Maria Wikman

Affibody-mediated tumour targeting of HER-2 expressing xenografts in mice

PurposeTargeted delivery of radionuclides for diagnostic and therapeutic applications has until recently largely been limited to receptor ligands, antibodies and antibody-derived molecules. Here, we present a new type of molecule, a 15-kDa bivalent affibody called (ZHER2:4)2, with potential for such applications. The (ZHER2:4)2 affibody showed high apparent affinity (KD=3xa0nM) towards the oncogene product HER-2 (also called p185/neu or c-erbB-2), which is often overexpressed in breast and ovarian cancers. The purpose of this study was to investigate the in vivo properties of the new targeting agent. MethodsThe biodistribution and tumour uptake of the radioiodinated (ZHER2:4)2 affibody was studied in nude mice carrying tumours from xenografted HER-2 overexpressing SKOV-3 cells.ResultsThe radioiodinated (ZHER2:4)2 affibody was primarily excreted through the kidneys, and significant amounts of radioactivity were specifically targeted to the tumours. The blood-borne radioactivity was, at all times, mainly in the macromolecular fraction. A tumour-to-blood ratio of about 10:1 was obtained 8xa0h post injection, and the tumours could be easily visualised with a gamma camera at this time point.ConclusionThe results indicate that the (ZHER2:4)2 affibody is an interesting candidate for applications in nuclear medicine, such as radionuclide-based tumour imaging and therapy.

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

Selection and characterization of an HIV-1 gp120-binding affibody ligand

To evaluate the possibility of generating novel proteins binding to highly glycosylated viral proteins, affibody ligands were selected by bacteriophage display technology to the HIV‐1 envelope glycoprotein gp120 (glycoprotein 120), from a combinatorial protein library based on the 58‐amino‐acid‐residue staphylococcal Protein A domain. The predominant variant from the bacteriophage selection was produced in Escherichia coli and characterized by biosensor analyses. Both univalent and bivalent affibody molecules were shown to bind selectively to the gp120 target molecule in a biosensor analysis. The dissociation equilibrium constants (KD) were determined to be approx. 100 nM for the univalent affibody and 10 nM for the bivalent affibody, confirming the stronger gp120 binding of the bivalent affibody ligand. The affibody constructs were further introduced into the Ad5 (adenovirus type 5) fibre gene, and the recombinant fibres were shown to bind selectively to gp120 in a biosensor analysis and to gp160 transiently expressed in African‐green‐monkey (Cercopithecus aethiops) kidney cells. Neither the affibody ligand nor the Ad5 fibres showed any virus neutralization activity, suggesting that the affibody bound to a non‐neutralizing site on gp120. To investigate the binding site for the affibody ligand on gp120, CD4 (cluster of differentiation 4) and a panel of mAbs (monoclonal antibodies) known to bind to gp120 were allowed to compete with the affibody ligand in a biosensor study. Two mAbs, 670‐30D and 697‐30D, were found to compete with gp120 for overlapping binding sites. Although neutralization effects were not achieved in this initial investigation, the successful selection of a gp120‐binding affibody ligand indicates that future affibody‐based strategies might evolve to complement antibody‐based efforts for HIV‐1 therapy. Strategies for directed selection of affibody ligands binding to neutralizing epitopes and the potential of using adenovirus for gene‐therapy‐mediated efforts are discussed.

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (KD≈10−15 M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His6‐tagged SA fusion protein (His6–SA) was bound to Ni2+‐loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA‐coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53‐amino‐acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood‐stage antigen Pf155/RESA, and a 232‐amino‐acid segment (SRS2′) from the central region (from Pro‐97 to Lys‐328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen‐containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high‐titre antigen‐specific antibody responses on immunization of mice. For the particular target immunogen SRS2′, the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

Achieving directed immunostimulating complexes incorporation

In recent years, several studies have been reported with the common aim of generating general expression systems for straightforward production and subsequent coupling of expressed antigens to an adjuvant system. Here, we describe a series of such efforts with a common theme of using gene fusion technology for association of recombinant antigens to immunostimulating complexes (iscoms). In the early stages of vaccine development, uniform antigen preparations are crucial to allow the comparison of immune responses to different antigens, or even subdomains thereof, and we believe that the described systems constitute an important development in this context.