Natural killer (NK) cells are an essential part of the innate immune system as they can recognize and eliminate various cells in distress, such as virus-infected and tumor cells. Several promising therapeutics based on NK cell function are currently being developed and under preclinical and clinical investigation. This study aimed to evaluate the efficacy of an affibody-derived bispecific NK cell engager engineered to target CD16a on NK cells and BCMA on multiple myeloma (MM) cells, both in vitro and in vivo. In vitro, efficient lysis of MM cells was observed across a panel of cell lines with varying BCMA expression using NK cells from healthy donors. The bispecific engager also achieved MM cell clearance in a 48-hour real-time cytotoxicity assay at a 2:1 effector-to-target ratio when combined with ADAPT-NK cells—ex vivo expanded adaptive NK cells from healthy donors (Haroun-Izquierdo A et al., J Immunother Cancer 2022). The engager did not induce IL-6 release measured in the presence of PBMCs and target cells. In vivo, a humanized mouse model of MM was used to assess preclinical efficacy of the engager. In this model, human MM.1S cells - tagged with GFP and luciferase (BPS Bioscience) and xenograft-derived from mouse bone marrow - were combined with ADAPT-NK cells. The NSG-Tg(huIL-15) mice (The Jackson Laboratory) were given an intravenous injection of 0.5 million bone marrow-derived MM.1S cells, and tumor growth was followed by bioluminescence imaging (BLI). At study day 4, the dosing of the engager was started and continued daily until study day 18. Belantamab was used as a positive control and administered every fourth day, for a total of four doses, beginning on study day 4. ADAPT-NK cells were administered on study days 4, 8 and 13. Blood samples were obtained for flow cytometry throughout the study, and the mice were sacrificed on study day 33 when bone marrow was harvested for flow cytometry analysis. Body weight developed similarly in all groups. Inhibition of tumor growth was observed by BLI from day 18 onward in the engager-treated group in comparison to vehicle controls. In the ex vivo BLI, lower tumor burden was observed in bones, liver, lungs and spleen in the engager treated group compared to the control. Flow cytometry of bone marrow showed fewer BCMA+CD138+ cells, indicating MM cells, in the engager- and belantamab-treated groups compared to vehicle controls. These findings support the potential of the bispecific NK cell engager combined with ADAPT-NK cells as a treatment for MM. The study also demonstrates the utility of the humanized MM mouse model for evaluating NK cell engagers using BLI and flow cytometry.

Affibody affinity proteins are engineered scaffold proteins that can be selected by molecular display techniques for high-affinity, specific binding to various molecular targets. Conjugation of cytotoxic payloads (radionuclides or drugs) to affibodies has enabled successful treatment of human xenografts in mice. In this study, we evaluated a novel affibody-based system for in vivo targeting of tumours expressing the pancarcinoma antigen carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5). A CEACAM5-binding affibody head-to-tail dimer, C9-C9, was fused with the albumin-binding domain ABD035 to extend its blood residence time. Two variants were designed and evaluated, one with ABD035 placed at the N-terminus of the construct (ABD035-C9-C9) and the other with ABD035 at the C-terminus (C9-C9-ABD035). The constructs were site-specifically labelled with 99mTc, employing a C-terminal EYEC tetrapeptide extension to allow in vivo tracking. The radiolabelled affibody retained specific binding to CEACAM5-expressing cells in vitro. When compared in vivo to non-ABD035-fused C9-C9, both constructs showed a 10-fold decrease in renal uptake, a 50-fold increase in blood concentration, and a 9-fold increase in tumour uptake 4 h after injection in mice bearing human CEACAM5-expressing xenografts. The tumour uptake was CEACAM5-specific and depended on the target's expression level. The biodistribution was dependent on the placement of ABD035, and the construct with ABD035 at the N-terminus, [99mTc]Tc-ABD035-C9-C9, had a longer half-life in blood and significantly higher uptake in tumours. In conclusion, a fusion of C9-C9 with ABD035 and optimisation of the molecular architecture provides an affibody-based platform for targeting CEACAM5-expressing malignant tumours.

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.

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics.

We report development and characterization of small non-immunoglobulin affibody affinity proteins directed to the highly glycosylated human carcinoembryonic antigen-related adhesion molecule 5 (CEACAM5, CEA), and their use in immunohistochemical (IHC) analyses of human pancreatic cancer samples and for in vivo tumor imaging. A total of nineteen unique anti-CEA affibodies were identified from large phage display libraries constructed using combinatorial protein engineering of a small 58 amino acid three-helix bundle protein domain. Molecular modeling suggested that all enriched clones share a binding surface with several clustered tryptophan residues interacting with a hydrophobic patch in the N1 domain of CEA centered around a phenylalanine residue. One variant, designated as C9, exhibited the highest affinity in biosensor analyses and was reformatted into a 15 kDa homodimer expressed in Escherichia coli. The biotinylated form, C9-C9-Bio, was evaluated for its IHC performance on matched frozen and formalin-fixed, paraffin-embedded (FFPE) sections of human pancreatic cancer samples (n = 7). Compared to clinical-grade monoclonal antibodies II-7 and CEA31, as well as a polyclonal reagent, C9-C9-Bio demonstrated highly sensitive CEA detection with minimal background staining. Statistical analyses including intraclass correlation and Bland-Altman assessments revealed excellent agreement between C9-C9-Bio and the two monoclonal antibodies in FFPE tissue samples. Further, a ^99mTc[Tc]-labeled C9-C9 construct showed CEA-dependent binding to human cancer cell lines in vitro, and selectively bound to CEA-expressing BxPC3 xenografts in mice when investigated as a tracer for in vivo imaging, allowing for a visualization of tumors after four hours. In summary, these findings highlight the potential use of the easily produced CEA-binding C9 affibody for various clinical applications, including IHC and medical imaging, and as a targeting moiety for directing various therapeutic modalities to CEA-expressing tumors.

BACKGROUND: Heterozygous loss-of-function mutations in the gene encoding progranulin (GRN) are causative in around 5-10% of frontotemporal dementia (FTD) cases. These mutations lead to a more than 50% reduction of progranulin levels in the plasma and cerebrospinal fluid of mutation carriers compared to healthy controls. Increasing the progranulin levels in FTD-GRN patients via inhibition of sortilin-mediated progranulin degradation has shown promise as a therapeutic strategy, as exemplified by Alector's latozinemab, currently in phase 3 clinical trials. Here we detail the development of an anti-sortilin affibody-based miniprotein as a non-immunoglobulin alternative, offering potential advantages due to its small size and cost-effective production.

METHOD: Sortilin-binding affibodies were selected by phage display and subsequently genetically fused to short peptides derived from the progranulin C-terminus. The resulting miniproteins were characterized in terms of affinity, structure, stability, and their ability to increase extracellular progranulin levels in a clearance assay using progranulin-secreting, sortilin-expressing U-251 MG cells.

RESULT: A set of moderate-affinity sortilin-binding affibodies were obtained from phage display selections. Following genetic fusion with short peptides derived from the progranulin C-terminus and optimization of the fusion constructs, a lead candidate with 185 pM affinity for sortilin was obtained. The affibody-peptide fusion, but not its parental affibody or peptide, was capable of elevating extracellular progranulin levels in vitro with similar potency as latozinemab.

CONCLUSION: A sortilin-binding affibody-based miniprotein was developed and optimized, performing on par with latozinemab in in vitro functional studies. Affibody-based miniproteins provide a promising alternative to immunoglobulins in cases such as the present, where Fc functions are undesirable, and long-term high-dose antibody treatments risk becoming prohibitively expensive.

This review outlines the historical development and versatile applications of one of the most well-studied bacterial proteins, namely the immunoglobulin (Ig)-binding staphylococcal protein A (SpA) of Staphylococcus aureus. Each segment of the SpA operon, from the 5’ promoter region and signal peptide to the 3’ cell wall anchoring region, has been exploited for various innovative applications in areas such as immunology and biotechnology. We provide an overview of selected applications and concepts that have had a significant impact on life science research, and some that have also led to significant commercial implications. In the 1980s, the SpA promoter and signal sequence were utilized in Escherichia coli for recombinant production of various proteins, yielding product secretion to the culture medium and thereby simplifying product recovery. The five homologous Ig-binding domains of SpA gained tremendous interest in the late 1980s, largely due to the rise of monoclonal antibodies (mAbs) for therapeutic use, prompting a growing demand for effective affinity ligands to facilitate their purification. Over the years, these Ig-binding domains have been extensively investigated and re-engineered to bind proteins other than antibodies, leading in the mid-1990s to the development of the affibody affinity protein technology. Today, affibody molecules are being investigated in late-stage clinical trials as potential protein therapeutics for various indications. Finally, the cell wall anchoring regions of SpA inspired the development of a surface display system for Staphylococcus carnosus, which has emerged as a technology platform in combinatorial protein engineering for work with large peptide, antibody and affibody libraries.

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.

Affinity protein-oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins. Herein, we report a novel site-selective conjugation method for furnishing affinity protein-oligonucleotide conjugates in a 93% yield within fifteen minutes. Using SPR, we explore how the choice of affinity protein, conjugation strategy, and DNA length impact target binding and reveal the deleterious effects of non-specific conjugation methods. Furthermore, we show that these adverse effects can be minimised by employing our site-selective conjugation strategy, leading to improved performance in an immuno-PCR assay. Finally, we investigate the interactions between affinity protein-oligonucleotide conjugates and live cells, demonstrating the benefits of site-selective conjugation. This work provides critical insight into the importance of conjugation strategy when constructing affinity protein-oligonucleotide conjugates.