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...