Engineering an Antibody V Gene-Selective Vaccine.
Ontology highlight
ABSTRACT:
SUBMITTER: Ronsard L
PROVIDER: S-EPMC8459710 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.
| S-EPMC9068625 | 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:Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.
| S-EPMC6927761 | biostudies-literature
Project description:Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGF?1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGF?1 binding affinity was reduced by ?50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGF?1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGF?1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.
| S-EPMC5916551 | biostudies-literature
Project description:Efficacious HIV-1 vaccination requires elicitation of long-lived antibody responses. However, our understanding of how different vaccine types elicit durable antibody responses is lacking. To assess the impact of vaccine type on antibody responses, we measured IgG isotypes against four consensus HIV antigens from 2 weeks to 10 years post HIV-1 vaccination and used mixed effects models to estimate half-life of responses in four human clinical trials. Compared to protein-boosted regimens, half-lives of gp120-specific antibodies were longer but peak magnitudes were lower in Modified Vaccinia Ankara (MVA)-boosted regimens. Furthermore, gp120-specific B cell transcriptomics from MVA-boosted and protein-boosted vaccines revealed a distinct signature at a peak (2 weeks after last vaccination) including CD19, CD40, and FCRL2-5 activation along with increased B cell receptor signaling. Additional analysis revealed contributions of RIG-I-like receptor pathway and genes such as SMAD5 and IL-32 to antibody durability. Thus, this study provides novel insights into vaccine induced antibody durability and B-cell receptor signaling.
| S-EPMC7398916 | biostudies-literature
Project description:Chemotherapy strategies thus far reported can result in both side effects and drug resistance. To address both of these issues at the cellular level, we report a molecular engineering strategy, which employs polymeric aptamers to induce selective cytotoxicity inside target cells. The polymeric aptamers, composed of both multiple cell-based aptamers and a high ratio of dye-labeled short DNA, exploit the target recognition capability of the aptamer, enhanced cell internalization via multivalent effects, and cellular disruption by the polymeric conjugate. Importantly, the polymer backbone built into the conjugate is cytotoxic only inside cells. As a result, selective cytotoxicity is achieved equally in both normal cancer cells and drug-resistant cells. Control assays have confirmed the nontoxicity of the aptamer itself, but they have also shown that the physical properties of the polymer backbone contribute to target cell cytotoxicity. Therefore, our approach may shed new light on drug design and drug delivery.
| S-EPMC3190636 | biostudies-literature
Project description:Bispecific antibodies show great promise as intrinsic combination therapies, but often suffer from poor physiochemical properties, many times related to poor heterodimerization. De Nardis et al. 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 and autoimmune diseases to infectious diseases.
| S-EPMC5582862 | biostudies-literature
Project description:Vaccines are very effective at preventing infectious disease but not all recipients mount a protective immune response to vaccination. Recently, gene expression profiles of PBMC samples in vaccinated individuals have been used to predict the development of protective immunity. However, the magnitude of change in gene expression that separates vaccine responders and nonresponders is likely to be small and distributed across networks of genes, making the selection of predictive and biologically relevant genes difficult. Here we apply a new approach to predicting vaccine response based on coordinated upregulation of sets of biologically informative genes in postvaccination gene expression profiles. We found that enrichment of gene sets related to proliferation and immunoglobulin genes accurately segregated high responders to influenza vaccination from low responders and achieved a prediction accuracy of 88% in an independent clinical trial. Many of the genes in these gene sets would not have been identified using conventional, single-gene level approaches because of their subtle upregulation in vaccine responders. Our results demonstrate that gene set enrichment method can capture subtle transcriptional changes and may be a generally useful approach for developing and interpreting predictive models of the human immune response.
| S-EPMC3973429 | biostudies-literature
Project description:Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process.
| S-EPMC5618251 | biostudies-literature
Project description:We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (Tm) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher Tm compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.
| S-EPMC7181397 | biostudies-literature