PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.

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Beta-glucans are large polysaccharides produced by a range of prokaryotic and eukaryotic organisms. They have potential immunostimulatory properties and have been used with therapeutic intent as anti-microbial and anti-tumour agents. A range of other potentially beneficial effects have been described, and oral forms of beta-glucans are widely available over-the-counter and online. Parenteral formulations are popular in parts of Asia and are the subject of ongoing trials, worldwide. Beta-glucans are also potential contaminants of pharmaceutical products, and high levels have been described in some blood products. However, little is known about the clinical effects of such contamination, considerable uncertainty exists over the level at which immunostimulation may occur, and there are no guidelines available on acceptable levels. We encountered beta-glucan contamination of one of our products, and we suspect that others may encounter similar issues since the origin of beta-glucan contamination includes commonly used filters and solutions applied in the manufacture of biotherapeutic agents. It is likely that regulators will increasingly enquire about beta-glucan levels in pharmaceutical products, especially those with an immunomodulatory mechanism of action. Here, we review the literature on beta-glucans in pharmaceutical products and propose an acceptable level for therapeutic agents for parenteral use.

The presence of impurities or contaminants in biological products such as monoclonal antibodies (mAb) could affect efficacy or cause adverse reactions in patients. ICH guidelines (Q6A and Q6B) are in place to regulate the level of impurities within clinical drug products. An impurity less often reported and, therefore, lacking regulatory guideline is beta‐glucan. Beta‐glucans are polysaccharides of d‐glucose monomers linked by (1‐3) beta‐glycosidic bonds, and are produced by prokaryotic and eukaryotic organisms, including plants. They may enter manufacturing processes via raw materials such as cellulose‐based membrane filters or sucrose. Here we report the detection of beta‐glucan contamination of a monoclonal IgE antibody (MOv18), manufactured in our facility for a first‐in‐human, first‐in‐class clinical trial in patients with cancer. Since beta‐glucans have potential immunostimulatory properties and can cause symptomatic infusion reactions, it was of paramount importance to identify the source of beta‐glucans in our product and to reduce the levels to clinically insignificant concentrations. We identified beta‐glucans in sucrose within the formulation buffer and within the housing storage buffer of the virus removal filter. We also detected low level beta‐glucan contamination in two of four commercially available antibodies used in oncology. Both formulation buffers contained sucrose. We managed to reduce levels of beta‐glucan in our product 10‐fold, by screening all sucrose raw material, filtering the sucrose by Posidyne® membrane filtration, and by incorporating extra wash steps when preparing the virus removal filter. The beta‐glucan levels now lie within a range that is unlikely to cause clinically significant immunological effects. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1494–1502, 2016

Molecular imagings of hEGF receptor 2 (HER2) using radiolabeled tracers has the potential to determine the extent of HER2-positive disease and could be of great clinical value. HER2 overexpression affects 20-25% of breast cancer patients, conferring a worse prognosis. HER2 status determines choice and response to therapy but can change in response to treatment and during disease progression. Anti-HER2 agents in development for molecular imaging include immunoglobulins (trastuzumab and pertuzumab), immunoglobulin fragments, F(ab´)2, diabodies, nanobodies and nonimmunoglobulin scaffolds, affibody and designed ankyrin-repeat proteins. Clinical assessment of radiolabeled trastuzumab and anti-HER2 affibody molecule demonstrated potential to identify new lesions but both agents lacked sensitivity and highlighted the need for improved pharmacokinetics. New tracers in the pipeline showed preclinical promise and could potentially improve sensitivity.