Evaluation of Approaches for the Assessment of HER2 Expression in Breast Cancer by Radionuclide Imaging Using the Scaffold Protein [99mTc]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, [99mTc]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 [99mTc]Tc-ADAPT6 in a planned Phase II study. HER2 expression was evaluated in primary tumours and metastatic axillary lymph nodes (mALNs). SPECT/CT imaging of twenty treatment-naive breast cancer patients was performed 2 h after injection of [99mTc]Tc-ADAPT6. The imaging data were compared with the data concerning HER2 expression obtained by immunohistochemical evaluation of samples obtained by core biopsy. Maximum Standard Uptake Values (SUVmax) afforded the best performance for both primary tumours and mALNs (areas under the receiver operating characteristic curve (ROC AUC) of 1.0 and 0.97, respectively). Lesion-to-spleen ratios provided somewhat lower performance. However, the ROC AUCs were still over 0.90 for both primary tumours and mALNs. Thus, lesion-to-spleen ratios should be further evaluated to find if these could be applied to imaging using stand-alone SPECT cameras that do not permit SUV calculations.

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. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

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. High-throughput systems with automated panning already in use often require high-priced equipment. Moreover, the bottleneck of the selection process is usually the screening and characterization. Therefore, having a high-throughput panning procedure without a scaled screening platform does not necessarily increase the discovery rate. Here, we present an easy-to-use high-throughput selection system with automated panning using cost-efficient equipment integrated into a workflow with high-throughput sequencing and a tailored screening step using biolayer-interferometry. The workflow has been developed for selections using two recombinant libraries, ADAPT (Albumin-binding domain-derived affinity proteins) and CaRA (Calcium-regulated affinity) and has been evaluated for three new targets. The newly established semi-automated system drastically reduced the hands-on time and increased robustness while the selection outcome, when compared to manual handling, was very similar in deep sequencing analysis and generated binders in the nanomolar affinity range. The developed selection system has shown to be highly versatile and has the potential to be applied to other binding domains for the discovery of new protein binders.

Direct Intra-Patient Comparison of Scaffold Protein-Based Tracers, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3, for Imaging of HER2-Positive Breast Cancer.

Previous Phase I clinical evaluations of the radiolabelled scaffold proteins [99mTc]Tc-ADAPT6 and DARPin [99mTc]Tc-(HE)3-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 [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3. Eleven treatment-naïve female patients (26-65 years) with HER2-positive primary and metastatic breast cancer were included in the study. Each patient was intravenously injected with [99mTc]Tc-ADAPT6, followed by an [99mTc]Tc-(HE)3-G3 injection 3-4 days later and chest SPECT/CT was performed. All primary tumours were clearly visualized using both tracers. The uptake of [99mTc]Tc-ADAPT6 in primary tumours (SUVmax = 4.7 ± 2.1) was significantly higher (p < 0.005) than the uptake of [99mTc]Tc-(HE)3-G3 (SUVmax = 3.5 ± 1.7). There was no significant difference in primary tumour-to-contralateral site values for [99mTc]Tc-ADAPT6 (15.2 ± 7.4) and [99mTc]Tc-(HE)3-G3 (19.6 ± 12.4). All known lymph node metastases were visualized using both tracers. The uptake of [99mTc]Tc-ADAPT6 in all extrahepatic soft tissue lesions was significantly (p < 0.0004) higher than the uptake of [99mTc]Tc-(HE)3-G3. In conclusion, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 are suitable for the visualization of HER2-positive breast cancer. At the selected time points, [99mTc]Tc-ADAPT6 has a significantly higher uptake in soft tissue lesions, which might be an advantage for the visualization of small metastases.

The GRPR Antagonist [99mTc]Tc-maSSS-PEG2-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 [99mTc]Tc-maSSS-PEG2-RM26 (based on [D-Phe6, Sta13, Leu14-NH2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 °C was monitored for 18 months. The biological properties of [99mTc]Tc-maSSS-PEG2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG2-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16-24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics.

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. These elution conditions can cause the formation of aggregates and thereby greatly compromise the yield, which can be a major problem for scFvs that are generally unstable molecules. Due to the costly and time-consuming production of biological drugs, like antibody fragments, we have engineered novel purification ligands that elute the scFvs in a calcium-dependent manner. The developed ligands are equipped with new, selective binding surfaces and were shown to efficiently elute all captured scFv at neutral pH with the use of a calcium chelator. Further, two of three ligands were proven not to bind to the CDRs of the scFv, indicating potential for use as generic affinity ligands to a range of different scFvs. Multimerization and optimization of the most promising ligand led to a 3-fold increase in binding capacity for the hexamer compared to the monomer, in addition to highly selective and efficient purification of a scFv with >95% purity in a single purification step. This calcium-dependent ligand could revolutionize the scFv industry, greatly facilitating the purification procedure and improving the quality of the final product.

Correlates of protection and viral load trajectories in omicron breakthrough infections in triple vaccinated healthcare workers.

Vaccination offers protection against severe COVID-19 caused by SARS-CoV-2 omicron but is less effective against infection. Characteristics such as serum antibody titer correlation to protection, viral abundance and clearance of omicron infection in vaccinated individuals are scarce. We present a 4-week twice-weekly SARS-CoV-2 qPCR screening in 368 triple vaccinated healthcare workers. Spike-specific IgG levels, neutralization titers and mucosal spike-specific IgA-levels were determined at study start and qPCR-positive participants were sampled repeatedly for two weeks. 81 (cumulative incidence 22%) BA.1, BA.1.1 and BA.2 infections were detected. High serum antibody titers are shown to be protective against infection (p < 0.01), linked to reduced viral load (p < 0.01) and time to viral clearance (p < 0.05). Pre-omicron SARS-CoV-2 infection is independently associated to increased protection against omicron, largely mediated by mucosal spike specific IgA responses (nested models lr test p = 0.02 and 0.008). Only 10% of infected participants remain asymptomatic through the course of their infection. We demonstrate that high levels of vaccine-induced spike-specific WT antibodies are linked to increased protection against infection and to reduced viral load if infected, and suggest that the additional protection offered by pre-omicron SARS-CoV-2 infection largely is mediated by mucosal spike-specific IgA.

Direct In Vivo Comparison of 99mTc-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, [99mTc]Tc-(HE)3-G3 and [99mTc]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue. The goal of this preclinical study was to select the best probe for stratification of patients and response monitoring. Biodistribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 expression or MDA-MB-468 xenografts with very low expression. Changes in accumulation of the probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 permitted high contrast imaging of HER2-expressing tumors and a clear discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [99mTc]Tc-(HE)3-G3 is capable of detecting the decrease of HER2 expression on response to trastuzumab therapy only 24 h after injection of the loading dose. This indicates that the [99mTc]Tc-(HE)3-G3 tracer would be better for monitoring early response to such treatment. The results of this study should be considered in planning of further clinical development of HER2 imaging probes.

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. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.

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. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in tumors. Administration of ADAPT6-ABD-mcDM1 provided a significant delay of tumor growth and increased the median survival of the mice, in comparison with both a non-targeting homologous construct (ADAPTNeg-ABD-mcDM1) and the vehicle-treated groups, without inducing toxicity to liver or kidneys. Moreover, the evaluation of imaging probes showed that small scaffold proteins, such as 99mTc(CO)3-ADAPT6 or the affibody molecule 99mTc-ZHER2:41071, are well suited as diagnostic companions for potential stratification of patients for ADAPT6-ABD-mcDM1-based therapy.

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. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.

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 = 2.9, range = 0.4-9.6 years) and time from last anti-CD20 infusion to vaccination (median = 190, range = 60-1032 days). RESULTS: We find that presence of B cells and/or rituximab in blood predict seroconversion better than time since last infusion. Using multiple logistic regression, presence of >0.5% B cells increased probability of seroconversion with an odds ratio (OR) of 5.0 (95% confidence interval [CI] = 1.0-28.1, p = 0.055), whereas the corresponding OR for ≥6 months since last infusion was 1.45 (95% CI = 0.20-10.15, p = 0.705). In contrast, detectable rituximab levels were negatively associated with seroconversion (OR = 0.05, 95% CI = 0.002-0.392, p = 0.012). Furthermore, naïve and memory IgG+ B cells correlated with antibody levels. Although retreatment with rituximab at 4 weeks or more after booster depleted spike-specific B cells, it did not noticeably affect the rate of decline in antibody titers. Interferon-γ and/or interleukin-13 T-cell responses to the spike S1 domain were observed in most patients, but with no correlation to spike antibody levels. CONCLUSIONS: These findings are relevant for providing individualized guidance to patients and planning of vaccination schemes, in turn optimizing benefit-risk with anti-CD20.

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 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa ; 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. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability.