Monoclonal antibody arming

Recently, monoclonal antibodies were attached to gas-filled microbubbles using this spacer coupHng technology. Testing in vitro, in a cell culture system, demonstrated selective accumulation of decafluorobutane-based lipid shell MP1950 microbubbles with covalently attached anti-lCAM-1 antibodies, onto the surface of activated endotheUum [8]. Anti-P-selectin antibodies attached to such microbubbles via an avidin-biotin scheme showed selective accumulation in the areas of inflammation and ischemia/reperfusion injury in an animal model [93]. In the latter example, biotin was also connected to the microbubble surface via a flexible polymer spacer arm. 

Recently, Sen et al. (2001) have described methods for generating highly effective HER-2-specific cytotoxic T cells by arming activated T cells with anti-CD3 X anti-HER-2 bispecific antibody. In this method OKT3 and 9184 anti-HER-2 monoclonal antibodies were conjugated and used to arm T cells that were subsequently tested for binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregate and selectively kill HER-2-positive breast cancer cells (MCF-7). 

More recently, this model has undergone substantial modification. First, it became apparent from biosynthetic studies on cells in culture that there were two distinct B chains, B1 and B2 (Cooper et al., 1981), which were not well resolved in laminin prepared from the EHS tumor. Analysis of the stoichiometry of the chains produced in these systems suggested that the laminin molecule contained one A, one Bl, and one B2 chain (Fig. 9). Studies with a monoclonal antibody specific for the A chain indicated that the A chain formed part of the short arm as well as the long arm of laminin (Palm et al., 1985). The assembly of the molecule appears to proceed in discrete steps, with the initial assembly of a B1-B2 dimer linked by disulfide bonds to which an A chain is added (Morita et al., 1985 Peters et al., 1985). 

Immobilized histidine on agarose beads by means of aminohexyl spacer arm has also been utilized to purify polyclonal217 and monoclonal antibodies.218 Adsorption of IgG on histidine-agarose occurs in the presence of 25 mM Tris-HCl buffer, pH 7.4, and elution is achieved using a solution of 0.2 M sodium chloride in the same buffer. In described conditions the binding capacity of antibodies was about 11 mg/mL of resin and purity of separated antibody was estimated by the authors at around 98% when combined with ethanol precipitation. Dissociation constant was also determined and reported between 2.4 X 10-6 and 4.6 X 10-6 M. 

The VEGF trap was later evaluated as an intravitreal injection in a phase I trial, structured without a placebo study arm. Three patients at each of 4 dose levels (0.05-, 0.15-, 0.5-, and 1.0-mg) have received the study medication. Preliminary results note that excess foveal thickness was reduced by at least 70% in 75% of patients, and acuity was stable or improved in 75% of subjects. Although this drug is in preliminary clinical trials, it offers an approach to VEGF inhibition that is different from aptamer or monoclonal antibody strategies. 

This study was followed up with reconstructions of CPMV complexed with Fab fragments from monoclonal antibodies 5B2 and 10B7 as well as IgCs from 5B2 [25]. The IgG was observed to bind in a monodentate fashion with only one Fab arm attached to the virus surface. Fab fragments from 5B2 and 10B7 bound to nearly identical positions and both were used to identify the 30 amino acids covered by the Fab arms. In this study, the unbound Fc and Fab arms were disordered and formed islands above the bound antibodies. 

Debaene, F., Wagner-Rousset, E., Colas, O., Ayoub, D., Corvaia, N., Van Dorsse-laer. A., Beck, A., Cianferani, S. (2013) Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and Bispecific Monoclonal Antibody Formation. Anal. Chem. 85 9785-9792. 

Protein A/G-Ig complex can be stabilized with the bifunctional crosslinker dimethyl pimelimidate (24, 25). In this form the antibody on the column is oriented with the Fab arms free from the matrix and available for antigen binding Protocol 4). Complete kits for immobilization of antibody on Protein A or G are marketed by Pierce (IgG Orientation Kit), but any source of Protein A-or Protein G-agarose will do. The differences between Proteins A and G lie in the specificity for different immimoglobulin isotypes and species. Protein G in particular binds to rat IgG isotypes that Protein A does not. The majority of monoclonal antibodies made by conventional ceU fusion will be IgGs of mouse or rat origin, so Table 2 shows the relative affinities of these two proteins for different rodent IgG isotypes. 

Geha R, Quinti I, Austen K, Cicardi M, Sheffer A, Rosen F Acquired Cl-inhibitor deficiency associated with anti-idiotypic antibody to monoclonal immunoglobuhns. N Engl J Med 1985 312 534-540. Donaldson V, Hess E, McAdams A Lupus-erythe-matosus-like disease in three umelated women with hereditary angioneurotic edema. Arm Intern Med 1977 86 312-313. 

---