Conformational changes in a Vernier zone region: Implications for antibody dual specificity

Arslan, Merve; Karadag, Dilara; Kalyoncu, Sibel

doi:10.1002/prot.25964

Cited by 6 publications

(3 citation statements)

References 64 publications

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“…Especially interesting is that the 71 H residue belongs to the Vernier-zone residues, which have been discussed to play a critical role in the humanization and for the rational design of antibodies in general as they can influence antibody specificity and affinity ( 65 , 75 , 76 ). Additionally, residue 71 H is part of the DE loop, also called H4 loop, which joins strands D and E on the heavy chain variable domain ( 9 ).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, also different residue types at position 71 H could be used to finetune the functions of antibodies and to balance the benefits of functional diversity by combining features of natural and engineered repertoires (73,74). Especially interesting is that the 71 H residue belongs to the Vernier-zone residues, which have been discussed to play a critical role in the humanization and for the rational design of antibodies in general as they can influence antibody specificity and affinity (65,75,76). Additionally, residue 71 H is part of the DE loop, also called H4 loop, which joins strands D and E on the heavy chain variable domain (9).…”

Section: Discussionmentioning

confidence: 99%

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Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

et al. 2021

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Characterizing and understanding the antibody binding interface have become a pre-requisite for rational antibody design and engineering. The antigen-binding site is formed by six hypervariable loops, known as the complementarity determining regions (CDRs) and by the relative interdomain orientation (VH–VL). Antibody CDR loops with a certain sequence have been thought to be limited to a single static canonical conformation determining their binding properties. However, it has been shown that antibodies exist as ensembles of multiple paratope states, which are defined by a characteristic combination of CDR loop conformations and interdomain orientations. In this study, we thermodynamically and kinetically characterize the prominent role of residue 71H (Chothia nomenclature), which does not only codetermine the canonical conformation of the CDR-H2 loop but also results in changes in conformational diversity and population shifts of the CDR-H1 and CDR-H3 loop. As all CDR loop movements are correlated, conformational rearrangements of the heavy chain CDR loops also induce conformational changes in the CDR-L1, CDR-L2, and CDR-L3 loop. These overall conformational changes of the CDR loops also influence the interface angle distributions, consequentially leading to different paratope states in solution. Thus, the type of residue of 71H, either an alanine or an arginine, not only influences the CDR-H2 loop ensembles, but co-determines the paratope states in solution. Characterization of the functional consequences of mutations of residue 71H on the paratope states and interface orientations has broad implications in the field of antibody engineering.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

et al. 2021

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“…In particular, residue 71 H [Kabat nomenclature ( 80 )], has been shown to co-determine the canonical conformation of the CDR-H2 loop, according to whether there is a bulky residue or a small side-chain present and thus bringing the CDR-H1 and CDR-H2 loops closer to each other ( 27 , 30 , 81 ). Especially interesting is that the 71 H residue is part of the Vernier-zone residues, which have been reported to play a critical role in the humanization process and for rational design of antibodies in general as they can influence antibody specificity and affinity ( 73 , 79 , 82 , 83 ). Differences in these framework residues might contribute to the distinct backbone and sidechain dynamics observed in the 5I15 and 5I19.…”

Section: Discussionmentioning

confidence: 99%

Germline-Dependent Antibody Paratope States and Pairing Specific VH-VL Interface Dynamics

et al. 2021

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Antibodies have emerged as one of the fastest growing classes of biotherapeutic proteins. To improve the rational design of antibodies, we investigate the conformational diversity of 16 different germline combinations, which are composed of 4 different kappa light chains paired with 4 different heavy chains. In this study, we systematically show that different heavy and light chain pairings strongly influence the paratope, interdomain interaction patterns and the relative VH-VL interface orientations. We observe changes in conformational diversity and substantial population shifts of the complementarity determining region (CDR) loops, resulting in distinct dominant solution structures and differently favored canonical structures. Additionally, we identify conformational changes in the structural diversity of the CDR-H3 loop upon different heavy and light chain pairings, as well as upon changes in sequence and structure of the neighboring CDR loops, despite having an identical CDR-H3 loop amino acid sequence. These results can also be transferred to all CDR loops and to the relative VH-VL orientation, as certain paratope states favor distinct interface angle distributions. Furthermore, we directly compare the timescales of sidechain rearrangements with the well-described transition kinetics of conformational changes in the backbone of the CDR loops. We show that sidechain flexibilities are strongly affected by distinct heavy and light chain pairings and decipher germline-specific structural features co-determining stability. These findings reveal that all CDR loops are strongly correlated and that distinct heavy and light chain pairings can result in different paratope states in solution, defined by a characteristic combination of CDR loop conformations and VH-VL interface orientations. Thus, these results have broad implications in the field of antibody engineering, as they clearly show the importance of considering paired heavy and light chains to understand the antibody binding site, which is one of the key aspects in the design of therapeutics.

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Structure-based humanization of a therapeutic antibody for multiple myeloma

Marino,

Daumke

2024

J Mol Med

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The optimal efficacy of xenogeneically generated proteins intended for application in humans requires that their own antigenicity be minimized. This necessary adaptation of antibodies to a humanized version poses challenges since modifications even distant from the binding sites can greatly influence antigen recognition and this is the primary feature that must be maintained during all modifications. Current strategies often rely on grafting and/or randomization/selection to arrive at a humanized variant retaining the binding properties of the original molecule. However, in terms of speed and efficiency, rationally directed approaches can be superior, provided the requisite structural information is available. We present here a humanization procedure based on the high-resolution X-ray structure of a chimaeric IgG against a marker for multiple myeloma. Based on in silico modelling of humanizing amino acid substitutions identified from sequence alignments, we devised a straightforward cloning procedure to rapidly evaluate the proposed sequence changes. Careful inspection of the structure allowed the identification of a potentially problematic amino acid change that indeed disrupted antigen binding. Subsequent optimization of the antigen binding loop sequences resulted in substantial recovery of binding affinity lost in the completely humanized antibody. X-ray structures of the humanized and optimized variants demonstrate that the antigen binding mode is preserved, with surprisingly few direct contacts to antibody atoms. These results underline the importance of structural information for the efficient optimization of protein therapeutics. Key messages Structure-based humanization of an IgG against BCMA, a marker for Multiple Myeloma. Identification of problematic mutations and unexpected modification sites. Structures of the modified IgG-antigen complexes verified predictions. Provision of humanized high-affinity IgGs against BCMA for therapeutic applications.

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Conformational changes in a Vernier zone region: Implications for antibody dual specificity

Cited by 6 publications

References 64 publications

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

Germline-Dependent Antibody Paratope States and Pairing Specific VH-VL Interface Dynamics

Structure-based humanization of a therapeutic antibody for multiple myeloma

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