Epstein-Barr virus (EBV) infection constitutes a prerequisite for multiple sclerosis (MS) development, and cross-reactivity between EBV nuclear antigen 1 (EBNA1) and anoctamin-2 (ANO2) antibodies was previously demonstrated in persons with MS (pwMS). Here, we show that ANO2-specific CD4+ T cells are more frequent in pwMS. Immunization of SJL/J mice with ANO2 or EBNA1 led to cross-reactive CD4+ T cell and antibody responses. ANO2 pre-immunization led to exacerbated experimental autoimmune encephalomyelitis (EAE), an effect mediated by CD4+ T cells, as confirmed by adoptive transfer experiments. T cell clones with cross-reactivity to EBNA1 and ANO2 could be isolated from natalizumab-treated pwMS, and sequencing of EBNA1-and ANO2-specific T cell receptors (TCRs) revealed a significant repertoire overlap. We thus report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS autoantigen ANO2, thereby establishing a link between EBV infection and neuroinflammation.

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.

Beta-2 microglobulin (β2m) is a small protein that forms the invariant subunit of the Major Histocompatibility Complex I. Monomeric β2m is stable under physiological conditions, however high local concentrations can induce misfolding, leading to amyloid deposition. This accumulation has been recently observed in the lysosomes of tumour-associated macrophages from patients affected by multiple myeloma. Such aggregation has been linked to inflammation and tumour progression. Stabilizing the native state of β2m could be the first step towards preventing this cancer-promoting process. To achieve this goal, the effect of affibody molecules, small and stress-resistant affinity proteins, was tested. Three affibodies molecules were selected against β2m. Affibody-β2m complex formation was initially assessed by size exclusion chromatography and subsequently confirmed by microscale thermophoresis and isothermal titration calorimetry. In parallel, in presence of one of the affibody (Zβ2m_01) a significant reduction in β2m aggregation was observed. The inhibition of amyloid formation was also confirmed by transmission electron microscopy. Taken together, these results indicate that Zβ2m_01 has the potential to act as β2m aggregation inhibitor under lysosomal-like pH values.

Overactive epidermal growth factor receptor (EGFR) signaling is often involved in driving different types of carcinomas. It is a well-studied target for targeted therapies, with both monoclonal antibodies and kinase inhibitors available for clinical use. Even though these drugs show a clinical benefit, most patients develop resistance over time. The development of new therapeutic modalities is therefore highly motivated. Herein, we describe a new type of drug candidate targeting EGFR, a so-called affibody-based drug conjugate. It consists of an EGFR-targeting affibody molecule, ZEGFR, expressed as a fusion to an albumin-binding domain for half-life extension, and coupled with the potent cytotoxic drug DM1 via a maleimidocaproyl linker. The resulting drug conjugate ZEGFR-ABD-mcDM1, showed strong binding to recombinant EGFR and EGFR-expressing cells. It was found to be highly potent in killing EGFR-expressing A431 cells with an IC50of 3.4 nM. In vivo, it showed moderate uptake in A431-derived xenografts with high EGFR expression. Collectively, the results from this study, demonstrate a potent and EGFR-specific drug candidate that holds promise for further development.

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.

The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated. It was, however, found that the spent medium of the HCDP inhibited the transfection of HEK293T cell aggregates, which was confirmed when testing with single-cell suspension culture. The formation of aggregates in shaken multi-well plates was also investigated to develop a screening system using the average power input as a scale-down criterion, which revealed that cell aggregates could be generated in 12-well plates, however with a larger size than in a STR. Taking into account the reported higher rAAV production of adherent cells in comparison with single cells for triple-plasmid transfection, HEK293T cell aggregates can possibly surpass single-cell suspension in space–time rAAV yield. The formation of HEK293T cell aggregates in a STR system offers a promising approach for scaling up and intensifying rAAV production by triple-plasmid transfection, in comparison with traditional 2D scale-up methods.

The popularity of rAAV vectors in gene therapy are placing a burden on current production systems. To improve the accessibility of these life changing treatments, increases in production yields and a reduction in the cost-of-goods are needed. Transient transfection is the most common way to introduce rAAV-encoding plasmids to producer cells but it suffers from significant drawbacks such as low and inconsistent yields as well as high cost due to its need for plasmid DNA. This study aims to address the low yield of transient transfection-based rAAV production through advanced methods in process characterization. Adherent and suspension cultures of a HEK293T cell line were triple-transfected for rAAV9 production using polyethylenimine (PEI). Samples were taken at various times post-transfection for analysis with bulk and single-cell transcriptomics. It was revealed that 46% of the cells lacked transcripts of genes from at least one plasmid, indicating that a significant proportion of the cells did not have the genes necessary for rAAV9 production. Among the remaining 54% of the cells expressing genes from all three plasmids, only 8% showed high plasmid gene expression. Flow cytometric analysis of intracellular rAAV9 confirmed these results by showing that only similar to 3% of cells contained assembled rAAV9 capsids. Titre analysis by qPCR of the supernatant and lysate of the producer cells indicated an average culture performance of 10(13) vg/L. Analysis of the single-cell transcriptomic data showed that a significant proportion of cells that had high plasmid gene expression were in the S-phase. Trajectory inference highlighted that genes involved in the G2-M phase transition, immune response, and protein unfolding were differentially expressed at the branch point between high and low plasmid expression. This study reveals a significant bottleneck in the transient transfection-based production of rAAV. With less than 5% of cells producing rAAV, significant improvements in titres can be achieved if this fraction can be increased. Moreover, regulation of the cell-cycle, inhibition of the immune response, and alleviating protein misfolding all potentially offer the key to enabling these life changing treatments to reach a wider audience.

Background: Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is overexpressed in several types of malignancies. Designed ankyrin repeat protein (DARPin) Ec1 is a 19 kDa engineered scaffold protein that binds with high affinity to EpCAM. Radiolabelled Ec1 might be used as a companion diagnostic for the selection of patients for personalized therapy. This study aimed to investigate the influence of different radiometal-chelator complexes on the biodistribution and imaging contrast of 68Ga-labelled Ec1. To investigate this, two macrocyclic chelators, 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the C-terminus of the Ec1. The previously developed DARPin Ec1 conjugated to 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA) was used as a comparator. Results: All Ec1 variants were successfully labelled with 68Ga. The use of NOTA and NODAGA provided twice higher radiochemical yield and improved label stability compared to DOTA. All labelled Ec1 variants bound to the EpCAM-expressing cells with nanomolar affinity and preserved targeting specificity in vitro and in vivo. Biodistribution studies in mice bearing EpCAM-expressing SKOV-3 xenografts showed that [68Ga]Ga-Ec1-NOTA had lower uptake in most normal organs while maintaining tumor uptake. Among all variants, [68Ga]Ga-Ec1-NOTA showed the lowest liver uptake, with no significant differences in tumor uptake. Additionally, [68Ga]Ga-Ec1-NOTA provided the highest tumor-to-blood ratio compared to [68Ga]Ga-Ec1-DOTA and [68Ga]Ga-Ec1-NODAGA. Conclusion: [68Ga]Ga-Ec1-NOTA is the preferred radioconjugate for PET imaging of EpCAM expression.