The influence of Bz-DOTA and CHX-A″-DTPA on the biodistribution of ABD-fused anti-HER2 Affibody molecules: implications for <Superscript>114m</Superscript>In-mediated targeting therapy

Purpose  Affibody molecules represent a novel class of high-affinity agents for radionuclide tumour targeting. Fusion of the Affibody molecules with an albumin-binding domain (ABD) enables modification of the blood kinetics of the Affibody molecules and reduction of the renal dose. ¹⁷⁷Lu-CHX-A″-DTPA-ABD-(Z_HER2:342)₂, an anti-HER2 Affibody molecule-ABD fusion protein has earlier demonstrated promising results in treatment of HER2-expressing micro-xenografts in mice. The use of the in vivo generator ^114mIn/¹¹⁴In as a label for ABD-fused Affibody molecules would create preconditions for efficient treatment of both micrometastases (due to conversion and Auger electrons of ^114mIn) and bulky tumours (due to high-energy beta particles from the daughter nuclide ¹¹⁴In). The goal of this study was to investigate if different chelators influence the biodistribution of ABD-(Z_HER2:342)₂ and to find an optimal chelator for attachment of ^114mIn to the Affibody molecule-ABD fusion protein. Methods  Isothiocyanate derivatives of Bz-DOTA and CHX-A″-DTPA were coupled to ABD-(Z_HER2:342)₂. The cellular processing of both conjugates was studied in vitro. The influence of chelators on the biodistribution was investigated in mice using double isotope (^114mIn and ¹¹¹In) labelling. Results  The apparent affinity of CHX-A″-DTPA-ABD-(Z_HER2:342)₂ and Bz-DOTA-ABD-(Z_HER2:342)₂ to the extracellular domain of HER2 was similar, 13.5 and 15.0 pM, respectively. It was found that both conjugates were internalized by SKOV-3 cells. The use of CHX-A″-DTPA provided better cellular retention of the radioactivity, better tumour accumulation of radioactivity and better tumour to organ dose ratios than Bz-DOTA-ABD-(Z_HER2:342)₂. Conclusion  CHX-A″-DTPA is more suitable for ^114mIn labelling of Affibody molecule-ABD fusion proteins for radionuclide therapy.     

Serum beta2-microglobulin in women with breast cancer and in age-matched, non-hospitalized controls.

Recently, reports have shown an increase of β₂-microglobulin in malignant disorders of different types including breast cancer. The present study was undertaken to evaluate further serum levels of β₂-microglobulin in 129 patients with breast cancer at various stages.As serum β₂-microglobulin concentration is significantly correlated to age, age-matched controls were used for comparisons.No significant difference was found in serum β₂-microglobulin levels in patients with breast cancer compared to age-matched controls, even when the levels were higher than reported for healthy individuals. The β₂-microglobulin concentration was not correlated to the TNM classification of the tumour.In order to compensate for the influence of renal function on serum β₂-microglobulin levels, the quotient between β₂-microglobulin and creatinine was calculated in 93 patients. The quotient was neither related to the stage of the disease nor to the tumour diameter. In 16 patients β₂-microglobulin was determined at different times in relation to mastectomy. There were no significant changes in circulating β₂-microglobulin levels after removal of the primary tumour.The present study underlines the need for age-matched controls, since serum β₂-microglobulin increases with age. The reason for such an increase could however only be speculated on, but may be explained by the high frequency of diseases causing known elevation in serum β₂-microglobulin such as infections of inflammatory disorders, liver diseases or renal dysfunction. Further studies must be undertaken in other malignancies before the role of serum β₂-microglobulin determination in such disorders can be stated.     

Nonviral and viral gene transfer into different subsets of human dendritic cells yield comparable efficiency of transfection

Among the many promising cancer immunotherapeutic strategies, dendritic cells (DC) have become of particular interest. This study aims to optimize a clinical grade protocol for culture and transfection of human DC. Monocytes and CD34(+) hematopoietic stem cells (HSC) from same donor were differentiated under serum-free conditions and analyzed for their susceptibility to several recently described nonviral transfection methods as compared with established virally mediated gene transfer. Nonviral gene transfer methods studied were square-wave electroporation, lipofection, and particle-mediated transfer of plasmid DNA or in vitro transcribed mRNA. We conclude that DNA is not suitable for transduction of DC using nonviral methods. In contrast, mRNA and square-wave electroporation reproducibly yields 60% and 50% transfected monocyte- and CD34(+)-derived DC, respectively, measured at protein level, without affecting the cell viability. Thus, the transfection efficiency of this method is comparable with the 40-90% transgene expression obtained using retroviral (RV) or adenoviral (AdV) vectors in CD34(+)- and monocyte-derived DC, respectively. In monocyte-derived DC, however, the amount of protein expressed per-cell basis was higher after AdV (MOI = 1000) compared with mRNA electroporation-mediated transfer. This is the first study directly demonstrating side-by-side that mRNA electroporation into DC of different origin indeed results in a comparable number of transduced cells as when using virus-mediated gene transfer.     

Ratio between serum IL-8 and pepsinogen A/C: a marker for atrophic body gastritis

BACKGROUND AND AIMS: Elevated serum gastrin and a low pepsinogen A/C ratio are well-recognized markers for atrophic body gastritis (ABG). We have shown that the presence of body atrophy is also associated with elevated serum pro-inflammatory cytokines. This study tested the hypothesis that serum cytokines provide additional information to gastrin and pepsinogens in screening for ABG. METHODS: Two hundred and twenty-six consecutive patients were investigated on referral for upper gastrointestinal endoscopy: 150 were patients with gastro-oesophageal reflux disease, receiving acid inhibitory medication either with proton pump inhibitors (n = 113) or with histamine2-receptor antagonists (n = 37), and 76 were nontreated controls, who had normal endoscopic findings. Gastric mucosal biopsies were sampled for histological examination (Sydney classification). Serum samples were analyzed for gastrin, chromogranin A (CgA), and pepsinogens A and C by RIA, and for the interleukins (IL)-1beta, IL-6, and IL-8 by ELISA. RESULTS: Subjects with ABG had significantly higher serum gastrin (P < 0.01) and serum CgA (P < 0.01) levels and significantly lower pepsinogen A/C ratios (P < 0.001) than those without ABG. Additionally, serum IL-1beta, IL-6 and, especially, IL-8 levels were significantly higher in the subjects with than in those without ABG (P < 0.0001, for all cytokines). To optimize the detection of body atrophy we defined the ABG index: the ratio between the simultaneously measured IL-8 and pepsinogen A/C. The area under the ROC curve for the ABG index was significantly greater than that for serum gastrin and for serum pepsinogen A/C alone (0.91 +/- 0.029 vs. 0.72 +/- 0.042, and vs. 0.83 +/- 0.031, P = 0.018 and P = 0.049). Using the ABG index at a cut-off value of 1.8 pg mL-1, 91% of the cases were classified correctly. CONCLUSIONS: The ratio between serum IL-8 and pepsinogen A/C accurately predicts the presence of ABG. We therefore propose the ABG index as a noninvasive screening test for ABG in population-based studies.     

Mature dendritic cells induce tumor-specific type 1 regulatory T cells

The aim of this study was to compare the tumor antigen-specific T-cell repertoire generated by transduced, human dendritic cells (DCs). The transductions were three commonly used antigen delivery procedures: adenovirus (AdV) infection, RNA electroporation, and liposome-mediated protein transfection. The DCs in each experimental group were transfected with similar efficacy and matured using TNF-alpha, anti-CD40, or lipopolysaccharide. Regardless of the gene transfer method or the maturation stimuli used, the DCs were indistinguishable with regard to surface phenotype and allostimulatory capacity. With the exception of the Adv transduced group, the T cells generated were tumor antigen specific, as characterized by high IFN-gamma production. The T cells generated upon stimulation with DCs subjected to AdV infection, and subsequently treated with TNF-alpha, exhibited tumor antigen specificity, but accompanied by reduced proliferation and IFNgamma production and increased IL-10 production. Moreover, these T cells exerted a suppressive effect on both autologous and allogeneic lymphocytes resembling type 1 regulatory T cells (Tr1). The authors show that mature DCs may induce tumor antigen-specific Tr1 cells by the appearance of high IL-10 and low IL-12. Similar results were also obtained with AdV-infected and TNF-matured DCs regardless of the transgene used. This work supports the conclusion that it can no longer be assumed that mature DCs induce only antitumor reactive T cells.     

Anionic linear aliphatic surfactants activate TRPV1: A possible endpoint for estimation of detergent induced eye nociception?

The transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca²⁺ influx in sensory C-fibres with secondary effects leading to neurogenic inflammation in the surrounding tissue. We have earlier reported specific activation of TRPV1 by surfactant-containing hygiene products. We have continued this project by investigating activation of the TRPV1 by shampoo and soap ingredients in low concentrations measured as intracellular Ca²⁺ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. As a TRPV1 specific control, the TRPV1 antagonist capsazepine was used. The response was quantified as the product induced Ca²⁺ influx during 2 min in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that anionic alkyl linear surfactant ingredients such as sodium lauryl sulphate, sodium laureth sulphate, ammonium lauryl sulphate, sodium C12-15 pareth sulphate and N-lauroylsarcosine concentration-dependently induced Ca²⁺ influx that could be addressed to TRPV1. The cationic surfactants benzalkonium chloride and cetylpyridinium chloride induced a Ca²⁺ influx that was not TRPV1 mediated as well as the zwitterionic surfactant cocamidopropyl betaine, the non-linear anionic surfactant sodium deoxycholate and the non-ionic surfactant Triton-X. These results reveal a new mechanistic pathway for surfactant-induced nociception.     

Biodistribution of 211At labeled HER-2 binding affibody molecules in mice

The size of affibody molecules makes them suitable as targeting agents for targeted radiotherapy with the alpha-emitter 211At, since their biokinetic properties match the short physical half-live of 211At. In this study, the potential for this approach was investigated in vivo. Two different HER-2 binding affibody molecules were radiolabeled with 211At using both the linker PAB (N-succinimidyl-para-astatobenzoate) and a decaborate-based linker, and the biodistribution in tumor-bearing nude mice was investigated. The influence of L-lysine and Na-thiocyanate on the 211At uptake in normal tissues was also studied. Based on the biokinetic information obtained, the absorbed dose was calculated for different organs. Compared with a previous biodistribution with 125I, the 211At biodistribution using the PAB linker showed higher uptake in lungs, stomach, thyroid and salivary glands, indicating release of free 211At. When the decaborate-based linker was used, the uptake in those organs was decreased, but instead, high uptake in kidneys and liver was found. The uptake, when using the PAB linker, could be significantly reduced in some organs by the use of L-lysine and/or Na-thiocyanate. In conclusion, affibody molecules have suitable blood-kinetics for targeted radionuclide therapy with 211At. However, the labeling chemistry affects the distribution in normal organs to a high degree and needs to be improved to allow clinical use.