Robert Waibel

Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M=Re, Tc) directed towards radiopharmaceutical application

Abstract A review on the synthesis and properties of the organometallic aqua-ion [M(OH2)3(CO)3]+ (M=Re, 99Tc, 99mTc), as relevant for radiopharmaceutical application, is presented. These important starting compounds can be prepared quantitatively, (a) on the no carrier added (n.c.a.) level (99mTc) in water, or (b) in organic solvents (Re, 99Tc) at atmospheric pressure in a short time and from [MO4]−. The main characteristics of these carbonyl complexes are the high substitution stability of the three CO ligands and the substitution lability of the coordinated water molecules. [M(OH2)3(CO)3]+ can be considered as a ‘semi aquo–ion’. On the macroscopic level, upon titration with OH−, hydroxo-bridged oligomers have been isolated and characterized. The formation of hydroxo-bridged complexes is a consequence of the considerable Bronstedt acidity of [M(OH2)3(CO)3]+, whereas on the n.c.a. level no such behavior was observed. Conditions and products of the water exchange by imidazole (im) and derivatives thereof (histamine, histidine) will be presented. The different mononuclear complexes with these ligands are of extraordinary inertness, which is the basis for potential applications in biology and nuclear medicine. Finally, as a basis for bioorganometallic chemistry, the adoption of the results from basic coordination chemistry to the labeling of biomolecules with an organometallic moiety will be exemplified with a selected penta-peptide and a recombinant single chain fragment.

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99mTc(I)–carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Efficient Tumor Targeting with High-Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.

Design of multivalent complexes using the barnase·barstar module

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185HER2-ECD 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5 EFFECTS ON TUMOR TARGETING

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Tumor Targeting of Mono-, Di-, and Tetravalent Anti-p185HER-2 Miniantibodies Multimerized by Self-associating Peptides

Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185HER-2 single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185HER-2 and on SK-OV-3 cells. The99mtechnetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.

New Derivatives of Vitamin B12 Show Preferential Targeting of Tumors

Rapidly growing cells show an increased demand for nutrients and vitamins. The objective of our work is to exploit the supply route of vitamin B12 to deliver new derivatives of this vital vitamin to hyperproliferative cells. To date, radiolabeled ((57)Co and (111)In) vitamin B12 derivatives showed labeling of tumor tissue but also undesired high accumulation of radioactivity in normal tissue. By abolishing the interaction of a tailored vitamin B12 derivative to its transport protein transcobalamin II and therefore interrupting transcobalamin II receptor and megalin mediated uptake in normal tissue, preferential accumulation of a radiolabeled vitamin in cancer tissue could be accomplished. We identified transcobalamin I on tumors as a possible new receptor for this preferential accumulation of vitamin-mediated targeting. The low systemic distribution of radioactivity and the high tumor to blood ratio opens the possibility of a more successful clinical application of vitamin B12 for imaging or therapy.

Anti‐neuroblastoma antibody chCE7 binds to an isoform of L1‐CAM present in renal carcinoma cells

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti‐neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 Mr cell surface protein. The protein was partially purified by immuno‐affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH2‐terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1‐CAM), and both were characterized by the NH2‐terminal deletion of 5 amino acids, comprising exon 2 of L1‐CAM. Reverse trancription‐polymerase chain reaction (RT‐PCR) analysis of the regions spanning exon 2 and exon 27 of L1‐CAM indicated that in neuroblastoma cells both transcripts for the full‐length and exon‐deleted forms are present, whereas in the renal carcinoma cell lines only the exon‐deleted L1‐CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1‐CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1‐CAM mRNA levels are correlated with protein expression. Int. J. Cancer 83: 401–408, 1999

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

Abstract New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 °C and retained their binding specificity.For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

Immunoscintigraphy of patients with head and neck carcinomas, with an anti-angiogenetic antibody fragment

Objective In a phase I/II clinical study, we investigated tumor targeting in patients with head and neck squamous cell carcinomas (SCC), using an antibody directed against the extra-domain-B of fibronectin (EDB), a marker of angiogenesis and tissue remodeling. Study Design And Setting Five patients with SCC were injected with the 123-iodine-radiolabeled L19(scFv)2 antibody and underwent scintigraphic detection with single photon emission tomography with computerized tomography (SPECT/CT). In addition, 18 F-fluorodeoxyglucose ( 18 FDG) positron emission tomography with computerized tomography (PET/CT) was performed. Results Successful targeting of the primary tumor could be achieved in 4 of 5 patients and was comparable to PET imaging. No side effects were observed. Conclusions Tumor targeting with the L19(scFv)2 antibody is also feasible for head and neck SCC. Significance These results may serve as a base for future therapeutical applications in human beings, with modified versions of the L19(scFv)2 antibody, designed to selectively deliver bioactive molecules into malignant tumors.