Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa

Ahmed, Alysia A.; Keremane, Sravya; Vielmetter, Jost; Björkman, Pamela J.

doi:10.1016/j.jsb.2016.02.001

Cited by 49 publications

(35 citation statements)

References 57 publications

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“…Numerous single mutations to the hIgG1 hinge, BC, C'E and FG loops are reported at positions 265, 267, 270, 297, 298 327, or 329 that reduced CD16A binding (reviewed in [ 35 ]). Some hIgG1 Fc combinations mutants bind CD16A with greater affinity than the wild type sequence and include variations in the C'E and FG loops, including G236A/S293A/A330L/I332E [ 36 ] and F243L/R292P/Y300L/V305I/P396L [ 37 ]. It is, however, unclear what the effect of each amino acid substitution, in particular Y300L found in mIgG2c, is on receptor binding.…”

Section: Discussionmentioning

confidence: 99%

Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1

Falconer

Barb

2018

PLoS ONE

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The structures of non-human antibodies are largely unstudied despite the potential for the identification of alternative structural motifs and physical properties that will benefit a basic understanding of protein and immune system evolution as well as highlight unexplored motifs to improve therapeutic monoclonal antibody. Here we probe the structure and receptor-binding properties of the mouse IgG2c crystallizable fragment (Fc) to compare to mouse IgG2b and human IgG1 Fcs. Models of mIgG2c Fc determined by x-ray crystallography with a complex-type biantennary (to 2.05 Å) or a truncated (1)GlcNAc asparagine-linked (N)-glycan attached (to 2.04 Å) show differences in key regions related to mouse Fc γ receptor IV (mFcγRIV) binding. Mouse IgG2c forms different non-bonded interactions between the BC, DE and FG loops than the highly-conserved mIgG2b and binds to FcγRIV with 4.7-fold greater affinity in the complex-type glycoform. Secondary structural elements surrounding the Asn297 site of glycosylation form longer beta strands in the truncated mIgG2c Fc glycoform when compared to mIgG2c with the complex-type N-glycan. Solution NMR spectroscopy of the N-linked (1)GlcNAc residues show differences between mIgG2b, 2c and hIgG1 Fc that correlate to receptor binding affinity. Mutations targeting differences in mIgG2 DE and FG loops decreased affinity of mIgG2c for FcγRIV and increased affinity of mIgG2b. Changes in NMR spectra of the mutant Fc proteins mirrored these changes in affinity. Our studies identified structural and functional differences in highly conserved molecules that were not predicted from primary sequence comparison.

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Section: Discussionmentioning

confidence: 99%

Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1

Falconer

Barb

2018

PLoS ONE

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“…Groups also adopted computational structure-based modelling coupled with high throughput protein screening in order to identify amino acid substitutions to enhance hFcγR-mediated effector functions. For example, the anti-CD52 hIgG1, alemtuzumab, with S239D/I332E or S239D/A330L/I332E mutations was found to have increased binding affinity to both allotypes of (A) Timeline of events detailing techniques used to discover variants with enhanced effector cell function and when the Fc variants were first described [40][41][42][43][44][45][46][47][48]. Letters refer to the common notation of the introduced amino acid substitutions as discussed in the main text.…”

Section: Amino Acid Substitutions To Enhance Effector Cell Functionmentioning

confidence: 99%

Fc-Engineering for Modulated Effector Functions—Improving Antibodies for Cancer Treatment

et al. 2020

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The majority of monoclonal antibody (mAb) therapeutics possess the ability to engage innate immune effectors through interactions mediated by their fragment crystallizable (Fc) domain. By delivering Fc-Fc gamma receptor (FcγR) and Fc-C1q interactions, mAb are able to link exquisite specificity to powerful cellular and complement-mediated effector functions. Fc interactions can also facilitate enhanced target clustering to evoke potent receptor signaling. These observations have driven decades-long research to delineate the properties within the Fc that elicit these various activities, identifying key amino acid residues and elucidating the important role of glycosylation. They have also fostered a growing interest in Fc-engineering whereby this knowledge is exploited to modulate Fc effector function to suit specific mechanisms of action and therapeutic purposes. In this review, we document the insight that has been generated through the study of the Fc domain; revealing the underpinning structure-function relationships and how the Fc has been engineered to produce an increasing number of antibodies that are appearing in the clinic with augmented abilities to treat cancer.

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“…Crucial amino acids for the low-affinity interaction of CD16A with a b the Fc part of IgG were previously identified and described. 51 Antibody 3G8 was reported to bind CD16A and CD16B and block IgG binding. 47 No blocking of binding of our antibodies to CD16A by IgGs was observed.…”

Section: Cd16 Binding Moietymentioning

confidence: 99%

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity

Ellwanger

Reusch

Fucek

et al. 2019

mAbs

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Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.

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Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa

Cited by 49 publications

References 57 publications

Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1

Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1

Fc-Engineering for Modulated Effector Functions—Improving Antibodies for Cancer Treatment

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity

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