Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies

Niederfellner, Gerhard; Lammens, Alfred; Mundigl, Olaf; Georges, Guy; Schaefer, W.; Schwaiger, Manfred; Franke, Andreas G.; Wiechmann, Kornelius; Jenewein, Stefan; Slootstra, Jerry W.; Timmerman, Peter; Brännström, Annika; Lindstrom, Frida; Mössner, Ekkehard; Umaña, Pablo; Hopfner, Karl‐Peter; Klein, Christian

doi:10.1182/blood-2010-09-305847

Cited by 199 publications

(209 citation statements)

References 15 publications

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“…Second, F83 mutation to the smaller side chain (A, V, or S) induces a more compact Fab conformation and increases structural stability. Third, LC-F83 mutation can affect the biological activity of an antibody-antigen interaction through its modification of the Fab elbow angle and interdomain dynamics (47)(48)(49). Furthermore, B-cell signaling is sensitive to small conformational changes in the B-cell receptor (50).…”

Section: Discussionmentioning

confidence: 99%

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Koenig¹,

Lee²,

Walters³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. Deciphering the molecular mechanism of SHMs in improving antibodies is critical for understanding the evolution of the immune repertoire. It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4-7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8-11).The role of SHM in antigen-distal FWR is less well understood, however. Previous studies have suggested that the antigen-distal FWR contributes primarily to the variable domain structural integrity; thus, mutations at these positions would be either detrimental to or neutral for antigen-binding function (12)(13)(14). Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15-21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.Here we took a systematic approach to assessing the mutability of the whole variable domain for maintaining or improving folding stability and antigen binding. We used a welloptimized anti-vascular endothelial cell growth factor (VEGF), G6.31, with high affinity (K d = 0.4 nM) and excellent thermostability [fragment antigen-binding (Fab) melting temperature (T m ) = 84°C]. G6.31 derives its HC and LC variable domains ...

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Section: Discussionmentioning

confidence: 99%

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Koenig¹,

Lee²,

Walters³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…19,20 The mechanism of action of anti-CD20 antibodies includes activation of complement-dependent cytotoxicity, antibodydependent cellular cytotoxicity (ADCC), and the induction of direct cell death, although the exact contribution of each component in vivo is unknown. 21,22 Obinutuzumab recognizes a CD20 epitope overlapping with that of rituximab, but it exhibits a different elbow hinge angle and binds CD20 in a different orientation compared with type I anti-CD20 antibodies, the latter of which may form the basis for the functional differences between type I and type II antibodies. 21 In addition, the Fc portion of obinutuzumab has been glycoengineered to reduce fucosylation, resulting in optimized affinity for the Fc␥RIIIa receptor and enhanced ADCC potency.…”

Section: Introductionmentioning

confidence: 99%

“…21,22 Obinutuzumab recognizes a CD20 epitope overlapping with that of rituximab, but it exhibits a different elbow hinge angle and binds CD20 in a different orientation compared with type I anti-CD20 antibodies, the latter of which may form the basis for the functional differences between type I and type II antibodies. 21 In addition, the Fc portion of obinutuzumab has been glycoengineered to reduce fucosylation, resulting in optimized affinity for the Fc␥RIIIa receptor and enhanced ADCC potency. 19,21 Importantly, GA101 as a type II antibody more potently induces direct cell death and may provide an advantage when combined with chemotherapy.…”

Section: Introductionmentioning

confidence: 99%

“…21 In addition, the Fc portion of obinutuzumab has been glycoengineered to reduce fucosylation, resulting in optimized affinity for the Fc␥RIIIa receptor and enhanced ADCC potency. 19,21 Importantly, GA101 as a type II antibody more potently induces direct cell death and may provide an advantage when combined with chemotherapy. 19 Obinutuzumab has demonstrated significantly increased ADCC compared with rituximab in in vitro models.…”

Section: Introductionmentioning

confidence: 99%

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A phase 1 study of obinutuzumab induction followed by 2 years of maintenance in patients with relapsed CD20-positive B-cell malignancies

Sehn

Assouline

Stewart

et al. 2012

Blood

140

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“…This does not fully explain why type II anti-CD20 mAb do not augment internalisation of CD20, which also interact with FcγRIIB expressed in cis. However, the crystal structure of the type II mAb GA101 (obinituzumab) indicates that type II antibodies bind CD20 in a different orientation to type I anti-CD20 mAb [38]. This difference may alter the affinity or density with which type I and II mAb interact with FcγR in cis.…”

Section: Role Of Lipid Raftsmentioning

confidence: 99%

Antibody modulation: Limiting the efficacy of therapeutic antibodies

Vaughan

Cragg

Beers

2015

Pharmacological Research

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Monoclonal antibodies (mAb) have revolutionised the way in which we treat disease.From cancer to autoimmunity, antibody therapy has been responsible for some of the most impressive clinical responses observed in the last 2 decades. A key component of this success has been their generally low levels of toxicity, and unique mechanisms of action. These two facets have allowed them to a) be integrated rapidly into clinical practice in combination with conventional radio-and chemotherapies and b) to avoid the resistance mechanisms typically observed with classical small molecule drugs, such as upregulation of drug efflux transporters, dysregulation of apoptosis and mutations in key target enzymes/pathways.Although success with mAb therapies has been impressive, they are also subject to their own resistance mechanisms. In this perspective we discuss the various ways in which mAb therapeutics can be inhibited, concentrating mainly on the ways in which they can be removed from the target cell surface -a process called modulation. This can be achieved either in a cis-fashion on a single cell or in trans, precipitated by engagement with a second phagocytic cell. The evidence for each of these processes will be discussed, in addition to possible therapeutic strategies that might be employed to inhibit or reverse them.Abbreviations: Fc gamma receptor, FcR; monoclonal antibody, mAb

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Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies

Cited by 199 publications

References 15 publications

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

A phase 1 study of obinutuzumab induction followed by 2 years of maintenance in patients with relapsed CD20-positive B-cell malignancies

Antibody modulation: Limiting the efficacy of therapeutic antibodies

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