EGFR- and HER2-Binding Affibody Molecules : Cellular studies of monomeric, dimeric and bispecific ligands

Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy. Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging. The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors

The HER2-binding Affibody Molecule (ZHER2:342)2 Increases Radiosensitivity in SKBR-3 cells

We have previously shown that the HER2-specific affibody molecule (ZHER2:342)2 inhibits proliferation of SKBR-3 cells. Here, we continue to investigate its biological effects in vitro by studying receptor dimerization and clonogenic survival following irradiation. We found that (ZHER2:342)2 sensitizes the HER2-overexpressing cell line SKBR-3 to ionizing radiation. The survival after exposure to (ZHER2:342)2 and 8 Gy (S8Gy 0.006) was decreased by a factor of 4 compared to the untreated (S8Gy 0.023). The low HER2-expressing cell line MCF-7 was more radiosensitive than SKBR-3 but did not respond to (ZHER2:342)2. Treatment by (ZHER2:342)2 strongly increased the levels of dimerized and phosphorylated HER2 already after 5 minutes of stimulation. The monomeric ZHER2:342 does not seem to be able to induce receptor phosphorylation and dimerization or sensitize cells to irradiation

Phosphorylated levels of EGFR, HER2 and HER3, as measured by ELISA.

<p>The relative levels for the treated samples were determined by comparing those with the untreated control (−) which was arbitrarily set as 1. Numbers indicate minutes of stimulation by (Z_HER2∶342)₂. EGF and NRG1-β1 was used for 5 minutes. A–C) SKBR-3 cell line. D–E) MCF-7 cell line. Mean values and standard deviation from at least two independent measurements are presented.</p

Survival after exposure to ionizing radiation.

<p>A–B) Clonogenic survival. Cells were treated with 10 nM (Z_HER2∶342)₂ for 2 hours prior to irradiation and allowed to recover for 16 hours before reseeding. Mean values and standard deviations are calculated on at least 7 replicates. The linear quadratic model was used for curve fitting and an unpaired t-test to test for significance. A) SKBR-3. Inset; after irradiation with 8 Gy the cells treated with (Z_HER2∶342)₂ had a significantly lower (<i>P</i><0.01) survival than the control group. B) MCF-7 cells. Inset; after irradiation with 8 Gy the cells treated with (Z_HER2∶342)₂ did not differ from the control group. C) Growth extrapolation method. SKBR-3 cells were treated with 16.6 nM (Z_HER2∶342)₂, Z_HER2∶342 or left untreated for two hours, irradiated and then reseeded. The figure shows the number of cells normalized to the starting value in each group after 28 days of cultivation in normal cell culture medium.</p

Receptor dimerization.

<p>SKBR-3 cells were treated with 10 nM (Z_HER2∶342)₂ for 5, 15, 60 and 120 minutes. Z_HER2∶342 was used at 100 nM for 1 h. Untreated cells (−), and cells treated with EGF and nrg1-β1 for 15 minutes were used as controls. The SKBR-3 cells were cross-linked by 5 nM BS³ for 30 minutes before cell lysis. Total cell lysates were then subjected to SDS-PAGE and western blot with antibodies specific for HER2 (A) and EGFR (B). (Z_HER2∶342)₂ treatment resulted in dimerization of HER2 even after 5 minutes of incubation. Numbers indicate minutes of stimulation.</p

The binding trace of ¹²⁵I-(Z_HER2∶342)₂ to SKBR-3 cells.

<p>The interaction was monitored in real-time at room temperature using LigandTracer Grey. A) In the first experiment two concentrations of ¹²⁵I-(Z_HER2∶342)₂ were added after each other. First, cells were incubated with 58 pM (Z_HER2∶342)₂ and thereafter, when equilibrium had been reached, more substance was added to a total concentration of 174 pM. B) In the second experiment, the off-rate after equilibrium of 174 nM exposure was followed for 24 hours. Data evaluation and estimation of the kinetic parameter K_D were performed using the software TraceDrawer, using a one-to-one binding model with depletion correction. CPS (Counts per second).</p

Analyzing alpha emitting isotopes of Pu, Am and Cm from NPP water samples : An intercomparison of Nordic radiochemical laboratories

Radioanalytical methods for the determination of isotopes of Pu, Am and Cm in water samples from nuclear power plants were compared and further developed in a Nordic project (Optimethod) through two intercomparison exercises among Nordic laboratories. With this intercomparison, the analytical performance of some laboratories was improved by modification of the analytical method and adopting new techniques. The obtained results from the two intercomparisons for alpha emitting transuranium isotopes are presented, and the lessons learnt from these intercomparison exercises are discussed.Peer reviewe

Prevalence of severe asthma : results from a population-based study

Introduction: Research on severe asthma based on population samples is scarce and defining severe asthma in epidemiological samples is a challenge. Aims and objectives: To investigate the prevalence and clinical features of severe asthma in a population-based study. Methods: In the West Sweden Asthma Study, a randomly selected sample (N=1172) and a separate asthma sample (N=744) underwent clinical examinations. Severe asthma required at least medium dose inhaled corticosteroids (ICS) plus a second controller or oral corticosteroids (OCS) based on GINA steps 4 and 5. The prevalence was calculated in the random sample and clinical features were assessed in the asthma sample. Asthma control was assessed with the GINA Symptom Control Tool. Results: In the random sample (mean age 50.4y, 54% women), 138 subjects had current asthma (12%) and 12 (1%) had severe asthma. In the asthma sample, 94.4% of the 72 subjects with severe asthma used ICS + LABA, 4.2% used ICS + inhaled anticholinergic and 1.4% used ICS + OCS. Subjects with severe asthma were older vs. non-severe asthma (N=672), 52.1y vs. 47.6y (p=0.02). They had both lower FEV1% of predicted, 88% vs. 97% (p<0.01) and lower FEV1/FVC%, 73% vs. 77% (p<0.01). Twice as many with severe asthma, 32% vs. 16%, had daily daytime symptoms (p<0.01) and only 19% vs. 44% had controlled asthma (p<0.01). They also had more exacerbations and emergency visits the last 12 months (p=0.01), but did not differ in terms of gender or allergy status. Conclusions: The prevalence of severe asthma on population level was 1%. Subjects with severe asthma had poorer lung function and asthma control and more symptoms than subjects with non-severe asthma

Severe Asthma in a General Population Study : Prevalence and Clinical Characteristics

Purpose: Current guidelines primarily use medication levels to distinguish severe asthma from other types of asthma. In addition, severe asthma must also be uncontrolled at high-intensity treatment or become uncontrolled if treatment level is decreased. To date, only a few studies have used this definition to investigate the prevalence and clinical characteristics of severe asthma in population-based samples. Therefore, the aim of this study was to evaluate the prevalence and clinical characteristics of individuals with severe asthma in the population-representative West Sweden Asthma Study. Materials and Methods: In this cross-sectional population-based study, a randomly selected sample (n=1172) and a separate asthma sample (n=744) underwent clinical examinations, completed a structured interview and responded to questionnaires. Severe asthma was defined as at least one feature of uncontrolled asthma despite treatment in line with the Global Initiative for Asthma (GINA) steps 4/5. This treatment level required a minimum medium dose of inhaled corticosteroids (ICS) plus a second controller or oral corticosteroids. Results: The prevalence of severe asthma was 1.1% in the adult random sample and 9.5% within the asthma sample. Individuals with severe asthma were older and had more symptoms, activity limitations, heart disease and blood neutrophils compared to those with other asthma. They also had lower lung function and despite these impairments, 32% did not have annual contact with a healthcare provider. Conclusion: The prevalence of severe asthma was higher compared to previous studies, and many individuals with severe asthma did not have regular contact with healthcare providers. Due to the high burden of symptoms and impairments for individuals with severe asthma, it is important that the healthcare system implement strategies to improve follow-up and evaluate these patients according to existing guidelines