Reduction−Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides

Abstract

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogenous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60−90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo, and are well tolerated at efficacious doses.Monoclonal antibodies (mAbs) have been used extensively as carriers of fluorophores, radionuclides, cytotoxic agents, and enzymes, yielding conjugates that find utility in therapeutic (1-3) and imaging applications (4,5), ELISA-based assays (6), as well as for the investigation of protein structure and dynamics (7). The methods employed for making mAbbased conjugates can be classified in two general categories: those that involve the random modification of mAb amino acid residues, and those that are highly regioselective. Examples of random modification procedures include the acylation of lysine ε-amino groups (8), alkylation of tyrosines (9), and amidation of carboxylates (10). The biological and functional properties of these conjugates are often acceptable, however random modification of mAbs may impair antigen binding and leads to conjugate heterogeneity.In the past several years, a number of selective methods have been described to introduce molecules of interest onto mAbs. The ability to control the location and stoichiometry of conjugation can significantly improve the properties of mAb conjugates in some applications. The greatest selectivities are obtained using recombinant technologies for the production of fusion proteins (11)(12)(13)(14). Selective modification has also been reported for such chemically based methods as reductive amination of oxidized mAb carbohydrates (15), photoaffinity labeling of unconventional mAb binding sites (16), and reduction-alkylation of antibody interchain disulfides (17,18 We have previously described the preparation of mAb-drug conjugates for use as antitumor agents (17,19). The potent a...

Brentuximab Vedotin (SGN-35) for Relapsed CD30-Positive Lymphomas

“…In vitro, the drug was found to be potent and selective against CD30-positive tumor-cell lines, and activity was observed in models of Hodgkin's lymphoma and ALCL in mice with severe combined immunodeficiency. [18][19][20] To assess the safety and clinical activity of brentuximab vedotin, we treated patients with relapsed or refractory CD30-positive hematologic cancers in a phase 1, open-label, dose-escalation trial. Furthermore, because serum levels of TARC have been shown to correlate with disease activity in patients with Hodgkin's lymphoma, 21, 22 we evaluated serum levels of TARC and various cytokines in patients in the expansion phase of the study.…”

Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

“…Engineered Anti-CD70 Antibody-Drug Conjugate eight potential attachment sites on solvent accessible cysteine residues (14). Using the analytical methods of Sun et al (14), we estimate that roughly half of the antibodies in our ADC preparations contain at least one hinge disulfide (data not shown).…”

“…ADCs were prepared following partial reduction of antibody interchain disulfide bonds with Tris(2-carboxyethyl)-phosphine (TCEP) as described (14) with the following modifications. Antibody preparations in PBS were concentrated to 10 mg/mL.…”

“…Conjugates were diluted 10-fold with 20 mmol/L sodium acetate (pH 5.0) and bound to the membrane, washed with additional sodium acetate buffer, and eluted with 3Â PBS. Drug loading was determined by reversephase high-performance liquid chromatography under reducing conditions (13,14) and by hydrophobic interaction chromatography (15).…”

A Self-Assembling Short Oligonucleotide Duplex Suitable for Pretargeting

“…Methods of conjugation of molecules to mAbs can be either random acylation of lysine e-amino groups, alkylation of tyrosine groups, amidation of carboxylates, or regioselective modifications (Sun et al, 2005). Here, oligo-F1-CHO and roligo-F1-CHO (see Fig.…”