Biology subjects

Kedari, A.

Publications and source records attributed to Kedari, A..

3 recordsLinked to original sources

bioRxiv2024

Structural landscape of engineered multivalent antibody fragments and their application as crystallization scaffolds

Multivalent recombinant antibody fragments, "multibodies", are produced by fusing antibody VH and VL domains and provide the ability to bind multiple antigens simultaneously. The oligomeric state of a multibody is believed to be determined by the length of the linker region between the V-domains, with longer linkers resulting in diabodies (60 kDa) and shorter linkers leading to the formation of triabodies (90 kDa), tetrabodies (120 kDa), and larger oligomers. In this work, we investigate this design space by engineering multibodies from the sequences of human mAbs CR57 and Imdevimab, and resolve their crystal structures at 2.25 [A] and 2.55 [A] resolution, respectively. Our results show that despite minimizing the length of the hinge region between the V-domains, these constructs form diabodies. This indicates that linker length is not the sole determinant of a multibodys oligomeric state, and additional factors such as the mAb origin species and light chain type must also be taken into account when designing multibodies. Moreover, we confirmed that the native paratope of the antibody is well- maintained in the diabody format, and conducted a proof-of-concept trial comparing the crystallization propensity of a diabody versus a Fab in antibody-antigen complex crystallization. Our results show that a diabody can promote crystallization more effectively than a Fab, demonstrating the potential of diabodies as crystallization scaffolds for antibody- antigen complexes.

molecular biology↗

bioRxiv2023

Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold

Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by trimeric glycoprotein (G) spikes, which also represent the primary target for the humoral immune response. RABV-G displays several antigenic sites targeted by neutralizing antibodies, including monoclonal antibodies (mAbs) which have been proposed as quality-controlled alternatives to traditional polyclonal rabies immunoglobulin treatment. In this study, we determine the epitope of a potently neutralizing human anti-rabies mAb, CR57, which we engineered into a diabody to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38 [A] resolution, and in complex with RABV-G domain III at 3.15 [A] resolution. CR57 is demonstrated to bind RABV through a predominantly hydrophobic interface, with essential interactions targeting a conserved six-residue peptide sequence KLCGVL on the RABV-G. Further, our structural analysis suggests that CR57 sterically hinders receptor recognition and the fusogenic transitions of the spike protein that are required for host-cell entry. Altogether, this investigation provides a structural perspective on rabies inhibition by a potent antibody and delineates a functionally significant region in the spike. This understanding could pave the way for the development of prophylactic antibodies and other therapeutic strategies. Author summaryRabies virus (RABV) and many other lyssaviruses possess the ability to invade the central nervous system, leading to fatal encephalitis in mammals. Initiation of the infectious cycle depends on host cell recognition and entry, which is mediated by viral surface glycoprotein (G) spikes and can be inhibited by spike-targeting neutralizing antibodies. In our study, we elucidated the crystal structure of an antigenic domain from RABV-G in complex with a diabody derived from the potently neutralizing antibody CR57. This investigation revealed the molecular interactions by which CR57 binds to RABV-G and outlined a site of vulnerability comprising a conserved peptide in RABV-G domain III, where antibody binding is likely to inhibit RABV by obstructing host cell entry. Insights into the binding modalities of antibodies like CR57 deepen our understanding of how RABV and other lyssaviruses are neutralized, aiding the development of potential therapeutics. Furthermore, our study showcases the utility of engineering antibodies into diabodies to obtain crystal structures of antibody-antigen complexes.

molecular biology↗

bioRxiv2023

Validation of an antigenic site targeted by monoclonal antibodies against Puumala virus

Identification of B-cell epitopes facilitates the development of vaccines, therapeutic antibodies and diagnostic tools. Previously, the binding site of the bank vole monoclonal antibody (mAb) 4G2 against Puumala virus (PUUV, an orthohantavirus in the Hantaviridae family of the Bunyavirales order) was predicted using a combination of methods, including pepscan, phage-display, and site-directed mutagenesis of vesicular stomatitis virus (VSV) particles pseudotyped with Gn and Gc glycoproteins from PUUV. These techniques led to the identification of the neutralization escape mutation F915A. To our surprise, a recent crystal structure of PUUV Gc in complex with Fab 4G2 revealed that residue F915 is distal from epitope of mAb 4G2. To clarify this issue and explore potential explanations for the inconsistency, we designed a mutagenesis experiment to probe the 4G2 epitope, with three PUUV pseudoviruses carrying amino acid changes E725A, S944F, and S946F, located within the structure-based 4G2 epitope in the Gc. These amino acid changes were able to convey neutralization escape from 4G2, and S944F and S946F also conveyed escape from neutralization by human mAb 1C9. Furthemore, our mapping of all the known neutralization evasion sites from hantaviral Gcs onto PUUV Gc revealed that over 60% of these sites reside within or close to the epitope of mAb 4G2, indicating that this region represents a crucial area targeted by neutralizing antibodies against various hantaviruses. The identification of this site of vulnerability could guide the creation of subunit vaccines against PUUV and other hantaviruses in the future.

molecular biology↗