hinge‐engineered monoclonal antibodies (mAbs) approved or in advanced clinical development.\nmAb name Target IgG backbone Fc modification Effect on mAb Therapy area Most advanced development stage\nAndecaliximab Matrix Metalloproteinase 9 (MMP9) IgG4 S228P Stabilize core hinge Oncology Phase III\nAnifrolumab Interferon alpha/beta receptor 1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2/F/G loop; plus ablate FcγR binding Immunology Phase III\nAtezolizumab PD‐L1 IgG1 Aglycosylated (N297A) Ablate FcγR binding Oncology Marketed\nBenralizumab Interleukin 5 IgG1 Afucosylated Selectively enhance FcγRIII interaction Respiratory dermatology; ear nose throat disorders; gastrointestinal; hematology; immunology; Marketed\nDurvalumab PD‐L1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2 F/G loop; plus ablate FcγR binding Oncology Marketed\nEvinacumab Angiopoietin‐related protein 3 IgG4 S228P Stabilize core hinge Metabolic disorders Phase III\nInebilizumab CD19 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIxekizumab Interleukin 17A IgG4 S228P Stabilize core hinge Dermatology; immunology; musculoskeletal disorders Marketed\nMargetuximab HER2 IgG1 F243L; L235V; R292P; Y300L; P396L Selectively enhance FcγRIII interaction Oncology Phase III\nMogamulizumab C–C chemokine receptor type 4 (CCR4) IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Marketed\nTafasitamab (MOR208 XmAb 5574) CD19 IgG1 S239D; I332E Selectively enhance FcγRIII interaction Oncology Phase III\nNivolumab PD‐1 IgG4 S228P Stabilize core hinge Infectious disease; oncology Marketed\nObinutuzumab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Immunology; oncology Marketed\nOcaratuzumab CD20 IgG1 P247I; A339Q Selectively enhance FcγRIII interaction Oncology Phase III\nPembrolizumab PD‐1 IgG4 S228P Stabilize core hinge Infection; oncology Marketed\nRoledumab Rhesus D IgG1 Afucosylated Selectively enhance FcγRIII interaction Hematological disorders Phase III\nSpesolimab (BI‐655130) IL‐36R IgG1 L234A; L235A Ablate FcγR binding Gastrointestinal; immunology Phase III\nTeplizumab CD3 IgG1 L234A; L235A Ablate FcγR binding Metabolic disorders Phase II\nTislelizumab PD‐1 IgG4 S228P; E233P; F234V; L235A; D265A; L309V; R409K Stabilize core hinge; mimic IgG2 lower hinge for restricted FcγR specificity; ablate FcγR binding; stabilize CH3 interaction Oncology Phase III\nToripalimab (JS 001) PD‐1 IgG4 S228P Stabilize core hinge Oncology Phase III\nUblituximab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIg, immunoglobulin.\nJohn Wiley \u0026 Sons, Ltd This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.\n\nModification of the Fc glycan\nThe typical complex N‐linked glycan attached to N297 of the heavy chain includes a core fucose.85 Antibodies that lack this fucose have approximately 50‐fold improved binding to FcγRIIIa and FcγRIIIb and importantly retain the weak, low‐affinity binding to the inhibitory FcγRIIb. Furthermore, this glycoengineering increased binding affinity of the modified IgG1 mAb for both FcγRIIIa V158 and F158 allelotypes.86, 87, 88 Afucosyl versions of the tumor targeting mAbs such as anti‐HER2, anti‐EGFR and anti‐CD20 had greater antitumor effects and increased survival,68, 88, 89 which is a reflection of the greatly increased, and selective, FcγRIII binding. Compared with their unmodified counterparts, the afucosyl mAbs showed dramatic improvement of FcγRIII‐related effector responses such as stronger NK cell‐mediated ADCC, or enhanced neutrophil‐mediated phagocytosis through FcγRIIIb and/or FcγRIIIa.23 However, certain neutrophil functions via FcγRIIa may be compromised.90, 91\nThere are six afucosylated antibodies in late‐stage clinical trials or approved for treatment (Table 4). Notable is obinutuzumab, an afucosyl anti‐CD20 mAb which nearly doubles progression‐free survival in chronic lymphocytic leukemia patients as compared with the fucose‐containing rituximab.68 This dramatic improvement in clinical utility reinforces the value of glycan engineering specifically and of Fc engineering generally in anticancer treatments.\n\nMutation of the Fc amino acids\nAlteration of the amino acids in the heavy‐chain Fc can alter IgG specificity and affinity for activating FcγRs. The anti‐CD19 antibody MOR208 (XmAb 5574) is currently in phase III trials for the treatment of chronic lymphocytic leukemia.92 It contains two mutations in its IgG1 Fc, S329D and I332E, which increases affinity to FcγRIIIa, particularly the “lower‐affinity” FcγRIIIa F158 allele. The mAb shows increased FcγRIII‐mediated ADCC and phagocytosis in vitro, and reduced lymphoma growth in mouse models.\nMargetuximab is an ADCC‐enhanced IgG1 Fc‐engineered variant of the approved anti‐HER2 mAb trastuzumab in phase III for HER2‐expressing cancers.66, 93 Alteration of five amino acids (L235V, F243L, R292P, Y300L and P396L) enhanced binding to FcγRIIIa which also had the additional effect of decreasing binding to the inhibitory FcγRIIb, and thereby increased its A‐to‐I FcγR ratio. This was apparent when compared with unmodified trastuzumab the margetuximab showed enhanced ADCC against HER2+ cells in vitro and demonstrated superior antitumor effects in an HER2‐expressing tumor model in mice.\nThe anti‐CD20 ocaratuzumab is an Fc‐engineered IgG1 mAb in late‐stage clinical trials for the treatment of a range of cancers, including non‐Hodgkin lymphoma and chronic lymphocytic leukemia.94 Two Fc mutations, P247I and A339Q, conferred about 20‐fold increase in binding to both major allelic variants of FcγRIIIa and elicited sixfold greater ADCC than unmodified IgG1.\nThus, the engineering of the Fc domain or glycan for improved FcγRIIIa binding is a powerful tool to create more potent and clinically effective anticancer mAbs.\n\nAttenuating and ablating FcγR‐related functions of IgG\nThere are circumstances where binding to FcγR is unnecessary or undesirable in the MOA of a therapeutic mAb. Unmodified IgG irrespective of its subclass or intended therapeutic effect has the potential to engage an FcγR which may lead to suboptimal therapeutic performance or to unexpected and catastrophic consequences.57, 59 Clearly reducing or eliminating FcγR interactions, when they are not required for therapeutic effect, may be desirable. Indeed, this had been addressed by the choice of IgG subclass or by modifying the Fc region. Indeed, most efforts in Fc engineering mAbs that have translated to an approved drug have focused on the reduction or elimination of FcγR interactions (Table 4).\nOne approach to minimize interactions with the activating FcγR has been the use of IgG4 or IgG2 backbones, which show a more restricted specificity for the activating FcγR and consequently have been traditionally, and simplistically, viewed as “inert” IgG subclasses. Notwithstanding the unexpected, and FcγR‐dependent, severe adverse reaction induced by the IgG4 TGN1412 mAb, the IgG4 or IgG2 backbones have been successfully used in many settings. Indeed, checkpoint inhibitors, such as mAbs targeting CTLA‐4 or the PD‐L1/PD‐1 interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an IgG4 backbone. Pembrolizumab, nivolumab and cemiplimab are all anti‐PD‐1 antibodies currently used for cancer therapy and have been formatted on an IgG4 backbone95, 96, 97 with a stabilized core hinge (S228P) to prevent half‐IgG4 exchange. Similarly, the checkpoint inhibitor tremelimumab is an anti‐CTLA‐4 antibody formatted on an IgG2 backbone to avoid potential ADCC killing of target cells.98\nHowever, the use of IgG2 and IgG4 as “inert” subclasses is problematic. Both bind to the activating receptors FcγRIIa‐H131 and FcγRI, respectively (Table 2), and initiate effector functions such as neutrophil activation and apoptosis induction.75, 99 Interestingly, in experimental systems, cross‐linking of anti‐PD‐1 IgG4‐based mAb by FcγRI switched its activity from blocking to activatory.10 Moreover, IgG4 binds to FcγRIIb, which may scaffold the therapeutic mAb. Although scaffolding may be beneficial in some contexts, for example, in immune agonism,43 it can be disastrous and unexpected in others as it was for the anti‐CD28 TGN1412 mAb.59 Thus, the IgG2 and IgG4 subclasses are not the optimum choice for “FcR‐inactive” mAbs, and so modifying the Fc is a more direct approach.\nThe complete removal of the heavy‐chain N‐linked glycan is well known to ablate all FcγR binding by dramatically altering the Fc conformation.36, 67, 101, 102 Atezolizumab, an IgG1 anti‐PD‐L1 checkpoint inhibitor mAb, utilizes this strategy and eliminates FcγR and also complement activation.13\nModification to the Fc amino acid sequence of the FcγR‐contact regions can also be used to reduce FcγR binding. A widely used modification of IgG1 is the substitution of leucine 234 and 235 in the lower hinge sequence (L234 L235 G236 G237) with alanine (L234A L235A). It is often referred to as the “LALA mutation” and effectively eliminates FcγR binding by more than 100 fold104, 105 and is used in teplizumab and spesolimab (Table 4).\nA separate strategy has used combinations of amino acid residues from the FcγR‐binding regions of IgG2 and IgG4, which have restricted FcγR specificity, together with other binding‐inactivating mutations. The lower hinge amino acids of the IgG1 mAbs durvalumab (anti‐PD‐L1) and anifrolumab (anti‐interferon‐α receptor; Table 4) were modified to mimic the lower hinge of IgG4 (L234F). They additionally incorporated L235E in the lower hinge and P331S in the F/G loop of the CH2 domain to ablate FcγR binding by disrupting two major FcγR contact sites7 and also coincidently decreasing C1q activation.16\nIgG4 mAbs have been similarly engineered to eliminate their interaction with FcγRI and FcγRIIb. The IgG4 anti‐PD‐1 antibody tislelizumab has had its FcγR contact residues in the lower hinge E233, F234, L235 substituted with the equivalent residues of IgG2 P, V, A (E233P, F234V, L235A) as well as the additional D265A mutation which disrupts a major FcγR contact in CH2. It also has substitutions in the core hinge S228P and the CH3 L309V and R409K to stabilize the H‐chain disulfides and CH3 interactions, respectively, thereby preventing half‐Ig exchange characteristic of natural IgG4. Collectively, these modifications create a stable IgG4 with no FcγR binding nor complement activation.17\nThus, Fc engineering is an effective way to remove FcγR effector functions and may be preferable to using unmodified IgG2 or IgG4 backbones that have a more restricted repertoire of FcγR interactions but which are still able to induce certain effector functions.\n\nImproving FcγRIIb interactions\nPreferential or specific Fc engagement of FcγRIIb over the activating FcγR offers several potential therapeutic advantages for new mAbs in distinct therapeutic settings.\n\nImproved recruitment of FcγRIIb immunoreceptor tyrosine inhibition motif‐dependent inhibitory function\nHarnessing the physiological inhibitory function of FcγRIIb by mAbs that target ITAM receptors has the potential to shut down ITAM‐dependent signaling pathways of major importance in antibody pathologies.32, 108 Such ITAM signaling receptors include the BCR complex on B cells which is active in systemic lupus erythematosus, the FcεRI on basophils and mast cell subsets in allergies or the activating‐type FcγR on a variety of innate leukocytes in antibody‐mediated tissue destruction. In such scenarios, the ITAM signaling receptor complex that is targeted by the therapeutic mAb must be co‐expressed on the cell surface with the inhibitory FcγRIIb. This permits coengagement with ITAM signaling receptor by the Fab of the mAb and inhibitory FcγRIIb by its Fc which is the critical requirement in the inhibitory MOA for such therapeutic mAbs (Figure 1).\nObexelimab (also known as XmAb5871; Table 4), currently in early clinical testing in inflammatory autoimmune disease, is an IgG1 mAb that targets CD19 of the BCR complex.19 It contains two Fc modifications, S267E and L328F (also known as “SELF” mutations), that selectively increased FcγRIIb binding by 400‐fold to about 1 nm, which results in powerful suppression of BCR signaling and the proliferation of primary B cells.19\nThe anti‐IgE mAb omalizumab is an IgG1 mAb approved for the treatment of allergic disorders.110, 111 A similar but Fc‐engineered IgG1 mAb XmAb7195, currently in early clinical testing, contains the affinity‐enhancing SELF modifications.112 Both mAbs sterically neutralize the interaction between IgE and its high‐affinity receptor FcεRI to prevent basophil and mast cell activation.113, 114 However, XmAb7195 exhibited more efficient removal (sweeping; discussed later) of circulating IgE and also inhibited B‐cell IgE production, presumably by binding to the IgE BCR on the B‐cell surface and coclustering with FcγRIIb via its affinity‐enhanced Fc domain.112 Thus, XmAb7195’s selective modulation of IgE production by IgE+ B cells in addition to its enhanced clearance of IgE may offer significantly improved therapeutic benefits in allergy therapy beyond simple IgE neutralization.112 The “SELF” mutations have also been used in agonistic mAbs (discussed later).\nOne cautionary note is that the arginine 131 (R131) of the IgG‐binding site in FcγRIIb is critical for the enhanced affinity binding of “SELF”‐mutated Fcs but it is also present in the activating‐type “high responder” FcγRIIa‐R131. Thus, antibodies modified with “SELF” have very‐high‐affinity binding to FcγRIIa‐R131 115 with a potentially increased risk of FcγRIIa‐dependent complications in patients expressing this allelic form, although, so far, none have been reported in clinical trials. However, an alternative set of six Fc mutations, termed “V12” (P238D, E233D, G237D, H268D, P271G and A330R), potently enhanced FcγRIIb binding without increasing FcγRIIa–R131 interaction.115\n\nEnhancing the sweeping of small immune complexes\nThe expression of FcγRIIb on LSEC and its action in the “sweeping” or removal of small immune complexes has opened up new possibilities for the application of FcγRIIb‐enhancing modifications.17 Antibodies or Fc fusion proteins, whose primary MOA is the neutralization of soluble molecules such as IgE or cytokines, are particularly attractive candidates for this approach. Proof‐of‐concept for this strategy has been demonstrated in experimental models.48 Indeed, this may be a significant component of the rapid disappearance of IgE from the circulation of patients treated with the anti‐IgE XmAb7195 containing the FcγRIIb enhancing “SELF” modifications, as described previously.\n\nImmune agonism through FcγR scaffolding\nAgonistic mAbs induce responses in target cells by stimulating signaling of their molecular target. Typically, this is to either enhance antitumor immunity by engaging costimulatory molecules on antigen‐presenting cells or T cells (i.e. CD40, 4‐1BB, OX40) or promote apoptosis by engaging death receptors on cancer cells (i.e. DR4, DR5, Fas).116\nThe role of FcγR in the action of these types of mAbs appears to be primarily as a scaffold. FcγRIIb is often the predominate receptor involved and the extent of its involvement is complex. In the case of CD40, the degree of FcγRIIb scaffolding potency is linked to the epitope location of the targeting mAb with greater potency seen for membrane proximal epitopes.43, 117 It is also noteworthy that depending on the epitope location, the scaffolding of anti‐CD40 mAbs may convert antagonist mAbs to agonistic.\nEngineering of the IgG1 Fc region for enhanced and/or specific binding to FcγRIIb can greatly improve agonistic function.72, 118, 119, 120 Such mutations induced significantly greater agonistic activity in an anti‐DR5 model through increased induction of apoptotic death and decreased tumor growth compared with unmodified IgG1.121 The “SELF” modifications that dramatically and selectively increase affinity for FcγRIIb have also been used to enhance immune agonism in an anti‐OX40 model.122\nThe incorporation of the \"V12\" Fc mutations into IgG1 specifically enhance FcγRIIb interaction 200‐fold, conferring the enhanced agonistic activity of an anti‐CD137 antibody and an anti‐OX40 mAb.115, 122"}

LitCovid-PubTator

LitCovid-PD-FMA-UBERON

LitCovid-PD-UBERON

LitCovid-PD-MONDO

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-sample-MedDRA

{"project":"LitCovid-sample-MedDRA","denotations":[{"id":"T17","span":{"begin":413,"end":418},"obj":"http://purl.bioontology.org/ontology/MEDDRA/10022891"},{"id":"T18","span":{"begin":11870,"end":11872},"obj":"http://purl.bioontology.org/ontology/MEDDRA/10022891"},{"id":"T19","span":{"begin":11874,"end":11888},"obj":"http://purl.bioontology.org/ontology/MEDDRA/10022891"}],"attributes":[{"id":"A17","pred":"meddra_id","subj":"T17","obj":"http://purl.bioontology.org/ontology/MEDDRA/10048064"},{"id":"A18","pred":"meddra_id","subj":"T18","obj":"http://purl.bioontology.org/ontology/MEDDRA/10021496"},{"id":"A19","pred":"meddra_id","subj":"T19","obj":"http://purl.bioontology.org/ontology/MEDDRA/10021496"}],"text":"IgG Subclasses: Structure and Properties\n\nThe molecular basis of IgG and FcγR interactions\nThe extracellular regions of the FcγR are structurally similar. Each low‐affinity FcγR has two ectodomains, whereas the high‐affinity FcγRI has a third domain but this is not directly involved in IgG binding.62\nThe interaction between the IgG subclasses and the FcγR is most comprehensively defined for human IgG1 by both X‐ray crystallographic7, 62, 63 and mutagenesis structure/function analyses.64, 65, 66 These studies defined key regions of the IgG sequence required for interaction with their FcγRs.\nCrystallographic analyses of the human IgG1‐Fc complexed with FcγRI, FcγRII or FcγRIII show that these interactions are similar in topology, and asymmetric in nature. The second extracellular domain of the FcγR inserts between the two heavy chains. Here it makes contacts with the lower hinge of both H chains and with residues of the adjacent BC loop of one CH2 domain and the FG loop of the other. The N‐linked glycan at asparagine 297 (N297) of the heavy chain is essential for the structural integrity of the IgG‐Fc by affecting the spacing and conformation of the CH2 domains. Indeed, its removal ablates FcγR binding.67 Of particular relevance to therapeutic mAb development is that the normal low‐affinity IgG interaction with FcγRIIIa is profoundly increased by the removal of the core fucose from the N297 Fc oligosaccharide.68\nNo crystallographic data are available for IgG2 or IgG4 Fc in complex with FcγR, but mutagenesis studies of the Fc and the FcγR revealed general similarity, but with critical differences, in the interaction of these subclasses with their cognate FcγR.\n\nUnique features of the IgG2 and IgG4 subclasses\nIn IgG1, the stable interaction of the two heavy chains results from the combined effects of stable covalent inter H‐chain disulfide bonds and strong noncovalent interaction of the two CH3 domains (Table 3). In stark contrast, in IgG2 and IgG4 the interaction of the CH3 domains of each H‐chain is weak. Residues 392, 397 and 409 (Eu numbering) profoundly affect the stability of these interactions. The difference at position 409 (R409 in IgG4 and K409 in IgG1) confers a 100‐fold decrease in stability of the interface between the two CH3 domains of IgG4 compared with that of IgG1 (Table 3).69\nFurthermore, the core hinge of IgG4 differs from IgG1 at position 228 (P228 in IgG1 and S228 in IgG4), resulting in unstable inter‐heavy‐chain disulfide bonds. This, together with the destabilizing amino acids in the CH3, confers the unique property of half‐antibody (Fab arm) exchange between different IgG4 antibodies,69 thereby creating monovalent, bispecific IgG4 antibodies in vivo.69, 70 The similarly unstable interactions between the CH3 domains in IgG2 are conferred by the interface residue M397; however, the stable inter‐H‐chain disulfide bonds of the core and upper hinge prevent half‐molecule exchange (Table 3).69\nIn addition, IgG2 uniquely has three disulfide bond conformers (Table 3). The distinct conformers are formed when (1) each light chain is attached to the Cys131 residue of CH1 in the heavy chain (IgG2‐A conformer), (2) both light chains attach to the upper hinge (IgG2‐B) or (3) one light chain is attached to the CH1 Cys131 and one to the upper hinge of the other heavy chain (IgG2‐AB).71 This results in distinct positioning of the Fabs relative to the Fc portions in the different conformers, which has implications for the interaction with antigen and the capacity of IgG2 to cross‐link target molecules in the absence of FcγR binding, for example, in an agonistic mAb setting.72\nIt should also be noted that IgG3 has not been used in therapeutic mAbs despite its unique biology. The main impediment to its use are its physicochemical properties such as susceptibility to proteolysis and propensity to aggregate that present challenges to industry‐scale production and stability but protein engineering is attempting to overcome these hurdles.73\n\nTherapeutic antibody design: improving mAb potency\nMany factors affecting FcγR‐dependent responses in vivo come into play during mAb therapy. The experience of three decades of clinical use of mAbs taken together with our extensive, albeit incomplete, knowledge of IgG and FcγR structure and immunobiology provides a war chest for the innovative development of new and highly potent mAbs through the manipulation of their interaction with the FcγR.\nTherapeutic mAb engineering strategies are directed by many factors including the biology of the target, the nature of the antigen, the desired MOA and possibly the anatomical location of the therapeutic effect,21 and thus to optimize potency for a desired response, the context of use is critical.\n\nThe nature of the IgG isotype\nDifferent capabilities for the recruitment and activation of the different immune effector functions are naturally found in the Fc regions of the human IgG subclasses. Thus, to achieve a desired MOA, the different IgG subclasses are important starting points for the selection and engineering of the optimal mAb Fc. IgG1 is, in many ways, a proinflammatory or “effector‐active” subclass, as it can initiate the complement cascade and is a “universal” FcγR ligand.74 Notwithstanding it is also a ligand for the inhibitory FcγRIIb, IgG1 elicits proinflammatory responses through all activating‐type FcγRs, including ADCC, ADCP and cytokine release.\nBecause of their more restricted FcγR‐binding profile, IgG2 and IgG4 have offered some choice in potentially avoiding FcR effector function without the need for Fc engineering. They have been used as the backbone for therapeutic mAbs either because recruitment of patients’ effector functions was unlikely to be necessary for the primary MOA of the mAb or is possibly detrimental to the desired therapeutic effect.75 However, the use of these unmodified “inert” subclasses is not without consequences and underscores the need for Fc engineering to modify FcγR interactions—See the “Attenuating and ablating FcγR related functions of IgG” section.\nThus, the choice of IgG subclass for therapeutic mAb engineering is an important first step for engineering of novel mAbs of improved specificity, potency and safety.\n\nFc engineering for enhanced anticancer therapeutics\nIgG1 is the predominant subclass used in the development of cytotoxic mAbs where induction of an activation‐type response, ADCC or phagocytosis, is considered desirable.45, 76, 77\nCytotoxic mAb cancer therapeutics can control disease progression by one or more mechanisms. Their MOAs include direct induction of apoptotic cell death of the cancer cell (anti‐CD20, anti‐CD52) or blocking receptor signaling (anti‐HER2, anti‐EGFR). They may also harness FcγR effector functions, including ADCC in the tumor microenvironment.78 The approved mAbs, rituximab (anti‐CD20), trastuzumab (anti‐HER2) and cetuximab (anti‐EGFR), are formatted on a human IgG1 backbone and all require activating‐type FcγR engagement for optimal therapeutic activity.79, 80 This presents an example where context of therapeutic use is critical for therapeutic mAb design. IgG1 antibodies bind both the activating FcγR (e.g. FcγRIIIa) and the inhibitory FcγRIIb. In some environments effector cells will coexpress FcγRIIb together with FcγRI, FcγRIIa and FcγIIIa, as may occur on a tumor‐infiltrating macrophage. Therapy with an IgG1 anti‐cancer cell mAb may then be compromised by the inhibitory action of FcγRIIb upon the ITAM signaling of the activating FcγR as both types of receptor would be coengaged on such an effector cell by the mAb bound to the target cell. This leads to reduced therapeutic mAb potency. Thus, the relative contributions of the activating (A) and inhibitory (I) FcγR to the response by an effector cell, the A‐to‐I ratio, may be an important determinant in clinical outcome of therapeutic mAb activity,76, 81, 82 that is, the higher the A‐to‐I ratio, the greater the proinflammatory response induced by the therapeutic mAb or conversely the lower the A‐to‐I ratio, the greater the inhibition or dampening of the proinflammatory response.\nThus, the challenge for the development of more potent FcγR effector mAbs is to overcome three major obstacles. First, improving activation potency by selectively enhancing interaction with activating‐type FcγR, particularly FcγRIIIa owing to its predominant role in ADCC‐mediated killing of tumor cells. Second, reducing binding interactions with the inhibitory FcγRIIb. These two approaches improve the FcγR A‐to‐I ratio of cytotoxic IgG1 mAbs. Third, overcoming the significant affinity difference in the interaction with the main FcγRIII allelic forms of FcγRIIIa‐V158 and FcγRIIIa‐F158 76, 83, 84 which appears to be an important source of patient variability in responses to therapeutic mAb treatment of cancer.\nAt the time of writing, some mAbs with improved potency are coming into clinical use. Their improved action has been achieved by modifying the N‐linked glycan or the amino acid sequence of the heavy‐chain Fc (Table 4).\nTable 4 Fc or hinge‐engineered monoclonal antibodies (mAbs) approved or in advanced clinical development.\nmAb name Target IgG backbone Fc modification Effect on mAb Therapy area Most advanced development stage\nAndecaliximab Matrix Metalloproteinase 9 (MMP9) IgG4 S228P Stabilize core hinge Oncology Phase III\nAnifrolumab Interferon alpha/beta receptor 1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2/F/G loop; plus ablate FcγR binding Immunology Phase III\nAtezolizumab PD‐L1 IgG1 Aglycosylated (N297A) Ablate FcγR binding Oncology Marketed\nBenralizumab Interleukin 5 IgG1 Afucosylated Selectively enhance FcγRIII interaction Respiratory dermatology; ear nose throat disorders; gastrointestinal; hematology; immunology; Marketed\nDurvalumab PD‐L1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2 F/G loop; plus ablate FcγR binding Oncology Marketed\nEvinacumab Angiopoietin‐related protein 3 IgG4 S228P Stabilize core hinge Metabolic disorders Phase III\nInebilizumab CD19 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIxekizumab Interleukin 17A IgG4 S228P Stabilize core hinge Dermatology; immunology; musculoskeletal disorders Marketed\nMargetuximab HER2 IgG1 F243L; L235V; R292P; Y300L; P396L Selectively enhance FcγRIII interaction Oncology Phase III\nMogamulizumab C–C chemokine receptor type 4 (CCR4) IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Marketed\nTafasitamab (MOR208 XmAb 5574) CD19 IgG1 S239D; I332E Selectively enhance FcγRIII interaction Oncology Phase III\nNivolumab PD‐1 IgG4 S228P Stabilize core hinge Infectious disease; oncology Marketed\nObinutuzumab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Immunology; oncology Marketed\nOcaratuzumab CD20 IgG1 P247I; A339Q Selectively enhance FcγRIII interaction Oncology Phase III\nPembrolizumab PD‐1 IgG4 S228P Stabilize core hinge Infection; oncology Marketed\nRoledumab Rhesus D IgG1 Afucosylated Selectively enhance FcγRIII interaction Hematological disorders Phase III\nSpesolimab (BI‐655130) IL‐36R IgG1 L234A; L235A Ablate FcγR binding Gastrointestinal; immunology Phase III\nTeplizumab CD3 IgG1 L234A; L235A Ablate FcγR binding Metabolic disorders Phase II\nTislelizumab PD‐1 IgG4 S228P; E233P; F234V; L235A; D265A; L309V; R409K Stabilize core hinge; mimic IgG2 lower hinge for restricted FcγR specificity; ablate FcγR binding; stabilize CH3 interaction Oncology Phase III\nToripalimab (JS 001) PD‐1 IgG4 S228P Stabilize core hinge Oncology Phase III\nUblituximab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIg, immunoglobulin.\nJohn Wiley \u0026 Sons, Ltd This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.\n\nModification of the Fc glycan\nThe typical complex N‐linked glycan attached to N297 of the heavy chain includes a core fucose.85 Antibodies that lack this fucose have approximately 50‐fold improved binding to FcγRIIIa and FcγRIIIb and importantly retain the weak, low‐affinity binding to the inhibitory FcγRIIb. Furthermore, this glycoengineering increased binding affinity of the modified IgG1 mAb for both FcγRIIIa V158 and F158 allelotypes.86, 87, 88 Afucosyl versions of the tumor targeting mAbs such as anti‐HER2, anti‐EGFR and anti‐CD20 had greater antitumor effects and increased survival,68, 88, 89 which is a reflection of the greatly increased, and selective, FcγRIII binding. Compared with their unmodified counterparts, the afucosyl mAbs showed dramatic improvement of FcγRIII‐related effector responses such as stronger NK cell‐mediated ADCC, or enhanced neutrophil‐mediated phagocytosis through FcγRIIIb and/or FcγRIIIa.23 However, certain neutrophil functions via FcγRIIa may be compromised.90, 91\nThere are six afucosylated antibodies in late‐stage clinical trials or approved for treatment (Table 4). Notable is obinutuzumab, an afucosyl anti‐CD20 mAb which nearly doubles progression‐free survival in chronic lymphocytic leukemia patients as compared with the fucose‐containing rituximab.68 This dramatic improvement in clinical utility reinforces the value of glycan engineering specifically and of Fc engineering generally in anticancer treatments.\n\nMutation of the Fc amino acids\nAlteration of the amino acids in the heavy‐chain Fc can alter IgG specificity and affinity for activating FcγRs. The anti‐CD19 antibody MOR208 (XmAb 5574) is currently in phase III trials for the treatment of chronic lymphocytic leukemia.92 It contains two mutations in its IgG1 Fc, S329D and I332E, which increases affinity to FcγRIIIa, particularly the “lower‐affinity” FcγRIIIa F158 allele. The mAb shows increased FcγRIII‐mediated ADCC and phagocytosis in vitro, and reduced lymphoma growth in mouse models.\nMargetuximab is an ADCC‐enhanced IgG1 Fc‐engineered variant of the approved anti‐HER2 mAb trastuzumab in phase III for HER2‐expressing cancers.66, 93 Alteration of five amino acids (L235V, F243L, R292P, Y300L and P396L) enhanced binding to FcγRIIIa which also had the additional effect of decreasing binding to the inhibitory FcγRIIb, and thereby increased its A‐to‐I FcγR ratio. This was apparent when compared with unmodified trastuzumab the margetuximab showed enhanced ADCC against HER2+ cells in vitro and demonstrated superior antitumor effects in an HER2‐expressing tumor model in mice.\nThe anti‐CD20 ocaratuzumab is an Fc‐engineered IgG1 mAb in late‐stage clinical trials for the treatment of a range of cancers, including non‐Hodgkin lymphoma and chronic lymphocytic leukemia.94 Two Fc mutations, P247I and A339Q, conferred about 20‐fold increase in binding to both major allelic variants of FcγRIIIa and elicited sixfold greater ADCC than unmodified IgG1.\nThus, the engineering of the Fc domain or glycan for improved FcγRIIIa binding is a powerful tool to create more potent and clinically effective anticancer mAbs.\n\nAttenuating and ablating FcγR‐related functions of IgG\nThere are circumstances where binding to FcγR is unnecessary or undesirable in the MOA of a therapeutic mAb. Unmodified IgG irrespective of its subclass or intended therapeutic effect has the potential to engage an FcγR which may lead to suboptimal therapeutic performance or to unexpected and catastrophic consequences.57, 59 Clearly reducing or eliminating FcγR interactions, when they are not required for therapeutic effect, may be desirable. Indeed, this had been addressed by the choice of IgG subclass or by modifying the Fc region. Indeed, most efforts in Fc engineering mAbs that have translated to an approved drug have focused on the reduction or elimination of FcγR interactions (Table 4).\nOne approach to minimize interactions with the activating FcγR has been the use of IgG4 or IgG2 backbones, which show a more restricted specificity for the activating FcγR and consequently have been traditionally, and simplistically, viewed as “inert” IgG subclasses. Notwithstanding the unexpected, and FcγR‐dependent, severe adverse reaction induced by the IgG4 TGN1412 mAb, the IgG4 or IgG2 backbones have been successfully used in many settings. Indeed, checkpoint inhibitors, such as mAbs targeting CTLA‐4 or the PD‐L1/PD‐1 interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an IgG4 backbone. Pembrolizumab, nivolumab and cemiplimab are all anti‐PD‐1 antibodies currently used for cancer therapy and have been formatted on an IgG4 backbone95, 96, 97 with a stabilized core hinge (S228P) to prevent half‐IgG4 exchange. Similarly, the checkpoint inhibitor tremelimumab is an anti‐CTLA‐4 antibody formatted on an IgG2 backbone to avoid potential ADCC killing of target cells.98\nHowever, the use of IgG2 and IgG4 as “inert” subclasses is problematic. Both bind to the activating receptors FcγRIIa‐H131 and FcγRI, respectively (Table 2), and initiate effector functions such as neutrophil activation and apoptosis induction.75, 99 Interestingly, in experimental systems, cross‐linking of anti‐PD‐1 IgG4‐based mAb by FcγRI switched its activity from blocking to activatory.10 Moreover, IgG4 binds to FcγRIIb, which may scaffold the therapeutic mAb. Although scaffolding may be beneficial in some contexts, for example, in immune agonism,43 it can be disastrous and unexpected in others as it was for the anti‐CD28 TGN1412 mAb.59 Thus, the IgG2 and IgG4 subclasses are not the optimum choice for “FcR‐inactive” mAbs, and so modifying the Fc is a more direct approach.\nThe complete removal of the heavy‐chain N‐linked glycan is well known to ablate all FcγR binding by dramatically altering the Fc conformation.36, 67, 101, 102 Atezolizumab, an IgG1 anti‐PD‐L1 checkpoint inhibitor mAb, utilizes this strategy and eliminates FcγR and also complement activation.13\nModification to the Fc amino acid sequence of the FcγR‐contact regions can also be used to reduce FcγR binding. A widely used modification of IgG1 is the substitution of leucine 234 and 235 in the lower hinge sequence (L234 L235 G236 G237) with alanine (L234A L235A). It is often referred to as the “LALA mutation” and effectively eliminates FcγR binding by more than 100 fold104, 105 and is used in teplizumab and spesolimab (Table 4).\nA separate strategy has used combinations of amino acid residues from the FcγR‐binding regions of IgG2 and IgG4, which have restricted FcγR specificity, together with other binding‐inactivating mutations. The lower hinge amino acids of the IgG1 mAbs durvalumab (anti‐PD‐L1) and anifrolumab (anti‐interferon‐α receptor; Table 4) were modified to mimic the lower hinge of IgG4 (L234F). They additionally incorporated L235E in the lower hinge and P331S in the F/G loop of the CH2 domain to ablate FcγR binding by disrupting two major FcγR contact sites7 and also coincidently decreasing C1q activation.16\nIgG4 mAbs have been similarly engineered to eliminate their interaction with FcγRI and FcγRIIb. The IgG4 anti‐PD‐1 antibody tislelizumab has had its FcγR contact residues in the lower hinge E233, F234, L235 substituted with the equivalent residues of IgG2 P, V, A (E233P, F234V, L235A) as well as the additional D265A mutation which disrupts a major FcγR contact in CH2. It also has substitutions in the core hinge S228P and the CH3 L309V and R409K to stabilize the H‐chain disulfides and CH3 interactions, respectively, thereby preventing half‐Ig exchange characteristic of natural IgG4. Collectively, these modifications create a stable IgG4 with no FcγR binding nor complement activation.17\nThus, Fc engineering is an effective way to remove FcγR effector functions and may be preferable to using unmodified IgG2 or IgG4 backbones that have a more restricted repertoire of FcγR interactions but which are still able to induce certain effector functions.\n\nImproving FcγRIIb interactions\nPreferential or specific Fc engagement of FcγRIIb over the activating FcγR offers several potential therapeutic advantages for new mAbs in distinct therapeutic settings.\n\nImproved recruitment of FcγRIIb immunoreceptor tyrosine inhibition motif‐dependent inhibitory function\nHarnessing the physiological inhibitory function of FcγRIIb by mAbs that target ITAM receptors has the potential to shut down ITAM‐dependent signaling pathways of major importance in antibody pathologies.32, 108 Such ITAM signaling receptors include the BCR complex on B cells which is active in systemic lupus erythematosus, the FcεRI on basophils and mast cell subsets in allergies or the activating‐type FcγR on a variety of innate leukocytes in antibody‐mediated tissue destruction. In such scenarios, the ITAM signaling receptor complex that is targeted by the therapeutic mAb must be co‐expressed on the cell surface with the inhibitory FcγRIIb. This permits coengagement with ITAM signaling receptor by the Fab of the mAb and inhibitory FcγRIIb by its Fc which is the critical requirement in the inhibitory MOA for such therapeutic mAbs (Figure 1).\nObexelimab (also known as XmAb5871; Table 4), currently in early clinical testing in inflammatory autoimmune disease, is an IgG1 mAb that targets CD19 of the BCR complex.19 It contains two Fc modifications, S267E and L328F (also known as “SELF” mutations), that selectively increased FcγRIIb binding by 400‐fold to about 1 nm, which results in powerful suppression of BCR signaling and the proliferation of primary B cells.19\nThe anti‐IgE mAb omalizumab is an IgG1 mAb approved for the treatment of allergic disorders.110, 111 A similar but Fc‐engineered IgG1 mAb XmAb7195, currently in early clinical testing, contains the affinity‐enhancing SELF modifications.112 Both mAbs sterically neutralize the interaction between IgE and its high‐affinity receptor FcεRI to prevent basophil and mast cell activation.113, 114 However, XmAb7195 exhibited more efficient removal (sweeping; discussed later) of circulating IgE and also inhibited B‐cell IgE production, presumably by binding to the IgE BCR on the B‐cell surface and coclustering with FcγRIIb via its affinity‐enhanced Fc domain.112 Thus, XmAb7195’s selective modulation of IgE production by IgE+ B cells in addition to its enhanced clearance of IgE may offer significantly improved therapeutic benefits in allergy therapy beyond simple IgE neutralization.112 The “SELF” mutations have also been used in agonistic mAbs (discussed later).\nOne cautionary note is that the arginine 131 (R131) of the IgG‐binding site in FcγRIIb is critical for the enhanced affinity binding of “SELF”‐mutated Fcs but it is also present in the activating‐type “high responder” FcγRIIa‐R131. Thus, antibodies modified with “SELF” have very‐high‐affinity binding to FcγRIIa‐R131 115 with a potentially increased risk of FcγRIIa‐dependent complications in patients expressing this allelic form, although, so far, none have been reported in clinical trials. However, an alternative set of six Fc mutations, termed “V12” (P238D, E233D, G237D, H268D, P271G and A330R), potently enhanced FcγRIIb binding without increasing FcγRIIa–R131 interaction.115\n\nEnhancing the sweeping of small immune complexes\nThe expression of FcγRIIb on LSEC and its action in the “sweeping” or removal of small immune complexes has opened up new possibilities for the application of FcγRIIb‐enhancing modifications.17 Antibodies or Fc fusion proteins, whose primary MOA is the neutralization of soluble molecules such as IgE or cytokines, are particularly attractive candidates for this approach. Proof‐of‐concept for this strategy has been demonstrated in experimental models.48 Indeed, this may be a significant component of the rapid disappearance of IgE from the circulation of patients treated with the anti‐IgE XmAb7195 containing the FcγRIIb enhancing “SELF” modifications, as described previously.\n\nImmune agonism through FcγR scaffolding\nAgonistic mAbs induce responses in target cells by stimulating signaling of their molecular target. Typically, this is to either enhance antitumor immunity by engaging costimulatory molecules on antigen‐presenting cells or T cells (i.e. CD40, 4‐1BB, OX40) or promote apoptosis by engaging death receptors on cancer cells (i.e. DR4, DR5, Fas).116\nThe role of FcγR in the action of these types of mAbs appears to be primarily as a scaffold. FcγRIIb is often the predominate receptor involved and the extent of its involvement is complex. In the case of CD40, the degree of FcγRIIb scaffolding potency is linked to the epitope location of the targeting mAb with greater potency seen for membrane proximal epitopes.43, 117 It is also noteworthy that depending on the epitope location, the scaffolding of anti‐CD40 mAbs may convert antagonist mAbs to agonistic.\nEngineering of the IgG1 Fc region for enhanced and/or specific binding to FcγRIIb can greatly improve agonistic function.72, 118, 119, 120 Such mutations induced significantly greater agonistic activity in an anti‐DR5 model through increased induction of apoptotic death and decreased tumor growth compared with unmodified IgG1.121 The “SELF” modifications that dramatically and selectively increase affinity for FcγRIIb have also been used to enhance immune agonism in an anti‐OX40 model.122\nThe incorporation of the \"V12\" Fc mutations into IgG1 specifically enhance FcγRIIb interaction 200‐fold, conferring the enhanced agonistic activity of an anti‐CD137 antibody and an anti‐OX40 mAb.115, 122"}

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{"project":"LitCovid-sample-PD-NCBITaxon","denotations":[{"id":"T39","span":{"begin":394,"end":399},"obj":"Species"},{"id":"T40","span":{"begin":630,"end":635},"obj":"Species"},{"id":"T41","span":{"begin":4937,"end":4942},"obj":"Species"},{"id":"T42","span":{"begin":6942,"end":6947},"obj":"Species"},{"id":"T43","span":{"begin":11995,"end":12003},"obj":"Species"},{"id":"T44","span":{"begin":14220,"end":14225},"obj":"Species"},{"id":"T46","span":{"begin":14823,"end":14827},"obj":"Species"}],"attributes":[{"id":"A39","pred":"ncbi_taxonomy_id","subj":"T39","obj":"NCBItxid:9606"},{"id":"A40","pred":"ncbi_taxonomy_id","subj":"T40","obj":"NCBItxid:9606"},{"id":"A41","pred":"ncbi_taxonomy_id","subj":"T41","obj":"NCBItxid:9606"},{"id":"A42","pred":"ncbi_taxonomy_id","subj":"T42","obj":"NCBItxid:9606"},{"id":"A43","pred":"ncbi_taxonomy_id","subj":"T43","obj":"NCBItxid:2697049"},{"id":"A44","pred":"ncbi_taxonomy_id","subj":"T44","obj":"NCBItxid:10090"},{"id":"A45","pred":"ncbi_taxonomy_id","subj":"T44","obj":"NCBItxid:10088"},{"id":"A46","pred":"ncbi_taxonomy_id","subj":"T46","obj":"NCBItxid:10095"},{"id":"A47","pred":"ncbi_taxonomy_id","subj":"T46","obj":"NCBItxid:10088"}],"namespaces":[{"prefix":"NCBItxid","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"text":"IgG Subclasses: Structure and Properties\n\nThe molecular basis of IgG and FcγR interactions\nThe extracellular regions of the FcγR are structurally similar. Each low‐affinity FcγR has two ectodomains, whereas the high‐affinity FcγRI has a third domain but this is not directly involved in IgG binding.62\nThe interaction between the IgG subclasses and the FcγR is most comprehensively defined for human IgG1 by both X‐ray crystallographic7, 62, 63 and mutagenesis structure/function analyses.64, 65, 66 These studies defined key regions of the IgG sequence required for interaction with their FcγRs.\nCrystallographic analyses of the human IgG1‐Fc complexed with FcγRI, FcγRII or FcγRIII show that these interactions are similar in topology, and asymmetric in nature. The second extracellular domain of the FcγR inserts between the two heavy chains. Here it makes contacts with the lower hinge of both H chains and with residues of the adjacent BC loop of one CH2 domain and the FG loop of the other. The N‐linked glycan at asparagine 297 (N297) of the heavy chain is essential for the structural integrity of the IgG‐Fc by affecting the spacing and conformation of the CH2 domains. Indeed, its removal ablates FcγR binding.67 Of particular relevance to therapeutic mAb development is that the normal low‐affinity IgG interaction with FcγRIIIa is profoundly increased by the removal of the core fucose from the N297 Fc oligosaccharide.68\nNo crystallographic data are available for IgG2 or IgG4 Fc in complex with FcγR, but mutagenesis studies of the Fc and the FcγR revealed general similarity, but with critical differences, in the interaction of these subclasses with their cognate FcγR.\n\nUnique features of the IgG2 and IgG4 subclasses\nIn IgG1, the stable interaction of the two heavy chains results from the combined effects of stable covalent inter H‐chain disulfide bonds and strong noncovalent interaction of the two CH3 domains (Table 3). In stark contrast, in IgG2 and IgG4 the interaction of the CH3 domains of each H‐chain is weak. Residues 392, 397 and 409 (Eu numbering) profoundly affect the stability of these interactions. The difference at position 409 (R409 in IgG4 and K409 in IgG1) confers a 100‐fold decrease in stability of the interface between the two CH3 domains of IgG4 compared with that of IgG1 (Table 3).69\nFurthermore, the core hinge of IgG4 differs from IgG1 at position 228 (P228 in IgG1 and S228 in IgG4), resulting in unstable inter‐heavy‐chain disulfide bonds. This, together with the destabilizing amino acids in the CH3, confers the unique property of half‐antibody (Fab arm) exchange between different IgG4 antibodies,69 thereby creating monovalent, bispecific IgG4 antibodies in vivo.69, 70 The similarly unstable interactions between the CH3 domains in IgG2 are conferred by the interface residue M397; however, the stable inter‐H‐chain disulfide bonds of the core and upper hinge prevent half‐molecule exchange (Table 3).69\nIn addition, IgG2 uniquely has three disulfide bond conformers (Table 3). The distinct conformers are formed when (1) each light chain is attached to the Cys131 residue of CH1 in the heavy chain (IgG2‐A conformer), (2) both light chains attach to the upper hinge (IgG2‐B) or (3) one light chain is attached to the CH1 Cys131 and one to the upper hinge of the other heavy chain (IgG2‐AB).71 This results in distinct positioning of the Fabs relative to the Fc portions in the different conformers, which has implications for the interaction with antigen and the capacity of IgG2 to cross‐link target molecules in the absence of FcγR binding, for example, in an agonistic mAb setting.72\nIt should also be noted that IgG3 has not been used in therapeutic mAbs despite its unique biology. The main impediment to its use are its physicochemical properties such as susceptibility to proteolysis and propensity to aggregate that present challenges to industry‐scale production and stability but protein engineering is attempting to overcome these hurdles.73\n\nTherapeutic antibody design: improving mAb potency\nMany factors affecting FcγR‐dependent responses in vivo come into play during mAb therapy. The experience of three decades of clinical use of mAbs taken together with our extensive, albeit incomplete, knowledge of IgG and FcγR structure and immunobiology provides a war chest for the innovative development of new and highly potent mAbs through the manipulation of their interaction with the FcγR.\nTherapeutic mAb engineering strategies are directed by many factors including the biology of the target, the nature of the antigen, the desired MOA and possibly the anatomical location of the therapeutic effect,21 and thus to optimize potency for a desired response, the context of use is critical.\n\nThe nature of the IgG isotype\nDifferent capabilities for the recruitment and activation of the different immune effector functions are naturally found in the Fc regions of the human IgG subclasses. Thus, to achieve a desired MOA, the different IgG subclasses are important starting points for the selection and engineering of the optimal mAb Fc. IgG1 is, in many ways, a proinflammatory or “effector‐active” subclass, as it can initiate the complement cascade and is a “universal” FcγR ligand.74 Notwithstanding it is also a ligand for the inhibitory FcγRIIb, IgG1 elicits proinflammatory responses through all activating‐type FcγRs, including ADCC, ADCP and cytokine release.\nBecause of their more restricted FcγR‐binding profile, IgG2 and IgG4 have offered some choice in potentially avoiding FcR effector function without the need for Fc engineering. They have been used as the backbone for therapeutic mAbs either because recruitment of patients’ effector functions was unlikely to be necessary for the primary MOA of the mAb or is possibly detrimental to the desired therapeutic effect.75 However, the use of these unmodified “inert” subclasses is not without consequences and underscores the need for Fc engineering to modify FcγR interactions—See the “Attenuating and ablating FcγR related functions of IgG” section.\nThus, the choice of IgG subclass for therapeutic mAb engineering is an important first step for engineering of novel mAbs of improved specificity, potency and safety.\n\nFc engineering for enhanced anticancer therapeutics\nIgG1 is the predominant subclass used in the development of cytotoxic mAbs where induction of an activation‐type response, ADCC or phagocytosis, is considered desirable.45, 76, 77\nCytotoxic mAb cancer therapeutics can control disease progression by one or more mechanisms. Their MOAs include direct induction of apoptotic cell death of the cancer cell (anti‐CD20, anti‐CD52) or blocking receptor signaling (anti‐HER2, anti‐EGFR). They may also harness FcγR effector functions, including ADCC in the tumor microenvironment.78 The approved mAbs, rituximab (anti‐CD20), trastuzumab (anti‐HER2) and cetuximab (anti‐EGFR), are formatted on a human IgG1 backbone and all require activating‐type FcγR engagement for optimal therapeutic activity.79, 80 This presents an example where context of therapeutic use is critical for therapeutic mAb design. IgG1 antibodies bind both the activating FcγR (e.g. FcγRIIIa) and the inhibitory FcγRIIb. In some environments effector cells will coexpress FcγRIIb together with FcγRI, FcγRIIa and FcγIIIa, as may occur on a tumor‐infiltrating macrophage. Therapy with an IgG1 anti‐cancer cell mAb may then be compromised by the inhibitory action of FcγRIIb upon the ITAM signaling of the activating FcγR as both types of receptor would be coengaged on such an effector cell by the mAb bound to the target cell. This leads to reduced therapeutic mAb potency. Thus, the relative contributions of the activating (A) and inhibitory (I) FcγR to the response by an effector cell, the A‐to‐I ratio, may be an important determinant in clinical outcome of therapeutic mAb activity,76, 81, 82 that is, the higher the A‐to‐I ratio, the greater the proinflammatory response induced by the therapeutic mAb or conversely the lower the A‐to‐I ratio, the greater the inhibition or dampening of the proinflammatory response.\nThus, the challenge for the development of more potent FcγR effector mAbs is to overcome three major obstacles. First, improving activation potency by selectively enhancing interaction with activating‐type FcγR, particularly FcγRIIIa owing to its predominant role in ADCC‐mediated killing of tumor cells. Second, reducing binding interactions with the inhibitory FcγRIIb. These two approaches improve the FcγR A‐to‐I ratio of cytotoxic IgG1 mAbs. Third, overcoming the significant affinity difference in the interaction with the main FcγRIII allelic forms of FcγRIIIa‐V158 and FcγRIIIa‐F158 76, 83, 84 which appears to be an important source of patient variability in responses to therapeutic mAb treatment of cancer.\nAt the time of writing, some mAbs with improved potency are coming into clinical use. Their improved action has been achieved by modifying the N‐linked glycan or the amino acid sequence of the heavy‐chain Fc (Table 4).\nTable 4 Fc or hinge‐engineered monoclonal antibodies (mAbs) approved or in advanced clinical development.\nmAb name Target IgG backbone Fc modification Effect on mAb Therapy area Most advanced development stage\nAndecaliximab Matrix Metalloproteinase 9 (MMP9) IgG4 S228P Stabilize core hinge Oncology Phase III\nAnifrolumab Interferon alpha/beta receptor 1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2/F/G loop; plus ablate FcγR binding Immunology Phase III\nAtezolizumab PD‐L1 IgG1 Aglycosylated (N297A) Ablate FcγR binding Oncology Marketed\nBenralizumab Interleukin 5 IgG1 Afucosylated Selectively enhance FcγRIII interaction Respiratory dermatology; ear nose throat disorders; gastrointestinal; hematology; immunology; Marketed\nDurvalumab PD‐L1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2 F/G loop; plus ablate FcγR binding Oncology Marketed\nEvinacumab Angiopoietin‐related protein 3 IgG4 S228P Stabilize core hinge Metabolic disorders Phase III\nInebilizumab CD19 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIxekizumab Interleukin 17A IgG4 S228P Stabilize core hinge Dermatology; immunology; musculoskeletal disorders Marketed\nMargetuximab HER2 IgG1 F243L; L235V; R292P; Y300L; P396L Selectively enhance FcγRIII interaction Oncology Phase III\nMogamulizumab C–C chemokine receptor type 4 (CCR4) IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Marketed\nTafasitamab (MOR208 XmAb 5574) CD19 IgG1 S239D; I332E Selectively enhance FcγRIII interaction Oncology Phase III\nNivolumab PD‐1 IgG4 S228P Stabilize core hinge Infectious disease; oncology Marketed\nObinutuzumab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Immunology; oncology Marketed\nOcaratuzumab CD20 IgG1 P247I; A339Q Selectively enhance FcγRIII interaction Oncology Phase III\nPembrolizumab PD‐1 IgG4 S228P Stabilize core hinge Infection; oncology Marketed\nRoledumab Rhesus D IgG1 Afucosylated Selectively enhance FcγRIII interaction Hematological disorders Phase III\nSpesolimab (BI‐655130) IL‐36R IgG1 L234A; L235A Ablate FcγR binding Gastrointestinal; immunology Phase III\nTeplizumab CD3 IgG1 L234A; L235A Ablate FcγR binding Metabolic disorders Phase II\nTislelizumab PD‐1 IgG4 S228P; E233P; F234V; L235A; D265A; L309V; R409K Stabilize core hinge; mimic IgG2 lower hinge for restricted FcγR specificity; ablate FcγR binding; stabilize CH3 interaction Oncology Phase III\nToripalimab (JS 001) PD‐1 IgG4 S228P Stabilize core hinge Oncology Phase III\nUblituximab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIg, immunoglobulin.\nJohn Wiley \u0026 Sons, Ltd This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.\n\nModification of the Fc glycan\nThe typical complex N‐linked glycan attached to N297 of the heavy chain includes a core fucose.85 Antibodies that lack this fucose have approximately 50‐fold improved binding to FcγRIIIa and FcγRIIIb and importantly retain the weak, low‐affinity binding to the inhibitory FcγRIIb. Furthermore, this glycoengineering increased binding affinity of the modified IgG1 mAb for both FcγRIIIa V158 and F158 allelotypes.86, 87, 88 Afucosyl versions of the tumor targeting mAbs such as anti‐HER2, anti‐EGFR and anti‐CD20 had greater antitumor effects and increased survival,68, 88, 89 which is a reflection of the greatly increased, and selective, FcγRIII binding. Compared with their unmodified counterparts, the afucosyl mAbs showed dramatic improvement of FcγRIII‐related effector responses such as stronger NK cell‐mediated ADCC, or enhanced neutrophil‐mediated phagocytosis through FcγRIIIb and/or FcγRIIIa.23 However, certain neutrophil functions via FcγRIIa may be compromised.90, 91\nThere are six afucosylated antibodies in late‐stage clinical trials or approved for treatment (Table 4). Notable is obinutuzumab, an afucosyl anti‐CD20 mAb which nearly doubles progression‐free survival in chronic lymphocytic leukemia patients as compared with the fucose‐containing rituximab.68 This dramatic improvement in clinical utility reinforces the value of glycan engineering specifically and of Fc engineering generally in anticancer treatments.\n\nMutation of the Fc amino acids\nAlteration of the amino acids in the heavy‐chain Fc can alter IgG specificity and affinity for activating FcγRs. The anti‐CD19 antibody MOR208 (XmAb 5574) is currently in phase III trials for the treatment of chronic lymphocytic leukemia.92 It contains two mutations in its IgG1 Fc, S329D and I332E, which increases affinity to FcγRIIIa, particularly the “lower‐affinity” FcγRIIIa F158 allele. The mAb shows increased FcγRIII‐mediated ADCC and phagocytosis in vitro, and reduced lymphoma growth in mouse models.\nMargetuximab is an ADCC‐enhanced IgG1 Fc‐engineered variant of the approved anti‐HER2 mAb trastuzumab in phase III for HER2‐expressing cancers.66, 93 Alteration of five amino acids (L235V, F243L, R292P, Y300L and P396L) enhanced binding to FcγRIIIa which also had the additional effect of decreasing binding to the inhibitory FcγRIIb, and thereby increased its A‐to‐I FcγR ratio. This was apparent when compared with unmodified trastuzumab the margetuximab showed enhanced ADCC against HER2+ cells in vitro and demonstrated superior antitumor effects in an HER2‐expressing tumor model in mice.\nThe anti‐CD20 ocaratuzumab is an Fc‐engineered IgG1 mAb in late‐stage clinical trials for the treatment of a range of cancers, including non‐Hodgkin lymphoma and chronic lymphocytic leukemia.94 Two Fc mutations, P247I and A339Q, conferred about 20‐fold increase in binding to both major allelic variants of FcγRIIIa and elicited sixfold greater ADCC than unmodified IgG1.\nThus, the engineering of the Fc domain or glycan for improved FcγRIIIa binding is a powerful tool to create more potent and clinically effective anticancer mAbs.\n\nAttenuating and ablating FcγR‐related functions of IgG\nThere are circumstances where binding to FcγR is unnecessary or undesirable in the MOA of a therapeutic mAb. Unmodified IgG irrespective of its subclass or intended therapeutic effect has the potential to engage an FcγR which may lead to suboptimal therapeutic performance or to unexpected and catastrophic consequences.57, 59 Clearly reducing or eliminating FcγR interactions, when they are not required for therapeutic effect, may be desirable. Indeed, this had been addressed by the choice of IgG subclass or by modifying the Fc region. Indeed, most efforts in Fc engineering mAbs that have translated to an approved drug have focused on the reduction or elimination of FcγR interactions (Table 4).\nOne approach to minimize interactions with the activating FcγR has been the use of IgG4 or IgG2 backbones, which show a more restricted specificity for the activating FcγR and consequently have been traditionally, and simplistically, viewed as “inert” IgG subclasses. Notwithstanding the unexpected, and FcγR‐dependent, severe adverse reaction induced by the IgG4 TGN1412 mAb, the IgG4 or IgG2 backbones have been successfully used in many settings. Indeed, checkpoint inhibitors, such as mAbs targeting CTLA‐4 or the PD‐L1/PD‐1 interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an IgG4 backbone. Pembrolizumab, nivolumab and cemiplimab are all anti‐PD‐1 antibodies currently used for cancer therapy and have been formatted on an IgG4 backbone95, 96, 97 with a stabilized core hinge (S228P) to prevent half‐IgG4 exchange. Similarly, the checkpoint inhibitor tremelimumab is an anti‐CTLA‐4 antibody formatted on an IgG2 backbone to avoid potential ADCC killing of target cells.98\nHowever, the use of IgG2 and IgG4 as “inert” subclasses is problematic. Both bind to the activating receptors FcγRIIa‐H131 and FcγRI, respectively (Table 2), and initiate effector functions such as neutrophil activation and apoptosis induction.75, 99 Interestingly, in experimental systems, cross‐linking of anti‐PD‐1 IgG4‐based mAb by FcγRI switched its activity from blocking to activatory.10 Moreover, IgG4 binds to FcγRIIb, which may scaffold the therapeutic mAb. Although scaffolding may be beneficial in some contexts, for example, in immune agonism,43 it can be disastrous and unexpected in others as it was for the anti‐CD28 TGN1412 mAb.59 Thus, the IgG2 and IgG4 subclasses are not the optimum choice for “FcR‐inactive” mAbs, and so modifying the Fc is a more direct approach.\nThe complete removal of the heavy‐chain N‐linked glycan is well known to ablate all FcγR binding by dramatically altering the Fc conformation.36, 67, 101, 102 Atezolizumab, an IgG1 anti‐PD‐L1 checkpoint inhibitor mAb, utilizes this strategy and eliminates FcγR and also complement activation.13\nModification to the Fc amino acid sequence of the FcγR‐contact regions can also be used to reduce FcγR binding. A widely used modification of IgG1 is the substitution of leucine 234 and 235 in the lower hinge sequence (L234 L235 G236 G237) with alanine (L234A L235A). It is often referred to as the “LALA mutation” and effectively eliminates FcγR binding by more than 100 fold104, 105 and is used in teplizumab and spesolimab (Table 4).\nA separate strategy has used combinations of amino acid residues from the FcγR‐binding regions of IgG2 and IgG4, which have restricted FcγR specificity, together with other binding‐inactivating mutations. The lower hinge amino acids of the IgG1 mAbs durvalumab (anti‐PD‐L1) and anifrolumab (anti‐interferon‐α receptor; Table 4) were modified to mimic the lower hinge of IgG4 (L234F). They additionally incorporated L235E in the lower hinge and P331S in the F/G loop of the CH2 domain to ablate FcγR binding by disrupting two major FcγR contact sites7 and also coincidently decreasing C1q activation.16\nIgG4 mAbs have been similarly engineered to eliminate their interaction with FcγRI and FcγRIIb. The IgG4 anti‐PD‐1 antibody tislelizumab has had its FcγR contact residues in the lower hinge E233, F234, L235 substituted with the equivalent residues of IgG2 P, V, A (E233P, F234V, L235A) as well as the additional D265A mutation which disrupts a major FcγR contact in CH2. It also has substitutions in the core hinge S228P and the CH3 L309V and R409K to stabilize the H‐chain disulfides and CH3 interactions, respectively, thereby preventing half‐Ig exchange characteristic of natural IgG4. Collectively, these modifications create a stable IgG4 with no FcγR binding nor complement activation.17\nThus, Fc engineering is an effective way to remove FcγR effector functions and may be preferable to using unmodified IgG2 or IgG4 backbones that have a more restricted repertoire of FcγR interactions but which are still able to induce certain effector functions.\n\nImproving FcγRIIb interactions\nPreferential or specific Fc engagement of FcγRIIb over the activating FcγR offers several potential therapeutic advantages for new mAbs in distinct therapeutic settings.\n\nImproved recruitment of FcγRIIb immunoreceptor tyrosine inhibition motif‐dependent inhibitory function\nHarnessing the physiological inhibitory function of FcγRIIb by mAbs that target ITAM receptors has the potential to shut down ITAM‐dependent signaling pathways of major importance in antibody pathologies.32, 108 Such ITAM signaling receptors include the BCR complex on B cells which is active in systemic lupus erythematosus, the FcεRI on basophils and mast cell subsets in allergies or the activating‐type FcγR on a variety of innate leukocytes in antibody‐mediated tissue destruction. In such scenarios, the ITAM signaling receptor complex that is targeted by the therapeutic mAb must be co‐expressed on the cell surface with the inhibitory FcγRIIb. This permits coengagement with ITAM signaling receptor by the Fab of the mAb and inhibitory FcγRIIb by its Fc which is the critical requirement in the inhibitory MOA for such therapeutic mAbs (Figure 1).\nObexelimab (also known as XmAb5871; Table 4), currently in early clinical testing in inflammatory autoimmune disease, is an IgG1 mAb that targets CD19 of the BCR complex.19 It contains two Fc modifications, S267E and L328F (also known as “SELF” mutations), that selectively increased FcγRIIb binding by 400‐fold to about 1 nm, which results in powerful suppression of BCR signaling and the proliferation of primary B cells.19\nThe anti‐IgE mAb omalizumab is an IgG1 mAb approved for the treatment of allergic disorders.110, 111 A similar but Fc‐engineered IgG1 mAb XmAb7195, currently in early clinical testing, contains the affinity‐enhancing SELF modifications.112 Both mAbs sterically neutralize the interaction between IgE and its high‐affinity receptor FcεRI to prevent basophil and mast cell activation.113, 114 However, XmAb7195 exhibited more efficient removal (sweeping; discussed later) of circulating IgE and also inhibited B‐cell IgE production, presumably by binding to the IgE BCR on the B‐cell surface and coclustering with FcγRIIb via its affinity‐enhanced Fc domain.112 Thus, XmAb7195’s selective modulation of IgE production by IgE+ B cells in addition to its enhanced clearance of IgE may offer significantly improved therapeutic benefits in allergy therapy beyond simple IgE neutralization.112 The “SELF” mutations have also been used in agonistic mAbs (discussed later).\nOne cautionary note is that the arginine 131 (R131) of the IgG‐binding site in FcγRIIb is critical for the enhanced affinity binding of “SELF”‐mutated Fcs but it is also present in the activating‐type “high responder” FcγRIIa‐R131. Thus, antibodies modified with “SELF” have very‐high‐affinity binding to FcγRIIa‐R131 115 with a potentially increased risk of FcγRIIa‐dependent complications in patients expressing this allelic form, although, so far, none have been reported in clinical trials. However, an alternative set of six Fc mutations, termed “V12” (P238D, E233D, G237D, H268D, P271G and A330R), potently enhanced FcγRIIb binding without increasing FcγRIIa–R131 interaction.115\n\nEnhancing the sweeping of small immune complexes\nThe expression of FcγRIIb on LSEC and its action in the “sweeping” or removal of small immune complexes has opened up new possibilities for the application of FcγRIIb‐enhancing modifications.17 Antibodies or Fc fusion proteins, whose primary MOA is the neutralization of soluble molecules such as IgE or cytokines, are particularly attractive candidates for this approach. Proof‐of‐concept for this strategy has been demonstrated in experimental models.48 Indeed, this may be a significant component of the rapid disappearance of IgE from the circulation of patients treated with the anti‐IgE XmAb7195 containing the FcγRIIb enhancing “SELF” modifications, as described previously.\n\nImmune agonism through FcγR scaffolding\nAgonistic mAbs induce responses in target cells by stimulating signaling of their molecular target. Typically, this is to either enhance antitumor immunity by engaging costimulatory molecules on antigen‐presenting cells or T cells (i.e. CD40, 4‐1BB, OX40) or promote apoptosis by engaging death receptors on cancer cells (i.e. DR4, DR5, Fas).116\nThe role of FcγR in the action of these types of mAbs appears to be primarily as a scaffold. FcγRIIb is often the predominate receptor involved and the extent of its involvement is complex. In the case of CD40, the degree of FcγRIIb scaffolding potency is linked to the epitope location of the targeting mAb with greater potency seen for membrane proximal epitopes.43, 117 It is also noteworthy that depending on the epitope location, the scaffolding of anti‐CD40 mAbs may convert antagonist mAbs to agonistic.\nEngineering of the IgG1 Fc region for enhanced and/or specific binding to FcγRIIb can greatly improve agonistic function.72, 118, 119, 120 Such mutations induced significantly greater agonistic activity in an anti‐DR5 model through increased induction of apoptotic death and decreased tumor growth compared with unmodified IgG1.121 The “SELF” modifications that dramatically and selectively increase affinity for FcγRIIb have also been used to enhance immune agonism in an anti‐OX40 model.122\nThe incorporation of the \"V12\" Fc mutations into IgG1 specifically enhance FcγRIIb interaction 200‐fold, conferring the enhanced agonistic activity of an anti‐CD137 antibody and an anti‐OX40 mAb.115, 122"}

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{"project":"LitCovid-sample-PD-UBERON","denotations":[{"id":"T19","span":{"begin":2604,"end":2607},"obj":"Body_part"},{"id":"T20","span":{"begin":3913,"end":3918},"obj":"Body_part"},{"id":"T21","span":{"begin":4333,"end":4338},"obj":"Body_part"},{"id":"T22","span":{"begin":9767,"end":9770},"obj":"Body_part"},{"id":"T23","span":{"begin":9771,"end":9775},"obj":"Body_part"},{"id":"T24","span":{"begin":9776,"end":9782},"obj":"Body_part"},{"id":"T25","span":{"begin":10167,"end":10189},"obj":"Body_part"},{"id":"T26","span":{"begin":10572,"end":10594},"obj":"Body_part"},{"id":"T27","span":{"begin":11826,"end":11848},"obj":"Body_part"},{"id":"T28","span":{"begin":21038,"end":21044},"obj":"Body_part"}],"attributes":[{"id":"A22","pred":"uberon_id","subj":"T22","obj":"http://purl.obolibrary.org/obo/UBERON_0001690"},{"id":"A20","pred":"uberon_id","subj":"T20","obj":"http://purl.obolibrary.org/obo/UBERON_0002542"},{"id":"A27","pred":"uberon_id","subj":"T27","obj":"http://purl.obolibrary.org/obo/UBERON_0001017"},{"id":"A26","pred":"uberon_id","subj":"T26","obj":"http://purl.obolibrary.org/obo/UBERON_0001017"},{"id":"A23","pred":"uberon_id","subj":"T23","obj":"http://purl.obolibrary.org/obo/UBERON_0000004"},{"id":"A28","pred":"uberon_id","subj":"T28","obj":"http://purl.obolibrary.org/obo/UBERON_0000479"},{"id":"A25","pred":"uberon_id","subj":"T25","obj":"http://purl.obolibrary.org/obo/UBERON_0001017"},{"id":"A19","pred":"uberon_id","subj":"T19","obj":"http://purl.obolibrary.org/obo/UBERON_0001460"},{"id":"A24","pred":"uberon_id","subj":"T24","obj":"http://purl.obolibrary.org/obo/UBERON_0000341"},{"id":"A21","pred":"uberon_id","subj":"T21","obj":"http://purl.obolibrary.org/obo/UBERON_0001443"}],"text":"IgG Subclasses: Structure and Properties\n\nThe molecular basis of IgG and FcγR interactions\nThe extracellular regions of the FcγR are structurally similar. Each low‐affinity FcγR has two ectodomains, whereas the high‐affinity FcγRI has a third domain but this is not directly involved in IgG binding.62\nThe interaction between the IgG subclasses and the FcγR is most comprehensively defined for human IgG1 by both X‐ray crystallographic7, 62, 63 and mutagenesis structure/function analyses.64, 65, 66 These studies defined key regions of the IgG sequence required for interaction with their FcγRs.\nCrystallographic analyses of the human IgG1‐Fc complexed with FcγRI, FcγRII or FcγRIII show that these interactions are similar in topology, and asymmetric in nature. The second extracellular domain of the FcγR inserts between the two heavy chains. Here it makes contacts with the lower hinge of both H chains and with residues of the adjacent BC loop of one CH2 domain and the FG loop of the other. The N‐linked glycan at asparagine 297 (N297) of the heavy chain is essential for the structural integrity of the IgG‐Fc by affecting the spacing and conformation of the CH2 domains. Indeed, its removal ablates FcγR binding.67 Of particular relevance to therapeutic mAb development is that the normal low‐affinity IgG interaction with FcγRIIIa is profoundly increased by the removal of the core fucose from the N297 Fc oligosaccharide.68\nNo crystallographic data are available for IgG2 or IgG4 Fc in complex with FcγR, but mutagenesis studies of the Fc and the FcγR revealed general similarity, but with critical differences, in the interaction of these subclasses with their cognate FcγR.\n\nUnique features of the IgG2 and IgG4 subclasses\nIn IgG1, the stable interaction of the two heavy chains results from the combined effects of stable covalent inter H‐chain disulfide bonds and strong noncovalent interaction of the two CH3 domains (Table 3). In stark contrast, in IgG2 and IgG4 the interaction of the CH3 domains of each H‐chain is weak. Residues 392, 397 and 409 (Eu numbering) profoundly affect the stability of these interactions. The difference at position 409 (R409 in IgG4 and K409 in IgG1) confers a 100‐fold decrease in stability of the interface between the two CH3 domains of IgG4 compared with that of IgG1 (Table 3).69\nFurthermore, the core hinge of IgG4 differs from IgG1 at position 228 (P228 in IgG1 and S228 in IgG4), resulting in unstable inter‐heavy‐chain disulfide bonds. This, together with the destabilizing amino acids in the CH3, confers the unique property of half‐antibody (Fab arm) exchange between different IgG4 antibodies,69 thereby creating monovalent, bispecific IgG4 antibodies in vivo.69, 70 The similarly unstable interactions between the CH3 domains in IgG2 are conferred by the interface residue M397; however, the stable inter‐H‐chain disulfide bonds of the core and upper hinge prevent half‐molecule exchange (Table 3).69\nIn addition, IgG2 uniquely has three disulfide bond conformers (Table 3). The distinct conformers are formed when (1) each light chain is attached to the Cys131 residue of CH1 in the heavy chain (IgG2‐A conformer), (2) both light chains attach to the upper hinge (IgG2‐B) or (3) one light chain is attached to the CH1 Cys131 and one to the upper hinge of the other heavy chain (IgG2‐AB).71 This results in distinct positioning of the Fabs relative to the Fc portions in the different conformers, which has implications for the interaction with antigen and the capacity of IgG2 to cross‐link target molecules in the absence of FcγR binding, for example, in an agonistic mAb setting.72\nIt should also be noted that IgG3 has not been used in therapeutic mAbs despite its unique biology. The main impediment to its use are its physicochemical properties such as susceptibility to proteolysis and propensity to aggregate that present challenges to industry‐scale production and stability but protein engineering is attempting to overcome these hurdles.73\n\nTherapeutic antibody design: improving mAb potency\nMany factors affecting FcγR‐dependent responses in vivo come into play during mAb therapy. The experience of three decades of clinical use of mAbs taken together with our extensive, albeit incomplete, knowledge of IgG and FcγR structure and immunobiology provides a war chest for the innovative development of new and highly potent mAbs through the manipulation of their interaction with the FcγR.\nTherapeutic mAb engineering strategies are directed by many factors including the biology of the target, the nature of the antigen, the desired MOA and possibly the anatomical location of the therapeutic effect,21 and thus to optimize potency for a desired response, the context of use is critical.\n\nThe nature of the IgG isotype\nDifferent capabilities for the recruitment and activation of the different immune effector functions are naturally found in the Fc regions of the human IgG subclasses. Thus, to achieve a desired MOA, the different IgG subclasses are important starting points for the selection and engineering of the optimal mAb Fc. IgG1 is, in many ways, a proinflammatory or “effector‐active” subclass, as it can initiate the complement cascade and is a “universal” FcγR ligand.74 Notwithstanding it is also a ligand for the inhibitory FcγRIIb, IgG1 elicits proinflammatory responses through all activating‐type FcγRs, including ADCC, ADCP and cytokine release.\nBecause of their more restricted FcγR‐binding profile, IgG2 and IgG4 have offered some choice in potentially avoiding FcR effector function without the need for Fc engineering. They have been used as the backbone for therapeutic mAbs either because recruitment of patients’ effector functions was unlikely to be necessary for the primary MOA of the mAb or is possibly detrimental to the desired therapeutic effect.75 However, the use of these unmodified “inert” subclasses is not without consequences and underscores the need for Fc engineering to modify FcγR interactions—See the “Attenuating and ablating FcγR related functions of IgG” section.\nThus, the choice of IgG subclass for therapeutic mAb engineering is an important first step for engineering of novel mAbs of improved specificity, potency and safety.\n\nFc engineering for enhanced anticancer therapeutics\nIgG1 is the predominant subclass used in the development of cytotoxic mAbs where induction of an activation‐type response, ADCC or phagocytosis, is considered desirable.45, 76, 77\nCytotoxic mAb cancer therapeutics can control disease progression by one or more mechanisms. Their MOAs include direct induction of apoptotic cell death of the cancer cell (anti‐CD20, anti‐CD52) or blocking receptor signaling (anti‐HER2, anti‐EGFR). They may also harness FcγR effector functions, including ADCC in the tumor microenvironment.78 The approved mAbs, rituximab (anti‐CD20), trastuzumab (anti‐HER2) and cetuximab (anti‐EGFR), are formatted on a human IgG1 backbone and all require activating‐type FcγR engagement for optimal therapeutic activity.79, 80 This presents an example where context of therapeutic use is critical for therapeutic mAb design. IgG1 antibodies bind both the activating FcγR (e.g. FcγRIIIa) and the inhibitory FcγRIIb. In some environments effector cells will coexpress FcγRIIb together with FcγRI, FcγRIIa and FcγIIIa, as may occur on a tumor‐infiltrating macrophage. Therapy with an IgG1 anti‐cancer cell mAb may then be compromised by the inhibitory action of FcγRIIb upon the ITAM signaling of the activating FcγR as both types of receptor would be coengaged on such an effector cell by the mAb bound to the target cell. This leads to reduced therapeutic mAb potency. Thus, the relative contributions of the activating (A) and inhibitory (I) FcγR to the response by an effector cell, the A‐to‐I ratio, may be an important determinant in clinical outcome of therapeutic mAb activity,76, 81, 82 that is, the higher the A‐to‐I ratio, the greater the proinflammatory response induced by the therapeutic mAb or conversely the lower the A‐to‐I ratio, the greater the inhibition or dampening of the proinflammatory response.\nThus, the challenge for the development of more potent FcγR effector mAbs is to overcome three major obstacles. First, improving activation potency by selectively enhancing interaction with activating‐type FcγR, particularly FcγRIIIa owing to its predominant role in ADCC‐mediated killing of tumor cells. Second, reducing binding interactions with the inhibitory FcγRIIb. These two approaches improve the FcγR A‐to‐I ratio of cytotoxic IgG1 mAbs. Third, overcoming the significant affinity difference in the interaction with the main FcγRIII allelic forms of FcγRIIIa‐V158 and FcγRIIIa‐F158 76, 83, 84 which appears to be an important source of patient variability in responses to therapeutic mAb treatment of cancer.\nAt the time of writing, some mAbs with improved potency are coming into clinical use. Their improved action has been achieved by modifying the N‐linked glycan or the amino acid sequence of the heavy‐chain Fc (Table 4).\nTable 4 Fc or hinge‐engineered monoclonal antibodies (mAbs) approved or in advanced clinical development.\nmAb name Target IgG backbone Fc modification Effect on mAb Therapy area Most advanced development stage\nAndecaliximab Matrix Metalloproteinase 9 (MMP9) IgG4 S228P Stabilize core hinge Oncology Phase III\nAnifrolumab Interferon alpha/beta receptor 1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2/F/G loop; plus ablate FcγR binding Immunology Phase III\nAtezolizumab PD‐L1 IgG1 Aglycosylated (N297A) Ablate FcγR binding Oncology Marketed\nBenralizumab Interleukin 5 IgG1 Afucosylated Selectively enhance FcγRIII interaction Respiratory dermatology; ear nose throat disorders; gastrointestinal; hematology; immunology; Marketed\nDurvalumab PD‐L1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2 F/G loop; plus ablate FcγR binding Oncology Marketed\nEvinacumab Angiopoietin‐related protein 3 IgG4 S228P Stabilize core hinge Metabolic disorders Phase III\nInebilizumab CD19 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIxekizumab Interleukin 17A IgG4 S228P Stabilize core hinge Dermatology; immunology; musculoskeletal disorders Marketed\nMargetuximab HER2 IgG1 F243L; L235V; R292P; Y300L; P396L Selectively enhance FcγRIII interaction Oncology Phase III\nMogamulizumab C–C chemokine receptor type 4 (CCR4) IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Marketed\nTafasitamab (MOR208 XmAb 5574) CD19 IgG1 S239D; I332E Selectively enhance FcγRIII interaction Oncology Phase III\nNivolumab PD‐1 IgG4 S228P Stabilize core hinge Infectious disease; oncology Marketed\nObinutuzumab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Immunology; oncology Marketed\nOcaratuzumab CD20 IgG1 P247I; A339Q Selectively enhance FcγRIII interaction Oncology Phase III\nPembrolizumab PD‐1 IgG4 S228P Stabilize core hinge Infection; oncology Marketed\nRoledumab Rhesus D IgG1 Afucosylated Selectively enhance FcγRIII interaction Hematological disorders Phase III\nSpesolimab (BI‐655130) IL‐36R IgG1 L234A; L235A Ablate FcγR binding Gastrointestinal; immunology Phase III\nTeplizumab CD3 IgG1 L234A; L235A Ablate FcγR binding Metabolic disorders Phase II\nTislelizumab PD‐1 IgG4 S228P; E233P; F234V; L235A; D265A; L309V; R409K Stabilize core hinge; mimic IgG2 lower hinge for restricted FcγR specificity; ablate FcγR binding; stabilize CH3 interaction Oncology Phase III\nToripalimab (JS 001) PD‐1 IgG4 S228P Stabilize core hinge Oncology Phase III\nUblituximab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIg, immunoglobulin.\nJohn Wiley \u0026 Sons, Ltd This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.\n\nModification of the Fc glycan\nThe typical complex N‐linked glycan attached to N297 of the heavy chain includes a core fucose.85 Antibodies that lack this fucose have approximately 50‐fold improved binding to FcγRIIIa and FcγRIIIb and importantly retain the weak, low‐affinity binding to the inhibitory FcγRIIb. Furthermore, this glycoengineering increased binding affinity of the modified IgG1 mAb for both FcγRIIIa V158 and F158 allelotypes.86, 87, 88 Afucosyl versions of the tumor targeting mAbs such as anti‐HER2, anti‐EGFR and anti‐CD20 had greater antitumor effects and increased survival,68, 88, 89 which is a reflection of the greatly increased, and selective, FcγRIII binding. Compared with their unmodified counterparts, the afucosyl mAbs showed dramatic improvement of FcγRIII‐related effector responses such as stronger NK cell‐mediated ADCC, or enhanced neutrophil‐mediated phagocytosis through FcγRIIIb and/or FcγRIIIa.23 However, certain neutrophil functions via FcγRIIa may be compromised.90, 91\nThere are six afucosylated antibodies in late‐stage clinical trials or approved for treatment (Table 4). Notable is obinutuzumab, an afucosyl anti‐CD20 mAb which nearly doubles progression‐free survival in chronic lymphocytic leukemia patients as compared with the fucose‐containing rituximab.68 This dramatic improvement in clinical utility reinforces the value of glycan engineering specifically and of Fc engineering generally in anticancer treatments.\n\nMutation of the Fc amino acids\nAlteration of the amino acids in the heavy‐chain Fc can alter IgG specificity and affinity for activating FcγRs. The anti‐CD19 antibody MOR208 (XmAb 5574) is currently in phase III trials for the treatment of chronic lymphocytic leukemia.92 It contains two mutations in its IgG1 Fc, S329D and I332E, which increases affinity to FcγRIIIa, particularly the “lower‐affinity” FcγRIIIa F158 allele. The mAb shows increased FcγRIII‐mediated ADCC and phagocytosis in vitro, and reduced lymphoma growth in mouse models.\nMargetuximab is an ADCC‐enhanced IgG1 Fc‐engineered variant of the approved anti‐HER2 mAb trastuzumab in phase III for HER2‐expressing cancers.66, 93 Alteration of five amino acids (L235V, F243L, R292P, Y300L and P396L) enhanced binding to FcγRIIIa which also had the additional effect of decreasing binding to the inhibitory FcγRIIb, and thereby increased its A‐to‐I FcγR ratio. This was apparent when compared with unmodified trastuzumab the margetuximab showed enhanced ADCC against HER2+ cells in vitro and demonstrated superior antitumor effects in an HER2‐expressing tumor model in mice.\nThe anti‐CD20 ocaratuzumab is an Fc‐engineered IgG1 mAb in late‐stage clinical trials for the treatment of a range of cancers, including non‐Hodgkin lymphoma and chronic lymphocytic leukemia.94 Two Fc mutations, P247I and A339Q, conferred about 20‐fold increase in binding to both major allelic variants of FcγRIIIa and elicited sixfold greater ADCC than unmodified IgG1.\nThus, the engineering of the Fc domain or glycan for improved FcγRIIIa binding is a powerful tool to create more potent and clinically effective anticancer mAbs.\n\nAttenuating and ablating FcγR‐related functions of IgG\nThere are circumstances where binding to FcγR is unnecessary or undesirable in the MOA of a therapeutic mAb. Unmodified IgG irrespective of its subclass or intended therapeutic effect has the potential to engage an FcγR which may lead to suboptimal therapeutic performance or to unexpected and catastrophic consequences.57, 59 Clearly reducing or eliminating FcγR interactions, when they are not required for therapeutic effect, may be desirable. Indeed, this had been addressed by the choice of IgG subclass or by modifying the Fc region. Indeed, most efforts in Fc engineering mAbs that have translated to an approved drug have focused on the reduction or elimination of FcγR interactions (Table 4).\nOne approach to minimize interactions with the activating FcγR has been the use of IgG4 or IgG2 backbones, which show a more restricted specificity for the activating FcγR and consequently have been traditionally, and simplistically, viewed as “inert” IgG subclasses. Notwithstanding the unexpected, and FcγR‐dependent, severe adverse reaction induced by the IgG4 TGN1412 mAb, the IgG4 or IgG2 backbones have been successfully used in many settings. Indeed, checkpoint inhibitors, such as mAbs targeting CTLA‐4 or the PD‐L1/PD‐1 interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an IgG4 backbone. Pembrolizumab, nivolumab and cemiplimab are all anti‐PD‐1 antibodies currently used for cancer therapy and have been formatted on an IgG4 backbone95, 96, 97 with a stabilized core hinge (S228P) to prevent half‐IgG4 exchange. Similarly, the checkpoint inhibitor tremelimumab is an anti‐CTLA‐4 antibody formatted on an IgG2 backbone to avoid potential ADCC killing of target cells.98\nHowever, the use of IgG2 and IgG4 as “inert” subclasses is problematic. Both bind to the activating receptors FcγRIIa‐H131 and FcγRI, respectively (Table 2), and initiate effector functions such as neutrophil activation and apoptosis induction.75, 99 Interestingly, in experimental systems, cross‐linking of anti‐PD‐1 IgG4‐based mAb by FcγRI switched its activity from blocking to activatory.10 Moreover, IgG4 binds to FcγRIIb, which may scaffold the therapeutic mAb. Although scaffolding may be beneficial in some contexts, for example, in immune agonism,43 it can be disastrous and unexpected in others as it was for the anti‐CD28 TGN1412 mAb.59 Thus, the IgG2 and IgG4 subclasses are not the optimum choice for “FcR‐inactive” mAbs, and so modifying the Fc is a more direct approach.\nThe complete removal of the heavy‐chain N‐linked glycan is well known to ablate all FcγR binding by dramatically altering the Fc conformation.36, 67, 101, 102 Atezolizumab, an IgG1 anti‐PD‐L1 checkpoint inhibitor mAb, utilizes this strategy and eliminates FcγR and also complement activation.13\nModification to the Fc amino acid sequence of the FcγR‐contact regions can also be used to reduce FcγR binding. A widely used modification of IgG1 is the substitution of leucine 234 and 235 in the lower hinge sequence (L234 L235 G236 G237) with alanine (L234A L235A). It is often referred to as the “LALA mutation” and effectively eliminates FcγR binding by more than 100 fold104, 105 and is used in teplizumab and spesolimab (Table 4).\nA separate strategy has used combinations of amino acid residues from the FcγR‐binding regions of IgG2 and IgG4, which have restricted FcγR specificity, together with other binding‐inactivating mutations. The lower hinge amino acids of the IgG1 mAbs durvalumab (anti‐PD‐L1) and anifrolumab (anti‐interferon‐α receptor; Table 4) were modified to mimic the lower hinge of IgG4 (L234F). They additionally incorporated L235E in the lower hinge and P331S in the F/G loop of the CH2 domain to ablate FcγR binding by disrupting two major FcγR contact sites7 and also coincidently decreasing C1q activation.16\nIgG4 mAbs have been similarly engineered to eliminate their interaction with FcγRI and FcγRIIb. The IgG4 anti‐PD‐1 antibody tislelizumab has had its FcγR contact residues in the lower hinge E233, F234, L235 substituted with the equivalent residues of IgG2 P, V, A (E233P, F234V, L235A) as well as the additional D265A mutation which disrupts a major FcγR contact in CH2. It also has substitutions in the core hinge S228P and the CH3 L309V and R409K to stabilize the H‐chain disulfides and CH3 interactions, respectively, thereby preventing half‐Ig exchange characteristic of natural IgG4. Collectively, these modifications create a stable IgG4 with no FcγR binding nor complement activation.17\nThus, Fc engineering is an effective way to remove FcγR effector functions and may be preferable to using unmodified IgG2 or IgG4 backbones that have a more restricted repertoire of FcγR interactions but which are still able to induce certain effector functions.\n\nImproving FcγRIIb interactions\nPreferential or specific Fc engagement of FcγRIIb over the activating FcγR offers several potential therapeutic advantages for new mAbs in distinct therapeutic settings.\n\nImproved recruitment of FcγRIIb immunoreceptor tyrosine inhibition motif‐dependent inhibitory function\nHarnessing the physiological inhibitory function of FcγRIIb by mAbs that target ITAM receptors has the potential to shut down ITAM‐dependent signaling pathways of major importance in antibody pathologies.32, 108 Such ITAM signaling receptors include the BCR complex on B cells which is active in systemic lupus erythematosus, the FcεRI on basophils and mast cell subsets in allergies or the activating‐type FcγR on a variety of innate leukocytes in antibody‐mediated tissue destruction. In such scenarios, the ITAM signaling receptor complex that is targeted by the therapeutic mAb must be co‐expressed on the cell surface with the inhibitory FcγRIIb. This permits coengagement with ITAM signaling receptor by the Fab of the mAb and inhibitory FcγRIIb by its Fc which is the critical requirement in the inhibitory MOA for such therapeutic mAbs (Figure 1).\nObexelimab (also known as XmAb5871; Table 4), currently in early clinical testing in inflammatory autoimmune disease, is an IgG1 mAb that targets CD19 of the BCR complex.19 It contains two Fc modifications, S267E and L328F (also known as “SELF” mutations), that selectively increased FcγRIIb binding by 400‐fold to about 1 nm, which results in powerful suppression of BCR signaling and the proliferation of primary B cells.19\nThe anti‐IgE mAb omalizumab is an IgG1 mAb approved for the treatment of allergic disorders.110, 111 A similar but Fc‐engineered IgG1 mAb XmAb7195, currently in early clinical testing, contains the affinity‐enhancing SELF modifications.112 Both mAbs sterically neutralize the interaction between IgE and its high‐affinity receptor FcεRI to prevent basophil and mast cell activation.113, 114 However, XmAb7195 exhibited more efficient removal (sweeping; discussed later) of circulating IgE and also inhibited B‐cell IgE production, presumably by binding to the IgE BCR on the B‐cell surface and coclustering with FcγRIIb via its affinity‐enhanced Fc domain.112 Thus, XmAb7195’s selective modulation of IgE production by IgE+ B cells in addition to its enhanced clearance of IgE may offer significantly improved therapeutic benefits in allergy therapy beyond simple IgE neutralization.112 The “SELF” mutations have also been used in agonistic mAbs (discussed later).\nOne cautionary note is that the arginine 131 (R131) of the IgG‐binding site in FcγRIIb is critical for the enhanced affinity binding of “SELF”‐mutated Fcs but it is also present in the activating‐type “high responder” FcγRIIa‐R131. Thus, antibodies modified with “SELF” have very‐high‐affinity binding to FcγRIIa‐R131 115 with a potentially increased risk of FcγRIIa‐dependent complications in patients expressing this allelic form, although, so far, none have been reported in clinical trials. However, an alternative set of six Fc mutations, termed “V12” (P238D, E233D, G237D, H268D, P271G and A330R), potently enhanced FcγRIIb binding without increasing FcγRIIa–R131 interaction.115\n\nEnhancing the sweeping of small immune complexes\nThe expression of FcγRIIb on LSEC and its action in the “sweeping” or removal of small immune complexes has opened up new possibilities for the application of FcγRIIb‐enhancing modifications.17 Antibodies or Fc fusion proteins, whose primary MOA is the neutralization of soluble molecules such as IgE or cytokines, are particularly attractive candidates for this approach. Proof‐of‐concept for this strategy has been demonstrated in experimental models.48 Indeed, this may be a significant component of the rapid disappearance of IgE from the circulation of patients treated with the anti‐IgE XmAb7195 containing the FcγRIIb enhancing “SELF” modifications, as described previously.\n\nImmune agonism through FcγR scaffolding\nAgonistic mAbs induce responses in target cells by stimulating signaling of their molecular target. Typically, this is to either enhance antitumor immunity by engaging costimulatory molecules on antigen‐presenting cells or T cells (i.e. CD40, 4‐1BB, OX40) or promote apoptosis by engaging death receptors on cancer cells (i.e. DR4, DR5, Fas).116\nThe role of FcγR in the action of these types of mAbs appears to be primarily as a scaffold. FcγRIIb is often the predominate receptor involved and the extent of its involvement is complex. In the case of CD40, the degree of FcγRIIb scaffolding potency is linked to the epitope location of the targeting mAb with greater potency seen for membrane proximal epitopes.43, 117 It is also noteworthy that depending on the epitope location, the scaffolding of anti‐CD40 mAbs may convert antagonist mAbs to agonistic.\nEngineering of the IgG1 Fc region for enhanced and/or specific binding to FcγRIIb can greatly improve agonistic function.72, 118, 119, 120 Such mutations induced significantly greater agonistic activity in an anti‐DR5 model through increased induction of apoptotic death and decreased tumor growth compared with unmodified IgG1.121 The “SELF” modifications that dramatically and selectively increase affinity for FcγRIIb have also been used to enhance immune agonism in an anti‐OX40 model.122\nThe incorporation of the \"V12\" Fc mutations into IgG1 specifically enhance FcγRIIb interaction 200‐fold, conferring the enhanced agonistic activity of an anti‐CD137 antibody and an anti‐OX40 mAb.115, 122"}

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{"project":"LitCovid-sample-PD-MAT","denotations":[{"id":"T13","span":{"begin":9767,"end":9770},"obj":"http://purl.obolibrary.org/obo/MAT_0000317"},{"id":"T14","span":{"begin":9767,"end":9770},"obj":"http://purl.obolibrary.org/obo/MAT_0000233"},{"id":"T15","span":{"begin":9767,"end":9770},"obj":"http://purl.obolibrary.org/obo/MAT_0000138"},{"id":"T16","span":{"begin":9771,"end":9775},"obj":"http://purl.obolibrary.org/obo/MAT_0000139"},{"id":"T17","span":{"begin":10167,"end":10189},"obj":"http://purl.obolibrary.org/obo/MAT_0000457"},{"id":"T18","span":{"begin":10175,"end":10189},"obj":"http://purl.obolibrary.org/obo/MAT_0000026"},{"id":"T19","span":{"begin":10572,"end":10594},"obj":"http://purl.obolibrary.org/obo/MAT_0000457"},{"id":"T20","span":{"begin":10580,"end":10594},"obj":"http://purl.obolibrary.org/obo/MAT_0000026"},{"id":"T21","span":{"begin":11826,"end":11848},"obj":"http://purl.obolibrary.org/obo/MAT_0000457"},{"id":"T22","span":{"begin":11834,"end":11848},"obj":"http://purl.obolibrary.org/obo/MAT_0000026"},{"id":"T23","span":{"begin":22316,"end":22321},"obj":"http://purl.obolibrary.org/obo/MAT_0000488"},{"id":"T24","span":{"begin":22810,"end":22815},"obj":"http://purl.obolibrary.org/obo/MAT_0000488"},{"id":"T25","span":{"begin":24970,"end":24978},"obj":"http://purl.obolibrary.org/obo/MAT_0000491"}],"text":"IgG Subclasses: Structure and Properties\n\nThe molecular basis of IgG and FcγR interactions\nThe extracellular regions of the FcγR are structurally similar. Each low‐affinity FcγR has two ectodomains, whereas the high‐affinity FcγRI has a third domain but this is not directly involved in IgG binding.62\nThe interaction between the IgG subclasses and the FcγR is most comprehensively defined for human IgG1 by both X‐ray crystallographic7, 62, 63 and mutagenesis structure/function analyses.64, 65, 66 These studies defined key regions of the IgG sequence required for interaction with their FcγRs.\nCrystallographic analyses of the human IgG1‐Fc complexed with FcγRI, FcγRII or FcγRIII show that these interactions are similar in topology, and asymmetric in nature. The second extracellular domain of the FcγR inserts between the two heavy chains. Here it makes contacts with the lower hinge of both H chains and with residues of the adjacent BC loop of one CH2 domain and the FG loop of the other. The N‐linked glycan at asparagine 297 (N297) of the heavy chain is essential for the structural integrity of the IgG‐Fc by affecting the spacing and conformation of the CH2 domains. Indeed, its removal ablates FcγR binding.67 Of particular relevance to therapeutic mAb development is that the normal low‐affinity IgG interaction with FcγRIIIa is profoundly increased by the removal of the core fucose from the N297 Fc oligosaccharide.68\nNo crystallographic data are available for IgG2 or IgG4 Fc in complex with FcγR, but mutagenesis studies of the Fc and the FcγR revealed general similarity, but with critical differences, in the interaction of these subclasses with their cognate FcγR.\n\nUnique features of the IgG2 and IgG4 subclasses\nIn IgG1, the stable interaction of the two heavy chains results from the combined effects of stable covalent inter H‐chain disulfide bonds and strong noncovalent interaction of the two CH3 domains (Table 3). In stark contrast, in IgG2 and IgG4 the interaction of the CH3 domains of each H‐chain is weak. Residues 392, 397 and 409 (Eu numbering) profoundly affect the stability of these interactions. The difference at position 409 (R409 in IgG4 and K409 in IgG1) confers a 100‐fold decrease in stability of the interface between the two CH3 domains of IgG4 compared with that of IgG1 (Table 3).69\nFurthermore, the core hinge of IgG4 differs from IgG1 at position 228 (P228 in IgG1 and S228 in IgG4), resulting in unstable inter‐heavy‐chain disulfide bonds. This, together with the destabilizing amino acids in the CH3, confers the unique property of half‐antibody (Fab arm) exchange between different IgG4 antibodies,69 thereby creating monovalent, bispecific IgG4 antibodies in vivo.69, 70 The similarly unstable interactions between the CH3 domains in IgG2 are conferred by the interface residue M397; however, the stable inter‐H‐chain disulfide bonds of the core and upper hinge prevent half‐molecule exchange (Table 3).69\nIn addition, IgG2 uniquely has three disulfide bond conformers (Table 3). The distinct conformers are formed when (1) each light chain is attached to the Cys131 residue of CH1 in the heavy chain (IgG2‐A conformer), (2) both light chains attach to the upper hinge (IgG2‐B) or (3) one light chain is attached to the CH1 Cys131 and one to the upper hinge of the other heavy chain (IgG2‐AB).71 This results in distinct positioning of the Fabs relative to the Fc portions in the different conformers, which has implications for the interaction with antigen and the capacity of IgG2 to cross‐link target molecules in the absence of FcγR binding, for example, in an agonistic mAb setting.72\nIt should also be noted that IgG3 has not been used in therapeutic mAbs despite its unique biology. The main impediment to its use are its physicochemical properties such as susceptibility to proteolysis and propensity to aggregate that present challenges to industry‐scale production and stability but protein engineering is attempting to overcome these hurdles.73\n\nTherapeutic antibody design: improving mAb potency\nMany factors affecting FcγR‐dependent responses in vivo come into play during mAb therapy. The experience of three decades of clinical use of mAbs taken together with our extensive, albeit incomplete, knowledge of IgG and FcγR structure and immunobiology provides a war chest for the innovative development of new and highly potent mAbs through the manipulation of their interaction with the FcγR.\nTherapeutic mAb engineering strategies are directed by many factors including the biology of the target, the nature of the antigen, the desired MOA and possibly the anatomical location of the therapeutic effect,21 and thus to optimize potency for a desired response, the context of use is critical.\n\nThe nature of the IgG isotype\nDifferent capabilities for the recruitment and activation of the different immune effector functions are naturally found in the Fc regions of the human IgG subclasses. Thus, to achieve a desired MOA, the different IgG subclasses are important starting points for the selection and engineering of the optimal mAb Fc. IgG1 is, in many ways, a proinflammatory or “effector‐active” subclass, as it can initiate the complement cascade and is a “universal” FcγR ligand.74 Notwithstanding it is also a ligand for the inhibitory FcγRIIb, IgG1 elicits proinflammatory responses through all activating‐type FcγRs, including ADCC, ADCP and cytokine release.\nBecause of their more restricted FcγR‐binding profile, IgG2 and IgG4 have offered some choice in potentially avoiding FcR effector function without the need for Fc engineering. They have been used as the backbone for therapeutic mAbs either because recruitment of patients’ effector functions was unlikely to be necessary for the primary MOA of the mAb or is possibly detrimental to the desired therapeutic effect.75 However, the use of these unmodified “inert” subclasses is not without consequences and underscores the need for Fc engineering to modify FcγR interactions—See the “Attenuating and ablating FcγR related functions of IgG” section.\nThus, the choice of IgG subclass for therapeutic mAb engineering is an important first step for engineering of novel mAbs of improved specificity, potency and safety.\n\nFc engineering for enhanced anticancer therapeutics\nIgG1 is the predominant subclass used in the development of cytotoxic mAbs where induction of an activation‐type response, ADCC or phagocytosis, is considered desirable.45, 76, 77\nCytotoxic mAb cancer therapeutics can control disease progression by one or more mechanisms. Their MOAs include direct induction of apoptotic cell death of the cancer cell (anti‐CD20, anti‐CD52) or blocking receptor signaling (anti‐HER2, anti‐EGFR). They may also harness FcγR effector functions, including ADCC in the tumor microenvironment.78 The approved mAbs, rituximab (anti‐CD20), trastuzumab (anti‐HER2) and cetuximab (anti‐EGFR), are formatted on a human IgG1 backbone and all require activating‐type FcγR engagement for optimal therapeutic activity.79, 80 This presents an example where context of therapeutic use is critical for therapeutic mAb design. IgG1 antibodies bind both the activating FcγR (e.g. FcγRIIIa) and the inhibitory FcγRIIb. In some environments effector cells will coexpress FcγRIIb together with FcγRI, FcγRIIa and FcγIIIa, as may occur on a tumor‐infiltrating macrophage. Therapy with an IgG1 anti‐cancer cell mAb may then be compromised by the inhibitory action of FcγRIIb upon the ITAM signaling of the activating FcγR as both types of receptor would be coengaged on such an effector cell by the mAb bound to the target cell. This leads to reduced therapeutic mAb potency. Thus, the relative contributions of the activating (A) and inhibitory (I) FcγR to the response by an effector cell, the A‐to‐I ratio, may be an important determinant in clinical outcome of therapeutic mAb activity,76, 81, 82 that is, the higher the A‐to‐I ratio, the greater the proinflammatory response induced by the therapeutic mAb or conversely the lower the A‐to‐I ratio, the greater the inhibition or dampening of the proinflammatory response.\nThus, the challenge for the development of more potent FcγR effector mAbs is to overcome three major obstacles. First, improving activation potency by selectively enhancing interaction with activating‐type FcγR, particularly FcγRIIIa owing to its predominant role in ADCC‐mediated killing of tumor cells. Second, reducing binding interactions with the inhibitory FcγRIIb. These two approaches improve the FcγR A‐to‐I ratio of cytotoxic IgG1 mAbs. Third, overcoming the significant affinity difference in the interaction with the main FcγRIII allelic forms of FcγRIIIa‐V158 and FcγRIIIa‐F158 76, 83, 84 which appears to be an important source of patient variability in responses to therapeutic mAb treatment of cancer.\nAt the time of writing, some mAbs with improved potency are coming into clinical use. Their improved action has been achieved by modifying the N‐linked glycan or the amino acid sequence of the heavy‐chain Fc (Table 4).\nTable 4 Fc or hinge‐engineered monoclonal antibodies (mAbs) approved or in advanced clinical development.\nmAb name Target IgG backbone Fc modification Effect on mAb Therapy area Most advanced development stage\nAndecaliximab Matrix Metalloproteinase 9 (MMP9) IgG4 S228P Stabilize core hinge Oncology Phase III\nAnifrolumab Interferon alpha/beta receptor 1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2/F/G loop; plus ablate FcγR binding Immunology Phase III\nAtezolizumab PD‐L1 IgG1 Aglycosylated (N297A) Ablate FcγR binding Oncology Marketed\nBenralizumab Interleukin 5 IgG1 Afucosylated Selectively enhance FcγRIII interaction Respiratory dermatology; ear nose throat disorders; gastrointestinal; hematology; immunology; Marketed\nDurvalumab PD‐L1 IgG1 L234F; L235E; P331S Mimic IgG4 hinge and its CH2 F/G loop; plus ablate FcγR binding Oncology Marketed\nEvinacumab Angiopoietin‐related protein 3 IgG4 S228P Stabilize core hinge Metabolic disorders Phase III\nInebilizumab CD19 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIxekizumab Interleukin 17A IgG4 S228P Stabilize core hinge Dermatology; immunology; musculoskeletal disorders Marketed\nMargetuximab HER2 IgG1 F243L; L235V; R292P; Y300L; P396L Selectively enhance FcγRIII interaction Oncology Phase III\nMogamulizumab C–C chemokine receptor type 4 (CCR4) IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Marketed\nTafasitamab (MOR208 XmAb 5574) CD19 IgG1 S239D; I332E Selectively enhance FcγRIII interaction Oncology Phase III\nNivolumab PD‐1 IgG4 S228P Stabilize core hinge Infectious disease; oncology Marketed\nObinutuzumab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Immunology; oncology Marketed\nOcaratuzumab CD20 IgG1 P247I; A339Q Selectively enhance FcγRIII interaction Oncology Phase III\nPembrolizumab PD‐1 IgG4 S228P Stabilize core hinge Infection; oncology Marketed\nRoledumab Rhesus D IgG1 Afucosylated Selectively enhance FcγRIII interaction Hematological disorders Phase III\nSpesolimab (BI‐655130) IL‐36R IgG1 L234A; L235A Ablate FcγR binding Gastrointestinal; immunology Phase III\nTeplizumab CD3 IgG1 L234A; L235A Ablate FcγR binding Metabolic disorders Phase II\nTislelizumab PD‐1 IgG4 S228P; E233P; F234V; L235A; D265A; L309V; R409K Stabilize core hinge; mimic IgG2 lower hinge for restricted FcγR specificity; ablate FcγR binding; stabilize CH3 interaction Oncology Phase III\nToripalimab (JS 001) PD‐1 IgG4 S228P Stabilize core hinge Oncology Phase III\nUblituximab CD20 IgG1 Afucosylated Selectively enhance FcγRIII interaction Central nervous system; oncology Phase III\nIg, immunoglobulin.\nJohn Wiley \u0026 Sons, Ltd This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.\n\nModification of the Fc glycan\nThe typical complex N‐linked glycan attached to N297 of the heavy chain includes a core fucose.85 Antibodies that lack this fucose have approximately 50‐fold improved binding to FcγRIIIa and FcγRIIIb and importantly retain the weak, low‐affinity binding to the inhibitory FcγRIIb. Furthermore, this glycoengineering increased binding affinity of the modified IgG1 mAb for both FcγRIIIa V158 and F158 allelotypes.86, 87, 88 Afucosyl versions of the tumor targeting mAbs such as anti‐HER2, anti‐EGFR and anti‐CD20 had greater antitumor effects and increased survival,68, 88, 89 which is a reflection of the greatly increased, and selective, FcγRIII binding. Compared with their unmodified counterparts, the afucosyl mAbs showed dramatic improvement of FcγRIII‐related effector responses such as stronger NK cell‐mediated ADCC, or enhanced neutrophil‐mediated phagocytosis through FcγRIIIb and/or FcγRIIIa.23 However, certain neutrophil functions via FcγRIIa may be compromised.90, 91\nThere are six afucosylated antibodies in late‐stage clinical trials or approved for treatment (Table 4). Notable is obinutuzumab, an afucosyl anti‐CD20 mAb which nearly doubles progression‐free survival in chronic lymphocytic leukemia patients as compared with the fucose‐containing rituximab.68 This dramatic improvement in clinical utility reinforces the value of glycan engineering specifically and of Fc engineering generally in anticancer treatments.\n\nMutation of the Fc amino acids\nAlteration of the amino acids in the heavy‐chain Fc can alter IgG specificity and affinity for activating FcγRs. The anti‐CD19 antibody MOR208 (XmAb 5574) is currently in phase III trials for the treatment of chronic lymphocytic leukemia.92 It contains two mutations in its IgG1 Fc, S329D and I332E, which increases affinity to FcγRIIIa, particularly the “lower‐affinity” FcγRIIIa F158 allele. The mAb shows increased FcγRIII‐mediated ADCC and phagocytosis in vitro, and reduced lymphoma growth in mouse models.\nMargetuximab is an ADCC‐enhanced IgG1 Fc‐engineered variant of the approved anti‐HER2 mAb trastuzumab in phase III for HER2‐expressing cancers.66, 93 Alteration of five amino acids (L235V, F243L, R292P, Y300L and P396L) enhanced binding to FcγRIIIa which also had the additional effect of decreasing binding to the inhibitory FcγRIIb, and thereby increased its A‐to‐I FcγR ratio. This was apparent when compared with unmodified trastuzumab the margetuximab showed enhanced ADCC against HER2+ cells in vitro and demonstrated superior antitumor effects in an HER2‐expressing tumor model in mice.\nThe anti‐CD20 ocaratuzumab is an Fc‐engineered IgG1 mAb in late‐stage clinical trials for the treatment of a range of cancers, including non‐Hodgkin lymphoma and chronic lymphocytic leukemia.94 Two Fc mutations, P247I and A339Q, conferred about 20‐fold increase in binding to both major allelic variants of FcγRIIIa and elicited sixfold greater ADCC than unmodified IgG1.\nThus, the engineering of the Fc domain or glycan for improved FcγRIIIa binding is a powerful tool to create more potent and clinically effective anticancer mAbs.\n\nAttenuating and ablating FcγR‐related functions of IgG\nThere are circumstances where binding to FcγR is unnecessary or undesirable in the MOA of a therapeutic mAb. Unmodified IgG irrespective of its subclass or intended therapeutic effect has the potential to engage an FcγR which may lead to suboptimal therapeutic performance or to unexpected and catastrophic consequences.57, 59 Clearly reducing or eliminating FcγR interactions, when they are not required for therapeutic effect, may be desirable. Indeed, this had been addressed by the choice of IgG subclass or by modifying the Fc region. Indeed, most efforts in Fc engineering mAbs that have translated to an approved drug have focused on the reduction or elimination of FcγR interactions (Table 4).\nOne approach to minimize interactions with the activating FcγR has been the use of IgG4 or IgG2 backbones, which show a more restricted specificity for the activating FcγR and consequently have been traditionally, and simplistically, viewed as “inert” IgG subclasses. Notwithstanding the unexpected, and FcγR‐dependent, severe adverse reaction induced by the IgG4 TGN1412 mAb, the IgG4 or IgG2 backbones have been successfully used in many settings. Indeed, checkpoint inhibitors, such as mAbs targeting CTLA‐4 or the PD‐L1/PD‐1 interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an IgG4 backbone. Pembrolizumab, nivolumab and cemiplimab are all anti‐PD‐1 antibodies currently used for cancer therapy and have been formatted on an IgG4 backbone95, 96, 97 with a stabilized core hinge (S228P) to prevent half‐IgG4 exchange. Similarly, the checkpoint inhibitor tremelimumab is an anti‐CTLA‐4 antibody formatted on an IgG2 backbone to avoid potential ADCC killing of target cells.98\nHowever, the use of IgG2 and IgG4 as “inert” subclasses is problematic. Both bind to the activating receptors FcγRIIa‐H131 and FcγRI, respectively (Table 2), and initiate effector functions such as neutrophil activation and apoptosis induction.75, 99 Interestingly, in experimental systems, cross‐linking of anti‐PD‐1 IgG4‐based mAb by FcγRI switched its activity from blocking to activatory.10 Moreover, IgG4 binds to FcγRIIb, which may scaffold the therapeutic mAb. Although scaffolding may be beneficial in some contexts, for example, in immune agonism,43 it can be disastrous and unexpected in others as it was for the anti‐CD28 TGN1412 mAb.59 Thus, the IgG2 and IgG4 subclasses are not the optimum choice for “FcR‐inactive” mAbs, and so modifying the Fc is a more direct approach.\nThe complete removal of the heavy‐chain N‐linked glycan is well known to ablate all FcγR binding by dramatically altering the Fc conformation.36, 67, 101, 102 Atezolizumab, an IgG1 anti‐PD‐L1 checkpoint inhibitor mAb, utilizes this strategy and eliminates FcγR and also complement activation.13\nModification to the Fc amino acid sequence of the FcγR‐contact regions can also be used to reduce FcγR binding. A widely used modification of IgG1 is the substitution of leucine 234 and 235 in the lower hinge sequence (L234 L235 G236 G237) with alanine (L234A L235A). It is often referred to as the “LALA mutation” and effectively eliminates FcγR binding by more than 100 fold104, 105 and is used in teplizumab and spesolimab (Table 4).\nA separate strategy has used combinations of amino acid residues from the FcγR‐binding regions of IgG2 and IgG4, which have restricted FcγR specificity, together with other binding‐inactivating mutations. The lower hinge amino acids of the IgG1 mAbs durvalumab (anti‐PD‐L1) and anifrolumab (anti‐interferon‐α receptor; Table 4) were modified to mimic the lower hinge of IgG4 (L234F). They additionally incorporated L235E in the lower hinge and P331S in the F/G loop of the CH2 domain to ablate FcγR binding by disrupting two major FcγR contact sites7 and also coincidently decreasing C1q activation.16\nIgG4 mAbs have been similarly engineered to eliminate their interaction with FcγRI and FcγRIIb. The IgG4 anti‐PD‐1 antibody tislelizumab has had its FcγR contact residues in the lower hinge E233, F234, L235 substituted with the equivalent residues of IgG2 P, V, A (E233P, F234V, L235A) as well as the additional D265A mutation which disrupts a major FcγR contact in CH2. It also has substitutions in the core hinge S228P and the CH3 L309V and R409K to stabilize the H‐chain disulfides and CH3 interactions, respectively, thereby preventing half‐Ig exchange characteristic of natural IgG4. Collectively, these modifications create a stable IgG4 with no FcγR binding nor complement activation.17\nThus, Fc engineering is an effective way to remove FcγR effector functions and may be preferable to using unmodified IgG2 or IgG4 backbones that have a more restricted repertoire of FcγR interactions but which are still able to induce certain effector functions.\n\nImproving FcγRIIb interactions\nPreferential or specific Fc engagement of FcγRIIb over the activating FcγR offers several potential therapeutic advantages for new mAbs in distinct therapeutic settings.\n\nImproved recruitment of FcγRIIb immunoreceptor tyrosine inhibition motif‐dependent inhibitory function\nHarnessing the physiological inhibitory function of FcγRIIb by mAbs that target ITAM receptors has the potential to shut down ITAM‐dependent signaling pathways of major importance in antibody pathologies.32, 108 Such ITAM signaling receptors include the BCR complex on B cells which is active in systemic lupus erythematosus, the FcεRI on basophils and mast cell subsets in allergies or the activating‐type FcγR on a variety of innate leukocytes in antibody‐mediated tissue destruction. In such scenarios, the ITAM signaling receptor complex that is targeted by the therapeutic mAb must be co‐expressed on the cell surface with the inhibitory FcγRIIb. This permits coengagement with ITAM signaling receptor by the Fab of the mAb and inhibitory FcγRIIb by its Fc which is the critical requirement in the inhibitory MOA for such therapeutic mAbs (Figure 1).\nObexelimab (also known as XmAb5871; Table 4), currently in early clinical testing in inflammatory autoimmune disease, is an IgG1 mAb that targets CD19 of the BCR complex.19 It contains two Fc modifications, S267E and L328F (also known as “SELF” mutations), that selectively increased FcγRIIb binding by 400‐fold to about 1 nm, which results in powerful suppression of BCR signaling and the proliferation of primary B cells.19\nThe anti‐IgE mAb omalizumab is an IgG1 mAb approved for the treatment of allergic disorders.110, 111 A similar but Fc‐engineered IgG1 mAb XmAb7195, currently in early clinical testing, contains the affinity‐enhancing SELF modifications.112 Both mAbs sterically neutralize the interaction between IgE and its high‐affinity receptor FcεRI to prevent basophil and mast cell activation.113, 114 However, XmAb7195 exhibited more efficient removal (sweeping; discussed later) of circulating IgE and also inhibited B‐cell IgE production, presumably by binding to the IgE BCR on the B‐cell surface and coclustering with FcγRIIb via its affinity‐enhanced Fc domain.112 Thus, XmAb7195’s selective modulation of IgE production by IgE+ B cells in addition to its enhanced clearance of IgE may offer significantly improved therapeutic benefits in allergy therapy beyond simple IgE neutralization.112 The “SELF” mutations have also been used in agonistic mAbs (discussed later).\nOne cautionary note is that the arginine 131 (R131) of the IgG‐binding site in FcγRIIb is critical for the enhanced affinity binding of “SELF”‐mutated Fcs but it is also present in the activating‐type “high responder” FcγRIIa‐R131. Thus, antibodies modified with “SELF” have very‐high‐affinity binding to FcγRIIa‐R131 115 with a potentially increased risk of FcγRIIa‐dependent complications in patients expressing this allelic form, although, so far, none have been reported in clinical trials. However, an alternative set of six Fc mutations, termed “V12” (P238D, E233D, G237D, H268D, P271G and A330R), potently enhanced FcγRIIb binding without increasing FcγRIIa–R131 interaction.115\n\nEnhancing the sweeping of small immune complexes\nThe expression of FcγRIIb on LSEC and its action in the “sweeping” or removal of small immune complexes has opened up new possibilities for the application of FcγRIIb‐enhancing modifications.17 Antibodies or Fc fusion proteins, whose primary MOA is the neutralization of soluble molecules such as IgE or cytokines, are particularly attractive candidates for this approach. Proof‐of‐concept for this strategy has been demonstrated in experimental models.48 Indeed, this may be a significant component of the rapid disappearance of IgE from the circulation of patients treated with the anti‐IgE XmAb7195 containing the FcγRIIb enhancing “SELF” modifications, as described previously.\n\nImmune agonism through FcγR scaffolding\nAgonistic mAbs induce responses in target cells by stimulating signaling of their molecular target. Typically, this is to either enhance antitumor immunity by engaging costimulatory molecules on antigen‐presenting cells or T cells (i.e. CD40, 4‐1BB, OX40) or promote apoptosis by engaging death receptors on cancer cells (i.e. DR4, DR5, Fas).116\nThe role of FcγR in the action of these types of mAbs appears to be primarily as a scaffold. FcγRIIb is often the predominate receptor involved and the extent of its involvement is complex. In the case of CD40, the degree of FcγRIIb scaffolding potency is linked to the epitope location of the targeting mAb with greater potency seen for membrane proximal epitopes.43, 117 It is also noteworthy that depending on the epitope location, the scaffolding of anti‐CD40 mAbs may convert antagonist mAbs to agonistic.\nEngineering of the IgG1 Fc region for enhanced and/or specific binding to FcγRIIb can greatly improve agonistic function.72, 118, 119, 120 Such mutations induced significantly greater agonistic activity in an anti‐DR5 model through increased induction of apoptotic death and decreased tumor growth compared with unmodified IgG1.121 The “SELF” modifications that dramatically and selectively increase affinity for FcγRIIb have also been used to enhance immune agonism in an anti‐OX40 model.122\nThe incorporation of the \"V12\" Fc mutations into IgG1 specifically enhance FcγRIIb interaction 200‐fold, conferring the enhanced agonistic activity of an anti‐CD137 antibody and an anti‐OX40 mAb.115, 122"}

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