Synthesis and evaluation of bifunctional tetrahydroxamate chelators for labeling antibodies with 89Zr for imaging with positron emission tomography

Two novel bifunctional tetrahydroxamate chelators 3 and 4 were synthesized and evaluated for labeling antibodies with ⁸⁹Zr for positron emission tomography imaging. Compared to previously reported tetrahydroxamate chelators 1 and 2 with an iminodiacetamide backbone, 3 and 4 were based on an extended iminodipropionamide and dipropylenetriamine backbone, respectively. Trastuzumab conjugates of 3 and 4 were efficiently labeled with ⁸⁹Zr (>95% radiochemical yield). The in vitro plasma stability of ⁸⁹Zr-4-Trastuzumab and especially ⁸⁹Zr-3-Trastuzumab was greatly improved over previously reported ⁸⁹Zr-1-Trastuzumab and ⁸⁹Zr-2-Trastuzumab, but their demetalation remained higher and faster than ⁸⁹Zr-deferoxamine (DFO)-Trastuzumab. These observations were confirmed by PET imaging and biodistribution in mice, with significant higher bone uptake for ⁸⁹Zr-4-Trastuzumab, followed by ⁸⁹Zr-3-Trastuzumab, and to a lesser extent for ⁸⁹Zr-DFO-Trastuzumab. Molecular modeling showed that 3 and 4 with an extended backbone could form eight-coordinate Zr-complexes as compared to only seven-coordinate Zr-complexes of 1 and 2. Our data suggest further elongation of linker length between hydroxamate motifs of this class of chelators is needed to reach a better Zr-coordination configuration and improve in vivo stability.     

Development of a pre-glycoengineered CHO-K1 host cell line for the expression of antibodies with enhanced Fc mediated effector function

Novel biotherapeutic glycoproteins, like recombinant monoclonal antibodies (mAbs) are widely used for the treatment of numerous diseases. The N-glycans attached to the constant region of an antibody have been demonstrated to be crucial for the biological efficacy. Even minor modifications of the N-glycan structure can dictate the potency of IgG effector functions such as the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Here, we present the development of a glycoengineered CHO-K1 host cell line (HCL), stably expressing β1,4-N-Acetylglucoseaminyltransferase III (GnT-III) and α-mannosidase II (Man-II), for the expression of a-fucosylated antibodies with enhanced Fc-mediated effector function. Glycoengineered HCLs were generated in a two-step strategy, starting with generating parental HCLs by stable transfection of CHO-K1 cells with GnT-III and Man-II. In a second step, parental HCLs were stably transfected a second time with these two transgenes to increase their copy number in the genetic background. Generated glycoengineered CHO-K1 cell lines expressing two different mAbs deliver antibody products with a content of more than 60% a-fucosylated glycans. In-depth analysis of the N-glycan structure revealed that the majority of the Fc-attached glycans of the obtained mAbs were of complex bisected type. Furthermore, we showed the efficient use of FcγRIIIa affinity chromatography as a novel method for the fast assessment of the mAbs a-fucosylation level. By testing different cultivation conditions for the pre-glycoengineered recombinant CHO-K1 clones, we identified key components essential for the production of a-fucosylated mAbs. The prevalent effect could be attributed to the trace element manganese, which leads to a strong increase of a-fucosylated complex- and hybrid-type glycans. In conclusion, the novel pre-glycoengineered CHO-K1 HCL can be used for the production of antibodies with high ratios of a-fucosylated Fc-attached N-glycans. Application of our newly developed FcγRIIIa affinity chromatography method during cell line development and use of optimized cultivation conditions can ultimately support the efficient development of a-fucosylated mAbs.     

Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells

In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.     

Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency

This paper analyses the performance of MAbMax^TM/Tricentric^TM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1_K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene sis, the eDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated.Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells,and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer,colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.     

Imaging of protein layers with an optical microscope for the characterization of peptide microarrays.

Solid-phase assays play a crucial role today in biological studies. These assays are based on the immobilization of probe molecules on a surface, which are able to capture specifically soluble receptors. In particular, peptide microarrays have emerged as powerful tools in a variety of applications. In this context, optical techniques that allow imaging of nanometer-thick biomolecular films, and thereby the characterization of microarrays, are of great interest. For this purpose, we used a recently disclosed wide-field optical imaging technique of surface nanostructures called Sarfus, which is based on the use of a standard optical microscope and antireflection substrates. We demonstrate here that this technique allows the imaging of the protein layers that result from the specific capture of antibodies by arrayed peptide probes with a spatial resolution of 0.45 microm. The relationship between the thickness of the antibody layer and peptide or antibody concentration was examined.     

New monoclonal antibodies against a recombinant second envelope protein of Hepatitis C virus

To study the immunological features of the hepatitis C virus (HCV) envelope protein (E2 protein), new specific monoclonal antibodies (mAbs) were generated. WKA/H rats were immunized with syngeneic cells infected with a vaccinia virus expressing the E2 protein and with soluble E2 protein obtained from Chinese hamster ovary cells with a plasmid-based expression system. By screening hybridoma cells obtained from spleen cells of the immunized rats, three specific mAbs were obtained. One mAb was reactive to a peptide corresponding to the hypervariable region 1 (HVR1) in E2 protein, while the others reacted to regions outside HVR1. The significance of these antibodies for the diagnosis of HCV infection as well as for analysis of the structure of the HCV E2 protein will be discussed.     

A novel pro-apoptosis protein PNAS-4 from <Emphasis Type="Italic">Xenopus laevis</Emphasis>: Cloning,expression, purification,and polyclonal antibody production

Human PNAS-4 was identified as a novel pro-apoptotic protein in mammalian cells. Here we report the cloning, expression, purification, and antibody production of a PNAS-4 homolog (named xPNAS-4) from Xenopus laevis, an extensively used model organism in exploring gene functions during embryonic development. Recombinant histidine-tagged xPNAS-4 protein was expressed in Escherichia coli as insoluble inclusion bodies. The inclusion bodies were subsequently dissolved in 8 M urea and purified to near homogeneity by Ni2+ affinity chromatography. The resulting denatured protein was refolded by stepwise dilution of urea concentration via dialysis. This procedure yielded about 4 mg refolded protein per liter of E. coli culture with a purity of 95%. The purified protein was identified by liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) and used to raise anti-xPNAS-4 polyclonal antibodies that were suitable for detecting the expression of PNAS-4 in X. laevis embryos by Western blotting. The availability of recombinant protein and specific polyclonal antibodies will provide a valuable tool in studying apoptotic mechanisms of this protein. To our knowledge, this is the first report to demonstrate the presence of PNAS-4 in X. laevis.     

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.