Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality

Tam, Susan H.; McCarthy, Stephen G.; Armstrong, Anthony A.; Somani, Sandeep; Wu, Sheng‐Jiun; Liu, Xuesong; Gervais, Alexis; Ernst, Robin; Saro, Dorina; Decker, Rose; Luo, Jinquan; Gilliland, Gary L.; Chiu, Mark L.; Scallon, Bernard J.

doi:10.3390/antib6030012

Cited by 32 publications

(34 citation statements)

References 79 publications

(131 reference statements)

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“…Hybrid IgG2/4 Fc regions with the Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4 can result in having less FcγR activity. Crystal structures and simulations of IgG1σ, IgG4σ1, and IgG4σ2 Fc variants reveal altered conformational preferences within the lower hinge and BC and FG loops relative to wild-type IgG, providing a structural rationalization for diminished Fc receptor engagement [406].…”

Section: Mutations That Modulate Effector Functionmentioning

confidence: 95%

“…In each of these cases, it is best not to stimulate unwanted cell and tissue damage or risk undesired effector cell activation, immune cell depletion, or FcγR cross-linking that might induce cytokine release through engagement of Fc-mediated effector functions [422]. The complexity of FcγR functional properties is increased by the varying densities of activating and inhibitory receptors on the different effector cell populations [406]. Likewise, since the threshold of activation can be variable with different patients, it would be prudent for safety considerations to develop antibodies with a more silent Fc region.…”

Section: Mutations That Modulate Effector Functionmentioning

confidence: 99%

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Antibody Structure and Function: The Basis for Engineering Therapeutics

et al. 2019

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Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.

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Section: Mutations That Modulate Effector Functionmentioning

confidence: 95%

Section: Mutations That Modulate Effector Functionmentioning

confidence: 99%

Antibody Structure and Function: The Basis for Engineering Therapeutics

et al. 2019

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“…[11][12][13] However, incorporation of novel mutations has notable drawbacks including the possibility of decreased stability and increased immunogenicity. 14,15 The glycan profile of IgG mAbs has also been linked to their immune functions, with low fucose IgG glycoforms having improved binding to FcγRIIIa and more potent ADCC. 16 A less established strategy for modulation of effector mechanisms is the duplication of the entire Fc C H 2-C H 3 domains.…”

mentioning

confidence: 99%

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

et al. 2019

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Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen‐binding fragments (Fabs) of an mAb allow for high‐avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half‐life. To augment the effector functions or half‐life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral‐like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6‐ to 130‐fold, resulting in apparent affinity increases of 7‐ to 42‐fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild‐type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

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“…In line with literature, all molecules comprising an IgG2 σ1 Fc interacted the least with all FcγRs. 39 It has been demonstrated that the limited interaction of IgG2 σ1 with FcγRs in vitro translated to low or no activity in several in vitro functional assays, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and antibody-dependent cellular phagocytosis. 39 This observation confirmed that T cell activity would not likely be mediated via Fc because applied Fc mutations minimized binding to FcγR at the functional concentration ranges of BsAb used in killing experiments although subsets of T cells can express FcγRs.…”

Section: Discussionmentioning

confidence: 99%

“…39 It has been demonstrated that the limited interaction of IgG2 σ1 with FcγRs in vitro translated to low or no activity in several in vitro functional assays, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and antibody-dependent cellular phagocytosis. 39 This observation confirmed that T cell activity would not likely be mediated via Fc because applied Fc mutations minimized binding to FcγR at the functional concentration ranges of BsAb used in killing experiments although subsets of T cells can express FcγRs. 40 Single-arm affinities to CD19 and CD3 were recorded within a range of < 3X for all molecules, revealing that neither the IgG subclass nor molecular format (antibody vs. BsAb) significantly affected affinity in vitro.…”

Section: Discussionmentioning

confidence: 99%

Influence of the bispecific antibody IgG subclass on T cell redirection

Kapelski

Cleiren

Attar

et al. 2019

mAbs

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T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1-or IgG4-F(ab) 2 are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab) 2 domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab) 2 region to the BsAb's functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab) 2 domains.

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Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality

Cited by 32 publications

References 79 publications

Antibody Structure and Function: The Basis for Engineering Therapeutics

Antibody Structure and Function: The Basis for Engineering Therapeutics

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Influence of the bispecific antibody IgG subclass on T cell redirection

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