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QC 20240109

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-99m]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-99m]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 [Tc-99m]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., Corresponding author: Vladimir Tolmachev

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. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy., QC 20240823

The present invention is within the field of protein engineering and purification. The invention relates to a target-binding polypeptide mutant of an IgG binding polypeptide, such as Protein A, Protein G, Protein L or Protein M, comprising a metal binding motif. More closely the invention relates to an Fc binding ligand comprising an engineered protein based on the Protein A derived Z domain, to which a calcium binding EF-loop has been introduced., QC 20240109

QC 20231123

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., Title in Web of Science: The GRPR Antagonist [Tc-99m]Tc-maSSS-PEG(2)-RM26 towards Phase I Clinical Trial: Kit Preparation, Characterization and Toxicity

Allosteric regulation of protein activity is a fundamental principle for the temporal and spatial control of cellular function. Transfer of such principles to rational design of proteins will pave the way for biotechnological applications were control of a given process with external cues is desirable. Through a semi-rational and directed evolution approach we have been able to design a calcium dependent molecular switch with distinct “on” and “off” states decided by the presence and absence of calcium, respectively. The design was established in the context of protein-protein interactions with antibodies which is a massive biotechnological application linked to purification of therapeutic antibodies. Our approach was to introduce a randomized calcium binding loop into the C2 domain of Streptococcal Protein G. The large ensemble of different sequences was displayed on the surface of E. coli and subjected to selective pressures for binding to a human FAB in the presence but not in the absence of calcium. From this directed evolution experiment we discovered evolved variants that contained calcium switches with distinct “on” and “off” behavior towards FAB binding. The molecular mechanism underlying the calcium switch was discovered from quantification of both structure and dynamics with NMR spectroscopy. We found the designed protein was partially disordered in the absence of calcium, and that the disordered segment corresponded to the calcium loop and part of the FAB interaction surface of the parental C2 domain. In presence of calcium both the calcium binding loop and the FAB surface became fully structured and as a consequence the FAB binding activity was restored. Therefore, the calcium-switch in our designed protein is dictated by a “coupled folding and binding” mechanism, a principle that has evolved over and over again under natural selection in for instance intrinsically disordered proteins.

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