Immobilizing calcium-dependent affinity ligand onto iron oxide nanoparticles for mild magnetic mAb separation.

Current downstream processing of monoclonal antibodies (mAbs) is limited in throughput and requires harsh pH conditions for mAb elution from Protein A affinity ligands. The use of an engineered calcium-dependent ligand (Z) in magnetic separation applications promises improvements due to mild elution conditions, fast processability, and process integration prospects. In this work, we synthesized and evaluated three magnetic nanoparticle types immobilized with the cysteine-tagged ligand Z-cys.

Prevalent and persistent new-onset autoantibodies in mild to severe COVID-19.

Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients at five time points over a 16-month period in 2020 and 2021 using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months.

Cooperative folding as a molecular switch in an evolved antibody binder.

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies.

Targeted HER2-positive cancer therapy using ADAPT6 fused to horseradish peroxidase.

Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects.

Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors.

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply.

Evaluation of Approaches for the Assessment of HER2 Expression in Breast Cancer by Radionuclide Imaging Using the Scaffold Protein [Tc]Tc-ADAPT6.

Due to its small size and high affinity binding, the engineered scaffold protein ADAPT6 is a promising targeting probe for radionuclide imaging of human epidermal growth factor receptor type 2 (HER2). In a Phase I clinical trial, [Tc]Tc-ADAPT6 demonstrated safety, tolerability and capacity to visualize HER2 expression in primary breast cancer. In this study, we aimed to select the optimal parameters for distinguishing between breast cancers with high and low expression of HER2 using [Tc]Tc-ADAPT6 in a planned Phase II study.

Distinct roles of vaccine-induced SARS-CoV-2-specific neutralizing antibodies and T cells in protection and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles.

An easy-to-use high-throughput selection system for the discovery of recombinant protein binders from alternative scaffold libraries.

Selection by phage display is a popular and widely used technique for the discovery of recombinant protein binders from large protein libraries for therapeutic use. The protein library is displayed on the surface of bacteriophages which are amplified using bacteria, preferably Escherichia coli, to enrich binders in several selection rounds. Traditionally, the so-called panning procedure during which the phages are incubated with the target protein, washed and eluted is done manually, limiting the throughput.

Direct Intra-Patient Comparison of Scaffold Protein-Based Tracers, [Tc]Tc-ADAPT6 and [Tc]Tc-(HE)-G3, for Imaging of HER2-Positive Breast Cancer.

Previous Phase I clinical evaluations of the radiolabelled scaffold proteins [Tc]Tc-ADAPT6 and DARPin [Tc]Tc-(HE)-G3 in breast cancer patients have demonstrated their safety and indicated their capability to discriminate between HER2-positive and HER2-negative tumours. The objective of this study was to compare the imaging of HER2-positive tumours in the same patients using [Tc]Tc-ADAPT6 and [Tc]Tc-(HE)-G3. Eleven treatment-naïve female patients (26-65 years) with HER2-positive primary and metastatic breast cancer were included in the study.

The GRPR Antagonist [Tc]Tc-maSSS-PEG-RM26 towards Phase I Clinical Trial: Kit Preparation, Characterization and Toxicity.

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [Tc]Tc-maSSS-PEG-RM26 (based on [D-Phe, Sta, Leu-NH]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models.

Calcium-dependent affinity ligands for the purification of antibody fragments at neutral pH.

The emerging formats of antibody fragments for biotherapeutics suffer from inadequate purification methods, delaying the advances of innovative therapies. One of the top therapeutic candidates, the single-chain variable fragment (scFv), requires the development of individual purification protocols dependent on the type of scFv. The available approaches that are based on selective affinity chromatography but do not involve the use of a purification tag, such as Protein L and Protein A chromatography, require acidic elution buffers.

Direct In Vivo Comparison of Tc-Labeled Scaffold Proteins, DARPin G3 and ADAPT6, for Visualization of HER2 Expression and Monitoring of Early Response for Trastuzumab Therapy.

Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratification of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies, thereby making treatment personalized and more efficient. Clinical evaluation in a phase I study demonstrated that injections of two scaffold protein-based imaging probes, [Tc]Tc-(HE)-G3 and [Tc]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue.

CaRA - A multi-purpose phage display library for selection of calcium-regulated affinity proteins.

Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium.

Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts: Efficacy and Selection of Companion Imaging Counterpart.

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials.

A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells.

New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins.

B-cell repopulation dynamics and drug pharmacokinetics impact SARS-CoV-2 vaccine efficacy in anti-CD20-treated multiple sclerosis patients.

Background And Purpose: Recent findings document a blunted humoral response to SARS-CoV-2 vaccination in patients on anti-CD20 treatment. Although most patients develop a cellular response, it is still important to identify predictors of seroconversion to optimize vaccine responses.

Methods: We determined antibody responses after SARS-CoV-2 vaccination in a real-world cohort of multiple sclerosis patients (n = 94) treated with anti-CD20, mainly rituximab, with variable treatment duration (median = 2.

Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies.

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 10  cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, Z ; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies.