Site-specific covalent modification of monoclonal antibodies: in vitro and in vivo evaluations.

Abstract

drugs (9, 26-28) in attempts to target these agents to tumors. These latter modifications all involve covalent attachment to tyrosines, e-amino side chains of lysines, carboxyl side chains of aspartic and glutamic acids, or sulfhydryl groups generated by reduction of cystines. Although each of these applications has shown promise, none, as yet, has reached the point of proven clinical utility. Since so many of the diagnostic and therapeutic applications envisioned for monoclonal antibodies require coupling of antibodies to other substances, there is a need for methods of covalent modification that can be used on a broad spectrum of different antibodies with minimal effect on antigen binding properties. This paper will present a strategy for site-specific covalent modification of antibodies based upon attachment through the oligosaccharide moiety of the molecule. Because of the restricted localization of the glycosylation sites on immunoglobulins, such an approach offers the potential advantage of modification of the antibodies at a site distal to the antigen combining site (29). Data will be presented that first compares the effect of various techniques of monoclonal antibody modification on the antigenbinding homogeneity and affinity of the conjugate. Second, the comparative in vivo biodistribution and tumor localization of additional radiolabeled monoclonal antibodies will be presented using nude mice bearing subcutaneous tumor xenografts. MATERIALS AND METHODSIn Vitro Binding. The mouse monoclonal anti-phosphocholine IgM, HPCM2 (30), was grown in BALB/c mice and purified from ascites by ammonium sulfate precipitation, followed by affinity chromatography (31). The following conjugates of HPCM2 were prepared.(i) For oligosaccharide attachments, the oligosaccharides moieties were oxidized to aldehydes by incubation in the dark with 10-30 mM NaIO4 in phosphate-buffered saline (0.15 M NaCl/0.01 M sodium phosphate) at pH 6.0 on ice for 1 hr. After passage through a Sephadex G-25 column equilibrated with phosphate-buffered saline at pH 6.0, oxidized antibody was incubated with a 270-fold molar excess of 1,6-diaminohexylethylenediaminedi-o-hydroxyphenylacetic acid (1,6-diaminohexyl-EDDHA) § for 1 hr at room temperature. Sodium cyanoborohydride (Aldrich) was added to a final concentration of 10 mM, and the solution was incubated for an additional 4 hr and then dialyzed at 4°C versus several changes of phosphate-buffered saline.(ii) For aspartic/glutamic acid attachments, the antibody was incubated in 20 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma) at pH 5.9 for 2 hr at room temperature. The 1,6-diaminohexyl-EDDHA was added to a final concentration of 10 mM, and the solution was incubated at room temperature for an additional 2 hr. Ten microliters of 1 M ethanolamine was added, and, after 1 hr the reaction mixture was dialyzed versus phosphate-buffered saline.(iii) For lysine attachments, the scheme for aspartic/glutamic acid modification was used except that EDDHA (5 mM; Sigma) replaced the 1,6-diamino...

Lysine-Directed Conjugation of Ethidium Homodimer to B72.3 Antibody: Retention of Immunoreactivity but Altered Tumor Targeting

“…For example, Sakahara et al (13), assessing the in vitro affinity of two MAb undergoing identical labeling procedures and of the same MAb radiolabeled with two different isotopes, found that the data did not reflect the in vivo behavior of these conjugates. Rodwell et al (9) compared the in vivo behavior of R9.75 MAb iodinated by either conventional tyrosine-directed electrophilic labeling or an iodinated tyrosine-containing peptide site-specifically attached to the oxidized oligosaccharides present on the F c region of the antibody. In these studies, the site-specifically radiolabeled antibody localized in tumors with an 18-fold greater efficiency than the corresponding conjugate modified nonselectively on its tyrosines, despite the fact that in vitro both radioimmunoconjugates had comparable binding properties with the antigen.…”

Antibody-targeted photolysis: in vitro studies with Sn(IV) chlorin e6 covalently bound to monoclonal antibodies using a modified dextran carrier.

“…For example, using direct attachment of radionuclide chelators to the carbohydrate of mAbs, Rodwell et al (16) reported chelator:mAb ratios of 5. To increase the ratio of active agent covalently coupled to mAb, polymeric carriers have been used as bridges between the active agent and the mAb (14,34,35 (Fig. 3) showed a sublethal damage region below 10 J/cm2 followed by logarithmic cell killing to nearly complete cell death at higher light doses.…”

“…Most direct coupling procedures have modified amino acid side chains (13,15) and produced structurally heterogenous populations of immunoconjugates with a range of binding properties (16). Efforts to direct the PS to a particular site on the mAb (e.g., carbohydrate) have produced more homogenous conjugates but appear limited in the number of PSs that can be coupled to a single mAb.…”

Development of a Platform of Antibody-Presenting Liposomes

“…The association of IgGs to liposomes is usually realized by chemical conjugation via hetero-bifunctional crosslinkers on the polar head of lipid molecules modified with a PEGylated reactive group [13]. This strategy of covalent coupling presents however several drawbacks, principally the lack of control of the site of protein modification which involves commonly their most accessible and/or most reactive protein amino groups, so that their molecular recognition properties may be altered due to chemical modification and/or non-optimal orientation [14]. The only method which ensures a favorable IgG orientation and preserves their binding domains involves IgG anchoring via carbohydrate residues located in their Fc domain to lipid molecules exposing hydrazide groups; this strategy presents however a low efficiency [15].…”