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Abstract Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8+ (10-15 fold) and CD4+ T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients.

Abstract Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

ABSTRACTCD40 agonism by systemic administration of CD40 monoclonal antibodies has been explored in clinical trials for immunotherapy of cancer, uncovering enormous potential, but also dosing challenges in terms of systemic toxicity. CD40-dependent activation of antigen presenting cells is dependent on crosslinking of the CD40 receptor. Here we exploited this requisite by coupling crosslinking to cancer-receptor density by dual-targeting of CD40 and platelet-derived growth factor receptor beta (PDGFRB), which is highly expressed in the stroma of various types of tumors. A novel PDGFRBxCD40 Fc-silenced bispecific AffiMab was developed to this end to test whether it is possible to activate CD40 in a PDGFRB-targeted manner. A PDGFRB-binding Affibody molecule was fused to each heavy chain of an Fc-silenced CD40 agonistic monoclonal antibody to obtain a bispecific “AffiMab”. Binding of the AffiMab to both PDGFRB and CD40 was confirmed by surface plasmon resonance, bio-layer interferometry and flow cytometry, through analysis of cells expressing respective target. In a reporter assay, the AffiMab displayed increased CD40 potency in the presence of PDGFRB-conjugated beads, in a manner dependent on PDGFRB amount/bead. To test the concept in immunologically relevant systems with physiological levels of CD40 expression, the AffiMab was tested in human monocyte-derived dendritic cells (moDCs) and B cells. Expression of activation markers was increased in moDCs specifically in the presence of PDGFRB-conjugated beads upon AffiMab treatment, while the Fc-silenced CD40 mAb did not stimulate CD40 activation. As expected, the AffiMab did not activate moDCs in the presence of unconjugated beads. Finally, in a co-culture experiment, the AffiMab activated moDCs and B cells in the presence of PDGFRB-expressing cells, but not in co-cultures with PDGFRB-negative cells. Collectively, these results suggest the possibility to activate CD40 in a PDGFRB-targeted manner in vitro. This encourages further investigation and the development of such an approach for the treatment of solid cancers.

Abstract Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

Summary: Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.

During the current COVID-19 pandemic, a need for evaluation of already available drugs for treatment of the disease is crucial. Hereby, based on literature review from the current pandemic and previous outbreaks with corona viruses we analyze the impact of the virus infection on cell stress responses and redox balance. High levels of mortality are noticed in elderly individuals infected with SARS-CoV2 and during the previous SARS-CoV1 outbreak. Elderly individuals maintain a chronic low level of inflammation which is associated with oxidative stress and inflammatory cytokine production, a condition that increases the severity of viral infections in this population. Coronavirus infections can lead to alterations of redox balance in infected cells through modulation of NAD + biosynthesis, PARP function along with altering proteasome and mitochondrial function in the cell thereby leading to enhanced cell stress responses which further exacerbate inflammation. ROS production can increase IL-6 production and lipid peroxidation resulting in cell damage. Therefore, early treatment with anti-oxidants such as NAC during COVID-19 can be a way to bypass the excessive inflammation and cell damage that lead to severe infection, thus early NAC as intervention should be evaluated in a clinical trial setting.

Summary: We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome—a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.

Summary: Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

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An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.