Lars Gedda

Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family

Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo‐ and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF‐receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein‐protein interactions and post‐translational modifications on a single‐cell basis.

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

HER2 (human epidermal‐growth‐factor receptor‐2; ErbB2) and EGFR (epidermal‐growth‐factor receptor) are overexpressed in various forms of cancer, and the co‐expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2‐binding affibody molecule was genetically fused in‐frame with a bivalent EGFR‐binding affibody molecule via a (G4S)3 [(Gly4‐Ser)3]‐encoding gene fragment. The encoded 30 kDa affibody construct (ZHER2)2–(G4S)3–(ZEGFR)2, with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow‐cytometric analysis, the latter using HER2‐ and EGFR‐overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real‐time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR‐overexpressing cells and soluble HER2; and (iii) a cell–cell interaction analysis where the bs affibody molecule bound HER2‐overexpressing SKBR‐3 cells and EGFR‐overexpressing A‐431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

Hsp90 is expressed and represents a therapeutic target in human oesophageal cancer using the inhibitor 17-allylamino-17-demethoxygeldanamycin.

Heat shock protein 90 (Hsp90) has been demonstrated to protect oncogenic variants of signalling molecules from degradation and may consequently serve as a therapeutic target for the treatment of oesophageal cancer for which adequate therapy is often lacking. We studied the expression of Hsp90 in tumour tissues of human oesophageal cancer and the impact of Hsp90 inhibition on oesophageal cancer cell lines using the drug 17-allylamino-17-demethoxygeldanamycin (17-AAG). Quantitative immunohistochemistry was performed on formalin-fixed paraffin-embedded tissues from patients with oesophageal cancer. In squamous cell carcinoma, a marked upregulation of Hsp90 could be noted in dysplastic epithelium and invasive cancer compared with normal epithelium. In adenocarcinoma, Hsp90 was expressed in neoplastic epithelium and also in normal non-neoplastic glands weakly. The inhibition of Hsp90 using 17-AAG led to a significant decrease in cell proliferation and viability in human oesophageal cancer cell lines. Using a clonogenic cell survival assay, Hsp90 inhibition significantly sensitised the cells for γ-photon irradiation. Heat shock protein 90 was found to be critical for proper signalling induced by both epidermal growth factor and insulin-like growth factor-1, in which the inhibition of signalling by 17-AAG correlated with the observed reduction in cell proliferation and viability. These results showed that Hsp90 was selectively expressed in oesophageal cancer tissue compared with the corresponding normal tissue, and the inhibition of Hsp90 resulted in decreased proliferation and viability as well as radiosensitisation of oesophageal cancer cells. Heat shock protein 90 represents a potential therapeutic target in the treatment of patients with oesophageal cancer, alone or in combination with radiotherapy.

Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with 125I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of 125I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with 125I-EGF.

Chemistry and biology of some low molecular weight boron compounds for Boron Neutron Capture Therapy

Boronated DNA targeting agents are especially attractive candidatesfor BNCT because they may deliver boron-10 tothe nuclei of tumor cells. Numerous boron-containing analogshave been synthesized and some have shown promisingresults in initial biological tests. One of themost challenging tasks in this special field ofresearch remains the finding of suitable targeting strategiesfor the selective delivery of boron rich DNA-intercalator/alkylatorto tumor cells. Synthetic and biological studies ofboron compounds suitable for DNA-binding are reviewed.The amino acid p-boronophenylalanine (BPA) is presently ofconsiderable clinical interest. Other boronated amino acids mightalso be candidates for BNCT either per se,as part of part of tumor-seeking peptides orconjugated to targeting macromolecules. A large number ofboronated L- and D-amino acids with varying liphophicilityand sterical requirements are now available for evaluation.Recent synthetic and biological studies of aromatic boronoaminoacids, carboranylamino acids and carboranyl amines are alsoreviewed.

[177Lu]Pertuzumab: Experimental Therapy of HER-2–Expressing Xenografts

Pertuzumab (Omnitarg) is a novel antibody against HER-2, domain II. HER-2 is a tyrosine kinase receptor that is overexpressed in several carcinomas, especially breast cancer. Pertuzumab, labeled with the low-energy beta emitter (177)Lu, might be a candidate for targeted radiotherapy of disseminated HER-2-positive micrometastases. The radiolabeled antibody [(177)Lu]pertuzumab showed favorable targeting properties in BALB/c (nu/nu) mice with HER-2-overexpressing xenografts. The absorbed dose in tumors was more than five times higher than the absorbed dose in blood and more than seven times the absorbed dose in any other normal organ. Experimental therapy showed that [(177)Lu]pertuzumab delayed tumor progression compared with controls (no treatment, P < 0.0001; nonlabeled pertuzumab antibody, P < 0.0001; and (177)Lu-labeled irrelevant antibody, P < 0.01). No adverse side effects of the treatment could be detected. Thus, the experimental results support the planning of clinical studies applying [(177)Lu]pertuzumab for therapy.

Strategy for boron neutron capture therapy against tumor cells with over-expression of the epidermal growth factor-receptor.

PURPOSE Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy. METHODS AND MATERIALS EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made. RESULTS The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF. CONCLUSION The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Ligand liposomes and boron neutron capture therapy.

Boron neutron capture therapy (BNCT) has been used both experimentally and clinically for the treatment of gliomas and melanomas, with varying results. However, the therapeutic effects on micro-invasive tumor cells are not clear. The two drugs that have been used clinically, p-boronophenylalanine, (BPA), and the sulfhydryl borane, (BSH), seem to be taken up preferentially in solid tumor areas but it is uncertain whether enough boron is taken up by micro-invasive tumor cells. To increase the selective uptake of boron by such cells, would be to exploit tumor transformation related cellular changes such as over-expression of growth factor receptors. However, the number of receptors varies from small to large and the uptake of large amounts of boron for each receptor interaction is necessary in order to deliver sufficient amounts of boron. Therefore, each targeting moiety must deliver large number of boron atoms. One possible way to meet these requirements would be to use receptor-targeting ligand liposomes, containing large number of boron atoms. This will be the subject of this review and studies of boron containing liposomes, with or without ligand, will be discussed. Two recent examples from the literature are ligand liposomes targeting either folate or epidermal growth factor (EGF) receptors on tumor cells. Other potential receptors on gliomas include PDGFR and EGFRvIII. Besides the appropriate choice of target receptor, it is also important to consider delivery of the ligand liposomes, their pharmacodynamics and pharmacokinetics and cellular processing, subjects that also will be discussed in this review.

A New Antibody Recognizing the vIII Mutation of Human Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) gene is frequently amplified and the receptor overexpressed in different types of human tumors. Furthermore, genomic rearrangements can cause expression of modified receptors, as one frequently occurring truncated form, EGFRvIII. This mutated receptor has previously been described and is formed by a 267-amino acid in-frame deletion and an insertion of a glycine in the fusion junction of the extracellular domain. EGFRvIII is a tumor-specific marker and therefore of interest for diagnostic and therapeutic applications. In this study we report on a new monoclonal antibody (Ua30:2) directed to the mutation site of EGFRvIII. The antibody was generated by immunization of mice with a synthetic peptide corresponding to the mutated sequence of the receptor. The affinity of Ua30:2 was found to be high [Kd = 45 nM (Biacore) and 80 nM (saturation analysis)]. Immunohistochemistry in tissue sections from human gliomas demonstrated a similar expression pattern for Ua30:2 as for the recently characterized antibodies L8A4 and DH8.3. The antibody binding was EGFRvIII specific with no measurable cross-reactivity to the wild-type receptor, wtEGFR, as analyzed both with displacement analysis, Western blots and immunohistochemistry. The new antibody is a candidate for radioimmunotargeting aiming at diagnostic and therapeutic applications.