Heide Kogelberg

Nanoparticles functionalised with recombinant single chain Fv antibody fragments (scFv) for the magnetic resonance imaging of cancer cells

Superparamagnetic iron oxide nanoparticles (SPIONs) can substantially improve the sensitivity of magnetic resonance imaging (MRI). We propose that SPIONs could be used to target and image cancer cells if functionalized with recombinant single chain Fv antibody fragments (scFv). We tested our hypothesis by generating antibody-functionalized (abf) SPIONs using a scFv specific for carcinoembryonic antigen (CEA), an oncofoetal cell surface protein. SPIONs of different hydrodynamic diameter and surface chemistry were investigated and targeting was confirmed by ELISA, cellular iron uptake, confocal laser scanning microscopy (CLSM) and MRI. Results demonstrated that abf-SPIONs bound specifically to CEA-expressing human tumour cells, generating selective image contrast on MRI. In addition, we observed that the cellular interaction of the abf-SPIONs was influenced by hydrodynamic size and surface coating. The results indicate that abf-SPIONs have potential for cancer-specific MRI.

New structural insights into lectin-type proteins of the immune system.

New structural data have emerged for the ligand-binding sites of C-type lectin domains and C-type lectin-like domains of receptors of the immune system. These include binding sites for oligosaccharide or polypeptide ligands, or both oligosaccharide and polypeptide ligands. The structural basis for the binding of a lectin domain of the beta-trefoil family to different sulfooligosaccharide sequences has been revealed. Lectin activity has been documented for a beta/alpha TIM barrel fold that does not have the chitinase activity of the prototype enzyme with this fold.

Inhibition of Adhesion of Plasmodium falciparum-Infected Erythrocytes by Structurally Defined Hyaluronic Acid Dodecasaccharides

ABSTRACT We recently reported that Plasmodium falciparum-infected erythrocytes (IRBCs) can adhere to hyaluronic acid (HA), which appears to be a receptor, in addition to chondroitin sulfate A (CSA), for parasite sequestration in the placenta. Further investigations of the nature and specificity of this interaction indicate that HA oligosaccharide fragments competitively inhibit parasite adhesion to immobilized purified HA in a size-dependent manner, with dodecasaccharides being the minimum size for maximum inhibition. Rigorously purified and structurally defined HA dodecasaccharides, free of contamination by CSA or other glycosaminoglycans, effectively inhibited IRBC adhesion to HA but not CSA, providing compelling evidence of a specific interaction between IRBCs and HA.

Engineering a Single Chain Fv Antibody to αvβ6 Integrin using the Specificity-Determining Loop of a Foot-and-Mouth Disease Virus

The alpha v beta 6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-beta1 and transforming growth factor-beta 3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein 1 (VP1) coat protein of the O(1) British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for alpha v beta 6, and we recently reported that a peptide derived from VP1 exhibited alpha v beta 6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to alpha v beta 6. A 17-mer peptide of VP1 was inserted into the complementarity-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to alpha v beta 6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with alpha v beta 6 but not with alpha 5 beta 1, alpha v beta 3, alpha v beta 5, alpha v beta 8 or CEA. B6-2 was internalized into alpha v beta 6-expressing cells and inhibited alpha v beta 6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of alpha v beta 6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block alpha v beta 6-mediated cancer cell invasion or to deliver and internalize toxins specifically to alpha v beta 6-expressing tumors.

NMR studies of mannitol-terminating oligosaccharides derived by reductive alkaline hydrolysis from brain glycoproteins

Interest in the characterisation of O-mannosyl glycan structures has been stimulated following the identification of mannitol-terminating oligosaccharides among the chains released from mammalian proteins in nervous and muscle tissues, and by the discovery of a putative human O-mannosyl transferase. Several mass spectrometry methods have been applied to structure elucidation particularly when low amounts of oligosaccharide are available for analysis. However, when sufficient amounts are available, a combination of through-bond homo- and heteronuclear, and of through-space homonuclear NMR experiments permit the complete identification of these oligosaccharide sequences. We describe here the assignment of 1H and 13C NMR chemical shifts from such experiments for four mannitol-terminating oligosaccharide alditols, GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)[Fucalpha-(1-->3)]GlcNAcbeta-(1-->2)Manol and NeuAcalpha-(2-->3)Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, that were released from brain glycopeptides by alkaline borohydride treatment.

Binding of Sucrose Octasulphate to the C‐Type Lectin‐Like Domain of the Recombinant Natural Killer Cell Receptor NKR‐P1A Observed by NMR Spectroscopy

NKR‐P1A is a C‐type lectin‐like receptor on natural killer cells believed to be involved in the cytotoxicity of these cells. Ligands for this protein are not known. Here, we describe the binding of a fully sulphated disaccharide, sucrose octasulphate, by the recombinant C‐type lectin‐like domain of NKR‐P1A. The binding was observed by NMR spectroscopy methods that have recently been described for the screening of compound libraries for bioaffinities, namely the 2D NOESY and saturation transfer difference NMR experiments. 1H titration studies indicate that the binding is specific. These findings raise the possibility that NKR‐P1A recognises sulphated natural ligands in common with certain other members of the C‐type lectin family.

Generation and Characterization of a Diabody Targeting the αvβ6 Integrin

The αvβ6 integrin is up-regulated in cancer and wound healing but it is not generally expressed in healthy adult tissue. There is increasing evidence that it has a role in cancer progression and will be a useful target for antibody-directed cancer therapies. We report a novel recombinant diabody antibody fragment that targets specifically αvβ6 and blocks its function. The diabody was engineered with a C-terminal hexahistidine tag (His tag), expressed in Pichia pastoris and purified by IMAC. Surface plasmon resonance (SPR) analysis of the purified diabody showed affinity in the nanomolar range. Pre-treatment of αvβ6-expressing cells with the diabody resulted in a reduction of cell migration and adhesion to LAP, demonstrating biological function-blocking activity. After radio-labeling, using the His-tag for site-specific attachment of 99mTc, the diabody retained affinity and targeted specifically to αvβ6-expressing tumors in mice bearing isogenic αvβ6 +/− xenografts. Furthermore, the diabody was specifically internalized into αvβ6-expressing cells, indicating warhead targeting potential. Our results indicate that the new αvβ6 diabody has a range of potential applications in imaging, function blocking or targeted delivery/internalization of therapeutic agents.