Tove L.J. Eriksson

Directed Evolution to Low Nanomolar Affinity of a Tumor-Targeting Epidermal Growth Factor Receptor-Binding Affibody Molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry

Noninvasive detection of epidermal growth factor receptor (EGFR) expression in malignant tumors by radionuclide molecular imaging may provide diagnostic information influencing patient management. The aim of this study was to evaluate a novel EGFR-targeting protein, the ZEGFR:1907 Affibody molecule, for radionuclide imaging of EGFR expression, to determine a suitable tracer format (dimer or monomer) and optimal label. Methods: An EGFR-specific Affibody molecule, ZEGFR:1907, and its dimeric form, (ZEGFR:1907)2, were labeled with 111In using benzyl-diethylenetriaminepentaacetic acid and with 125I using p-iodobenzoate. Affinity and cellular retention of conjugates were evaluated in vitro. Biodistribution of radiolabeled Affibody molecules was compared in mice bearing EGFR-expressing A431 xenografts. Specificity of EGFR targeting was confirmed by comparison with biodistribution of non–EGFR-specific counterparts. Results: Head-to-tail dimerization of the Affibody molecule improved the dissociation rate. In vitro, dimeric forms demonstrated superior cellular retention of radioactivity. For both molecular set-ups, retention was better for the 111In-labeled tracer than for the radioiodinated counterpart. In vivo, all conjugates accumulated specifically in xenografts and in EGFR-expressing tissues. The retention of radioactivity in tumors was better in vivo for dimeric forms; however, the absolute uptake values were higher for monomeric tracers. The best tracer, 111In-labeled ZEGFR:1907, provided a tumor-to-blood ratio of 100 (24 h after injection). Conclusion: The radiometal-labeled monomeric Affibody molecule ZEGFR:1907 has a potential for radionuclide molecular imaging of EGFR expression in malignant tumors.

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

Identification and Characterisation of Two Allergens from the Dust Mite Acarus siro, Homologous with Fatty Acid–Binding Proteins

Background: Dust mites are a major cause of allergic disease worldwide. The dust mite Acarus siro is an inducer of occupational allergy among farmers, but sensitisation has also been found in non–farming populations. Methods: A degenerate primer was designed to the N–terminal amino acid sequence of a 15–kD IgE–binding protein in A. siro extract. The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques. The protein was expressed in Escherichia coli with a 6–histidine tag at its C–terminus. Immunoblotting of the recombinant protein and whole extract was performed using patient sera. Results and conclusion: 15 and 17–kD allergens were identified in a fraction of A. siro extract. The cDNA of the 15–kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein. The calculated molecular weight of the cDNA–encoded protein is 14.2 kD. The predicted amino acid sequence has one potential N–glycosylation site at position 4–6 and a cytosolic fatty acid–binding protein signature at position 5–22. The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite Blomia tropicalis, as well as homology with several other fatty acid–binding proteins (FABPs) from different organisms. The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated. The amino acid sequence of the 17–kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.

A novel adjuvant-allergen complex, CBP-rFel d 1, induces up-regulation of CD86 expression and enhances cytokine release by human dendritic cells in vitro

Allergen‐specific immunotherapy is commonly performed with allergen extracts adsorbed to aluminium hydroxide (alum). The undesirable effects associated with the use of alum, including granuloma formation at the site of injection and stimulation of T helper 2 (Th2) cytokine production, has generated interest in alternative allergen carriers, one being carbohydrate‐based particles (CBPs). Here, we have investigated the in vitro effects of the recombinant major cat allergen Fel d 1 (rFel d 1) coupled to CBPs (CBP–rFel d 1) on human monocyte‐derived dendritic cells (MDDCs) obtained from healthy blood donors. A majority of the CD1a+ MDDCs internalized fluorescein isothiocyanate‐labelled CBP–rFel d 1, as demonstrated by flow cytometry and confocal laser‐scanning microscopy. Furthermore, an up‐regulation of the expression of the costimulatory molecule, CD86, on the MDDCs was induced by CBP–rFel d 1, but not by rFel d 1 or CBPs alone. Finally, three‐ and fourfold increases in the release of interleukin‐8 and tumour necrosis factor‐α, respectively, were observed when MDDCs were cultured in the presence of CBP‐rFel d 1. Altogether, our results indicate that the use of CBPs as an allergen carrier and adjuvant is a promising candidate for the improvement of allergen‐specific immunotherapy.

Evaluation of specific IgE to the recombinant group 2 mite allergens Lep d 2 and Tyr p 2 in the pharmacia CAP system

Background: Several recombinant allergens have been shown to be potentially useful for diagnosis of IgE–mediated allergy, but only a few recombinant allergens are at present commercially available in serological assays for detection of specific IgE. The aim of this study was to evaluate the IgE binding to the recombinant major dust mite allergens rLep d 2 and rTyr p 2 and compare it with the IgE binding to the commercial mite extracts Lepidoglyphus destructor and Tyrophagus putrescentiae in the Pharmacia RAST CAP System. Methods: The recombinant allergens rLep d 2 and rTyr p 2 were immobilised on ImmunoCAPs, and sera from 461 Swedish farmers who are frequently exposed to mites were analysed for specific IgE antibodies. Immunoblotting was performed to evaluate discrepancies between the results obtained with the recombinant and the commercial CAP assays. Results: The IgE values of each recombinant assay significantly correlated with the IgE values of the corresponding commercial CAP assay. The sensitivity of the rLep d 2 assay was 73.3% and that of the rTyr p 2 assay, 60.5% of that provided by the commercial L. destructor and T. putrescentiae assays. Two subjects out of 416, who tested negative in the commercial L. destructor assay, were positive to rLep d 2. The corresponding figures for rTyr p 2 and the T. putrescentiae extract were 5/418. The possibility that these subjects were sensitised to L. destructor and T. putrescentiae could not be excluded. Conclusions: The data suggest that it may be possible to use rLep d 2 and rTyr p 2 on ImmunoCAPs to detect and quantify IgE antibodies to these, the major allergens of L. destructor and T. putrescentiae. It appears likely that the addition of just a few more recombinant L. destructor and T. putrescentiae allergens in the CAP assay will be sufficient for in vitro diagnosis of IgE mediated allergy to L. destructor and T. putrescentiae.

T cell responses to recombinant isoforms, synthetic peptides and a mutant variant of Lep d 2, a major allergen from the dust mite Lepidoglyphus destructor

Background The dust mite Lepidoglyphus destructor is an important cause of allergic reactions to dust, especially in farming environments. Two isoforms, recombinant (r)Lep d 2.01 and rLep d 2.02, of the major allergen Lep d 2, have previously been expressed as recombinant proteins. These isoforms differ 10.4% at the amino acid level. Furthermore, a mutant form of Lep d 2.01 (rLep d 2.6Cys) with a highly reduced IgE reactivity, has also been produced.