Immunoreagents, most commonly antibodies, are integral components of lateral flow immunoassays. However, the use of antibodies comes with limitations, particularly relating to their reproducible production, and poor thermal and chemical stability. Here, we employ phage display to develop affibodies, a class of nonimmunoglobulin affinity proteins based on a small three-helix bundle scaffold, against SARS-CoV-2 Spike protein. Subsequently, we demonstrate the utility and viability of affibodies to directly replace antibodies in lateral flow immunoassays. In addition, we highlight several physiochemical advantages of affibodies, including their ability to withstand exposure to high temperature and humidity while maintaining superior performance compared to their antibody counterparts. Furthermore, we investigate the adsorption mechanism of affibodies to the surface of gold nanoparticle probes via a His6-tag, introduced to also facilitate recombinant purification. Through molecular dynamics simulations, we elucidate the structural and physical characteristics of affibody dimers which result in high-performing detection probes when immobilized on nanoparticle surfaces. This work demonstrates that affibodies can be used as direct replacements to antibodies in immunoassays and should be further considered as alternatives owing to their improved physiochemical properties and modular design.

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.

We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.

G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.

Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies targeting a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

The present invention is within the field of protein engineering and purification. The invention relates to a target-binding polypeptide mutant of an IgG binding polypeptide, such as Protein A, Protein G, Protein L or Protein M, comprising a metal binding motif. More closely the invention relates to an Fc binding ligand comprising an engineered protein based on the Protein A derived Z domain, to which a calcium binding EF-loop has been introduced.

Background: Enhanced levels of a dipeptide, WC-am, have been reported among elite controllers - patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WC-am.

Methods: Drug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strains were performed to evaluate the antiviral mechanism of WC-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WC-am.Results: The data suggest that WC-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WC-am also inhibited HIV-1 at 4-6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WC-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WC-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WC-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR).Conclusion: Naturally occurring in HIV-1 elite controllers, WC-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WC-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WC-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.