The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z HER2:342 showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging. (Cancer Res 2006; 66(8): 4339-48)

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The complete cDNA encoding a major allergen from the dust mite Tyrophagus putrescentiae, Tyr p 2, has been sequenced and expressed. A degenerate primer was designed to the N-terminal amino acid sequence of the 16-kDa protein. The complete cDNA sequence was achieved by using reverse transcriptase PCR, PCRϩ1, standard cloning and sequencing techniques. The cDNA of Tyr p 2 is 552 nucleotides in length from the start codon including 126 nucleotides after the stop codon up to the beginning of the poly(A) tail. The leader sequence consists of 15 amino acids. Regarding the predicted amino acid sequence, there are no potential N-glycosylation sites (N-X-S/T). The sequence showed similarity to group II allergens from other mite species, and some regions are completely conserved. To show that the cloned cDNA sequence was coding for an allergen, Tyr p 2 was expressed in Escherichia coli and shown to react with a T. putrescentiae-positive serum pool.Keywords : mite ; Tyrophagus putrescentiae; Tyr p 2 ; cDNA cloning ; protein expression.Dust mites are world wide the major cause of allergic dis-phoresis (CRIE). We have previously, by SDS/PAGE and immunoblotting, identified four allergens from T. putrescentiae eases, such as asthma and rhinitis [1]. The allergenic role of the dust mite Tyrophagus putrescentiae is an important inducer of whole extract in the molecular-mass range 16Ϫ33 kDa. The major allergen of T. putrescentiae was a 16-kDa component recogallergic asthma and allergic rhinitis among farmers [2Ϫ5], grain workers [6], bakers [7] and food industrial workers [8]. T. pu-nized by about 80% of sera RAST positive to this mite species [18]. We here describe the complete cDNA sequence encoding trescentiae has also been found in house dust and beds [9, 10], and sensitization to T. putrescentiae has been shown in urban this major allergen of T. putrescentiae. We have named it Tyr p 2 because of its similarity with group II allergens from other populations [11, 12]. Recently, Matsumoto and colleagues [13] reported systemic anaphylaxis caused by eaten food contami-dust mite species. nated with T. putrescentiae.Several allergens produced by dust mite species Dermato-MATERIALS AND METHODS phagoides have been cloned and their biological functions are Purification and N-terminal amino acid sequencing. known to some extent [14]. More recently, molecular characterWhole mite culture of T. putrescentiae (Allergon AB) was exization of allergens from the dust mite Lepidoglyphus destructor tracted and lyophilised as previously described [18]. 3.5 mg of has been performed, and the complete cDNA encoding two protein was then fractionated by the Micro Separation System isoallergens of the major allergen Lep d 2 (a group II allergen), 130 A and a reverse-phase (RP) 300 column (30 mm ϫ2.1 mm) has been sequenced [15]. It has been demonstrated that the 7 µ (Applied Biosystem, Inc.) with the gradient A (0.1% trigroup II allergens are of major importance in allergic disease, as fluoroacetic acid) and B (acetonitrile) : t ϭ 0, B ϭ 20%; t ϭ ove...

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L. destructor-sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and express new allergens from L. destructor and determine their recognition frequency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor-sensitized individuals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His 6 -tagged proteins. Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L. destructor-sensitized subjects, respectively.

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.

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 wildtype 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.The large increase in allergic diseases observed during the past decades has urged the development of safe and efficient treatments of allergy. Many new concepts based on targeting the underlying immunological causes of IgE-mediated allergy have been suggested (1-5). However, the only treatment that causes a long-lasting relief of symptoms is allergen-specific immunotherapy (ASIT).3 Today ASIT is based on the repeated administration of allergen extracts prepared from the allergen source. Although successful clinical outcomes are well documented, several problems are associated with allergen extractbased ASIT, including the risk of inducing local and systemic side effects and induction of new sensitizations (6, 7).Large batches of well defined single allergen components can be produced using recombinant techniques, making it feasible to solve problems linked to allergen extract-based ASIT. The most severe side effects of ASIT are caused by the binding of injected allergen to allergen-specific IgE on high affinity Fc⑀RI receptor-bearing effector cells, leading to cross-linking of the Fc⑀RI receptors, degranulation, and release of anaphylactogenic mediators. Therefore, allergens with reduced IgE binding capacity have been proposed to improve the safety of ASIT (8...