Single Binders Research Articles

A fine-tuned yeast surface-display/secretion platform enables the rapid discovery of neutralizing antibodies against Clostridioides difficile toxins

BackgroundNeutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19.ResultsIn this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable ‘switch’ through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB.ConclusionThe antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Pyrolysis mechanism of composite binder composed of coal tar pitch and phenolic resin for carbon materials

The composite binder consisting of coal tar pitch (CTP) and phenolic resin (PR) has both low viscosity and high char yield, and it has great potential in the preparation of advanced carbon materials. However, the lack of understanding of its synergetic behavior and mechanism hinders the optimization of the preparation process. In this work, the co-pyrolysis of CTP and PR was systematically investigated. The char yield of the composite binder was confirmed to be remarkably higher than the weighted average of the two single binders. Furthermore, the calculated activation energy for the pyrolysis of the composite binder was higher than that of CTP and PR after 400 °C. The interaction and structure evolution mechanisms of composite binder were clarified. Specifically, the ortho-position of the phenoxy carbon in CTP cross-link with the para-position of the phenoxy carbon in PR by the methylene bridge, and the free polycyclic aromatic hydrocarbons stack onto the as-crosslinked polycyclic aromatics driven by the pancake bonds.

Dynamics of Strength Gain in Sandy Soil Stabilised with Mixed Binders Evaluated by Elastic P-Waves during Compressive Loading.

This paper addresses the problem of stabilisation of poor subgrade soil for improving its engineering properties and stiffness. The study aim is to evaluate the effects from single and mixed binders on the gain of strength in sandy soil over the period of curing. We propose an effective non-destructive approach of using P-waves for identifying soil strength upon stabilisation. The growth of strength and stiffness is strongly dependent on time of curing and type of the stabilising agents which can include both single binders and their blended mixtures. The diverse effects from mixed binders on the properties of soil were evaluated, compared and analysed. We performed the experimental trials of five different binders for stabilisation of sandy soil using cement, lime, Ground Granulated Blast Furnace Slag (GGBFS), energy fly ash and bio fly ash. The methodology included soil stabilisation by binders during a total period of 90 days, strength test for the Unconfined Compressive Strength (UCS) and seismic tests on the stabilised samples. The dynamics of soil behaviour stabilised by different binders for days 7, 14, 28 and 90 was statistically analysed and compared. The optimisation of binder blending has been performed using mixture simplex lattice design with three binders in each case as independent variables. Using P-waves naturally exploited strength characteristics of soil samples and allowed us to compare the effects from the individual and blended binders over the complete period of curing with dominating mixes. The results indicate that strength growth in stabilised soil samples is nonlinear in both time and content of binders with dominating effects from slag which contributed the most to the compressive strength development, followed by cement.

Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.

Abstract 1546: Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy

Abstract IL1RAP (Interleukin 1 receptor accessory protein) is differentially expressed in both Ewing sarcoma (EWS) and select hematologic malignancies (CML, AML). Using single domain antibody-like binders generated against IL1RAP to create chimeric antigen receptors (CARs) we demonstrated highly specific cytokine production and cytolytic activity against multiple EWS cell lines in vitro. Preliminary in vivo functionality of IL1RAP CAR-T indicates that additional approaches will be required to increase the efficacy of IL1RAP CAR-T for orthotopic EWS tumor models. To optimize CAR-T cell therapy for EWS, we used model cell lines to define tumor- and stromal-derived factors that must be overcome to obtain effective treatment. The human cell line TC71 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with IL1RAP-specifc CAR-T cells. Tumor engraftment was not seen in the i.v. model, however the orthotopic i.m. model in NSG mice was successful, generating tumor and metastatic lesions surrounded by a collagen rich stromal element when CAR-T or control activated T cells were administered. This was not seen in control untreated tumors. Histochemical analysis of i.m. tumors demonstrated that T cells (CD3 positive) were confined to the tumor stromal boundary in proximity to cells of myeloid lineage (CD11b positive). We therefore analyzed cell surface and soluble factors produced by TC71 by flow cytometry, ELISA, and NanoString mRNA panels. TC71 was cultured alone or with interferon-gamma (IFNG), to model the presence of activated T cells and was found to constitutively express transcripts for both canonical and non-canonical chemokines: GPI, MIF, IL-32, TGFβ1; whereas CSF1, CXCL9, CXCL10 and CXCL11 were only induced by IFNG. MIF and TGF-β1 production were confirmed by ELISA. Flow cytometry revealed that numerous CAR-T targets (IL1RAP, CD276, EGFR and FGFR4) were expressed, as were the immune inhibitory molecules: PDL1, PDL2, Galectin-9, and CTLA-4. MHC class I and II and PDL1 were strongly induced by IFNG. We also observed transcripts for collagen related genes in TC71: COL16A1, 3A1, 5A3, LOXL1, P4HB and SERPINH1 both in vitro and in vivo. Our results indicate that tumor surface expression of immune inhibitory signals, PDL-1, PDL-2, CTLA4, as well as the soluble factors MIF, and TGF-b1, must be considered in armoring CAR-T for effective immune destruction of EWS, along with strategies designed to increase the transmigration of T cells through matrix proteins. Moreover, the chemokines produced by tumor alone, as well as those induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy which needs to be addressed for effective CAR-T therapy of this solid tumor. Citation Format: Rajesh Kumar, Peter Sullivan, Wei Li, Haifeng Zhang, Virginia Hoglund, Lingyang Wang, Yue Zhang, Poul H. Sorensen, Dimiter S. Dimitrov, Rimas J. Orentas. Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1546.

Performance of concrete with blended binders in ammonium-sulphate solution

Concrete elements in agricultural, wastewater treatment, mining, and industrial applications can be vulnerable to chemical attack by ammonium-based solutions. In particular, ammonium sulfate (commonly used as a fertilizer) is extremely deleterious to concrete. This is due to its dual acid–sulfate action, which may disintegrate the hydrated cement paste to various levels based on the prevailing exposure conditions and key mixture design parameters of concrete. The aim of this study was to investigate the response, in terms of physico-mechanical and microstructural features, of concrete comprising different types of cement (general use [GU] or Portland limestone cement [PLC]) with various combinations of supplementary cementitious materials (SCMs: fly ash, silica fume, and nanosilica) to a severe ammonium sulfate exposure. The study comprised 12 months of immersing test specimens in 5% ammonium sulfate solutions with a pH level of 6.0–8.0. The results revealed that the type of binder along with the dosage and nature of SCMs dictated different modes and levels of deterioration, and consequently the physico-mechanical trends of concrete were characterized by softening with (single binders) or without (blended binders) significant expansion. PLC may slightly improve the resistance of concrete to ammonium sulfate attack, whereas among the blended binders tested, binary binders comprising 5% silica fume, 5% nanosilica, or 30% fly ash improved the resistance of concrete to this type of chemical attack.

단일 고형화제를 이용한 중금속류 오염 토양의 고형화/안정화

Remediation of metal(loid)s-contaminated sites is crucial to protect human and ecosystem. Solidification and stabilization of metal(loid)s by the binder amendment is one of the cost-effective technologies. In this study, metal (loid)s in various field-contaminated soils obtained from steel-making, metal refinery and mining tillage were immobilized by the application of single binders such as diammonium phosphate (DAP), lime, and ladle slag. The efficiency of solidification and stabilization was evaluated by Toxicity Characteristic Leaching Procedure (TCLP) and the Standard, Measurements and Testing programme of European Union (SM&T) extraction processes. In terms of TCLP extraction, the binder was effective in order of lime > DAP > ladle slag. All binders were highly effective in the immobilization of Pb, Zn, Cu, Ni, and Cd. The increased immobilization efficiency is attributed to the increase in the Step III and IV fractions of the SM&T extraction. Lime and ladle slag were highly effective in the immobilization of the metal(loid)s, however, As release increased with DAP due to competition between the phosphate originated from DAP and arsenate. A further study is needed for the better immobilization of multi metal(loid)s using binary binders.

Chelating effect in short polymers for the design of bidentate binders of increased affinity and selectivity.

The design of new strong and selective binders is a key step towards the development of new sensing devices and effective drugs. Both affinity and selectivity can be increased through chelation and here we theoretically explore the possibility of coupling two binders through a flexible linker. We prove the enhanced ability of double binders of keeping their target with a simple model where a polymer composed by hard spheres interacts with a spherical macromolecule, such as a protein, through two sticky spots. By Monte Carlo simulations and thermodynamic integration we show the chelating effect to hold for coupling polymers whose radius of gyration is comparable to size of the chelated particle. We show the binding free energy of flexible double binders to be higher than that of two single binders and to be maximized when the binding sites are at distances comparable to the mean free polymer end-to-end distance. The affinity of two coupled binders is therefore predicted to increase non linearly and in turn, by targeting two non-equivalent binding sites, this will lead to higher selectivity.

General Bindings and Alpha-Equivalence in Nominal Isabelle

Nominal Isabelle is a definitional extension of the Isabelle/HOL theorem prover. It provides a proving infrastructure for reasoning about programming language calculi involving named bound variables (as opposed to de-Bruijn indices). In this paper we present an extension of Nominal Isabelle for dealing with general bindings, that means term constructors where multiple variables are bound at once. Such general bindings are ubiquitous in programming language research and only very poorly supported with single binders, such as lambda-abstractions. Our extension includes new definitions of alpha-equivalence and establishes automatically the reasoning infrastructure for alpha-equated terms. We also prove strong induction principles that have the usual variable convention already built in.

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven VH and seven VL (four Vκ and three Vλ) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2×109 members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the VH and VL CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.

High TiO 2 metadiabase dikes of the Hudson Highlands, New York and New Jersey; possible late Proterozoic rift rocks in the New York recess

<h3>ABSTRACT</h3> The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair (Sb#15 and Sb#68) that can bind simultaneously to the SARS-CoV-2 spike-RBD and efficiently neutralize pseudotyped and live-viruses by interfering with ACE2 interaction. Two spatially-discrete epitopes identified by cryo-EM translated into the rational design of bispecific and tri-bispecific fusions constructs, exhibiting up to 100- and 1000-fold increase in neutralization potency. Cryo-EM of the sybody-spike complex further revealed a novel <i>up-out</i> RBD conformation. While resistant viruses emerged rapidly in the presence of single binders, no escape variants were observed in presence of the bispecific sybody. The multivalent bispecific constructs further increased the neutralization potency against globally-circulating SARS- CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the development of clinically relevant therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.