Opposites attract in bispecific antibody engineering.
Ontology highlight
ABSTRACT:
SUBMITTER: van Gils MJ
PROVIDER: S-EPMC5582862 | biostudies-literature | 2017 Sep
REPOSITORIES: biostudies-literature
Publications
Opposites attract in bispecific antibody engineering.
van Gils Marit J MJ Sanders Rogier W RW
The Journal of biological chemistry 20170901 35
Bispecific antibodies show great promise as intrinsic combination therapies, but often suffer from poor physiochemical properties, many times related to poor heterodimerization. De Nardis <i>et al.</i> identify specific electrostatic interactions that facilitate efficient heterodimerization, resulting in bispecific antibodies with physiochemical properties very similar to those of naturally occurring antibodies. This provides a new platform for the treatment of an array of diseases from cancer a ...[more]
Similar Datasets
Project description:Frequent outbreaks of enterovirus A71 (EVA71) occur in the Asia-Pacific area, and these are closely associated with severe neurological symptoms in young children. No effective antiviral therapy is currently available for the treatment of EVA71 infection. The development of monoclonal antibodies (mAbs) has demonstrated promise as a novel therapy for the prevention and treatment of infectious diseases. Several medical conditions have been treated using bispecific or multi-specific antibodies that recognize two or more distinct epitopes simultaneously. However, bispecific or multi-specific antibodies often encounter protein expression and product stability problems. In this study, we developed an IgG-like bispecific antibody (E18-F1) comprising two anti-EVA71 antibodies: E18 mAb and llama-derived F1 single-domain antibody. E18-F1 was demonstrated to exhibit superior binding affinity and antiviral activity compared with E18 or F1. Additionally, E18-F1 not only improved survival rate, but also reduced clinical signs in human SCARB2 receptor (hSCARB2) transgenic mice challenged with a lethal dose of EVA71. Altogether, our results reveal that E18-F1 is a simple format bispecific antibody with promising antiviral activity for EVA71.
| S-EPMC8164134 | biostudies-literature
Project description:Gram-negative bacterial viability is greatly reduced by the disruption of heptose sugar addition during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane component. Heptosyltransferase I (HepI), a member of the GT-B structural subclass of glycosyltransferases, is therefore an essential enzyme for the biosynthesis of the LPS. The disruption of HepI also increases the susceptibility of bacteria to hydrophobic antibiotics, making HepI a potential target for drug development. In this work, the structural and dynamic properties of the catalytic cycle of HepI are explored. Previously, substrate-induced stabilization of HepI was observed and hypothesized to be assisted by interactions between the substrate and residues located on dynamic loops. Herein, positively charged amino acids were probed to identify binding partners of the negatively charged phosphates and carboxylates of Kdo2-lipid A and its analogues. Mutant enzymes were characterized to explore changes in enzymatic activities and protein stability. Molecular modeling of HepI in the presence and absence of ligands was then performed with the wild type and mutant enzyme to allow determination of the relative change in substrate binding affinity resulting from each mutation. Together, these studies suggest that multiple residues are involved in mediating substrate binding, and a lack of additivity of these effects illustrates the functional redundancy of these binding interactions. The redundancy of residues mediating conformational transitions in HepI illustrates the evolutionary importance of these structural rearrangements for catalysis. This work enhances the understanding of HepI's protein dynamics and mechanism and is a model for improving our understanding of glycosyltransferase enzymes.
| S-EPMC7582807 | biostudies-literature
Project description:AimCompetition for food among populations of closely related species and conspecifics that occur in both sympatry and parapatry can be reduced by interspecific and intraspecific spatial segregation. According to predictions of niche partitioning, segregation is expected to occur at habitat boundaries among congeners and within habitats among conspecifics, while negative relationships in the density of species or populations will occur in areas of overlap. We tested these predictions by modelling the winter distributions of two crested penguin species from three colonies in the south-western Atlantic.LocationPenguins were tracked from two large colonies on the Falkland Islands and one in South Georgia, from where they dispersed through the South Atlantic, Southern Ocean and south-eastern Pacific.MethodsForty macaroni penguins (Eudyptes chrysolophus) from South Georgia and 82 southern rockhopper penguins (Eudyptes chrysocome chrysocome) from two colonies in the Falkland Islands were equipped with global location sensors which log time and light, allowing positions to be estimated twice-daily, from April to August in 2011. Positions were gridded and converted into maps of penguin density. Metrics of overlap were calculated and density was related to remote-sensed oceanographic variables and competitor density using generalized additive models.ResultsMacaroni penguins from western South Georgia and southern rockhopper penguins from Steeple Jason Island, Falkland Islands, were spatially segregated by differences in their habitat preferences thus supporting our first prediction regarding interspecific segregation. However, southern rockhopper penguins from Beauchêne Island showed a marked spatial overlap with macaroni penguins as the two had similar habitat preferences and strong mutual associations when controlling for habitat. Contrary to our predictions relating to intraspecific segregation, southern rockhopper penguins from Beauchêne Island and Steeple Jason Island were segregated by differences in habitat selection.Main conclusionsMorphological differentiation probably allows macaroni penguins from South Georgia and southern rockhopper penguins from Beauchêne Island to coexist in areas of spatial overlap, whereas segregation of the two Falkland rockhopper penguin populations may have arisen from two distinct lineages retaining cultural fidelity to ancestral wintering areas.
| S-EPMC4255236 | biostudies-literature
Project description:T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and in vitro T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.
| S-EPMC11225918 | biostudies-literature
Project description:Although there are only two bispecific antibody (bsAb) drugs in the market, around 100 bsAb drug candidates are in clinical development. BsAbs have gained fast growing investment and attractions from the biopharmaceutical industry and academia in recent years. Antibody Engineering & Therapeutics 2019 (AET 2019) was held in San Diego, USA, from December 9th to 13th, 2019. This year's AET certainly reflected the trend. In this report, we selected eleven presentations from AET 2019 to highlight bsAbs' design and their potentials in cancer therapy. These presentations have discussed emerging strategies to improve bispecific antibody drugs in efficacy, safety, and production. As compared to CAR-Ts, some T cell-redirecting bsAbs may potentially achieve comparable efficacies with less side effects and toxicities, as evidenced with both preclinical and clinical data reviewed at the conference. Several approaches to reduce T cell engagers' toxicities including conditionally active bsAbs and IgM-based bsAbs were also presented and discussed at the conference. For the first time, The Antibody Society and the Chinese Antibody Society jointly held a special session at the AET.
| S-EPMC7363406 | biostudies-literature
Project description:The existing repertoire of HIV-1 patient derived broadly neutralising antibodies (bNAbs) that target the HIV-1 envelope glycoprotein (Env) present numerous and exciting opportunities for immune-based therapeutic and preventative strategies against HIV-1. Combination antibody therapy is required to ensure greater neutralization coverage and limit Env mediated escape mutations following treatment pressure. Engineered bispecific bNAbs (bibNAbs) assimilate the advantages of combination therapy into a single antibody molecule with several configurations reporting potency enhancement as a result of the increased avidity and simultaneous engagement of targeted epitopes. We report the engineering of a novel bibNAb (iMab-CAP256) comprising the highly potent, CAP256.VRC26.25 bNAb with anticipated extension in neutralization coverage through pairing with the host directed, anti-CD4 antibody, ibalizumab (iMab). Recombinant expression of parental monoclonal antibodies and the iMab-CAP256 bibNAb was performed in HEK293T (Human embryonic kidney 293 T antigen) cells, purified to homogeneity by Protein-A affinity chromatography followed by size exclusion chromatography. Antibody assembly and binding functionality of Fab moieties was confirmed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and ELISA, respectively. Breadth and potency were evaluated against a geographical diverse HIV-1 pseudovirus panel (n = 20). Overall, iMab-CAP256 demonstrated an expanded neutralizing coverage, neutralizing single, parental antibody resistant pseudovirus strains and an enhanced neutralization potency against all dual sensitive strains (average fold increase over the more potent parental antibody of 11.4 (range 2 to 31.8). Potency enhancement was not observed for the parental antibody combination treatment (iMab + CAP256) suggesting the presence of a synergistic relationship between the CAP256 and iMab paratope combination in this bibNAb configuration. In addition, iMab-CAP256 bibNAbs exhibited comparable efficacy to other bibNAbs PG9-iMab and 10E08-iMab previously reported in the literature. The enhanced neutralization coverage and potency of iMAb-CAP256 over the parental bNAbs should facilitate superior clinical performance as a therapeutic or preventative strategy against HIV-1.
| S-EPMC6842167 | biostudies-literature
Project description:Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.
| S-EPMC4367261 | biostudies-literature
Project description:Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.
| S-EPMC4938304 | biostudies-literature
Project description:Here we present a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. When expressed in mammalian cells and purified by one-step protein A chromatography, the bsAb retains parental affinities of each binding domain, exhibits IgG-like stability and demonstrates in vivo IgG-like tumor targeting and blood clearance. The extension of the C-terminus of the light chain of an IgG with an scFv or even a smaller peptide does appear to disrupt disulfide bond formation between the light and heavy chains; however, this does not appear to affect binding, stability or in vivo properties of the IgG. Thus, we demonstrate here that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. This format serves as a standardized platform for the construction of functional bsAbs.
| S-EPMC2841541 | biostudies-literature
Project description:Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy, but inefficiency in the current discovery has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model, we demonstrate that our single-cell platform possesses a high throughput screening efficiency of up to one and a half million variant library cells per run and can isolate rare functional clones at a low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones, including extremely rare ones (~ 0.001% abundance). We also discovered BiTEs that exhibit novel properties and insights to design variable preferences for functionality. We expect our single-cell platform to not only increase the discovery efficiency of new immunotherapeutics, but also enable identifying generalizable design principles based on an in-depth understanding of the inter-relationships between sequence, structure, and function.
| S-EPMC10082157 | biostudies-literature