Attachment of rhodosaminylanthracyclinone-type anthracyclines to the hinge-region of monoclonal antibodies

Abstract

We have found that a maleimidobenzoyl spacer attached to OH-4' of the rhodosamine moiety of rhodosaminylanthracyclinone-type anthracyclines is most suitable for the attachment of these drugs to carriers, providing important advantages: The spacer is selectively and most readily introduced into the rhodosamine moiety of the drugs, is stable enough for proper handling of the derivatives, and can easily be attached to thiol groups of carrier systems such as reduced monoclonal antibodies. The anthracyclines can be liberated from the conjugates by mere hydrolysis, requiring neither hydrolytic enzymes nor acidic pH. Liberation of the drugs can, moreover, be affected by the presence of the appropriate substituents Z on the phenylene ring of the spacer, thus allowing slowed or enhanced liberation of the cytostatically active drug. The corresponding p-maleimidobenzoyl derivatives of beta-rhodomycin I, N,N-dimethyldaunorubicin, and rodorubicin have been attached to thiol groups of the hinge region of reduced monoclonal antibody BW 494/32, directed against a pancreatic cancer associated glycoprotein antigen, resulting in MoAb BW 494/32 conjugates, carrying 4.8-6.8 mol of cytotoxic residues/mol of MoAb. Rodorubicin was similarly attached to MoAb BW 575/931/2, directed against a small cell lung cancer associated antigen and to MoAb BW 431/26, recognizing an epitope detectable on carcinoembryonic antigen. The results provide evidence that the newly developed method of coupling of anthracyclines to the hinge region of monoclonal antibodies may be of broader use.

Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion

“…Another feasible approach involves partial reduction of the immunoglobulin with mercaptoethanol or dithiothreitol (22,23), a reaction which cleaves the disulfide bonds between the heavy chains in the vicinity of the hinge between the Fab and Fc units. We have attempted to exploit this reaction for the site-directed (hinge-thiol) attachment of biotin (24), anthracyclines (25,26), enzymes, and antibodies (Hermentln et al, unpublished data) without any detectable loss of antibody specificity and immunoreactivity.…”

Synthesis, analysis and study of sulfide-linked high functionality resin beads

“…Furthermore, the internal symmetry of an anthracene-based reactant avoided the formation of stereoisomers in the cycloaddition step. 28,29 Dienophile and diene functional groups were both successfully coupled to polymer beads giving 12 and 13 respectively (Figure 6). By the same criterion, the anthracene-bearing polymer 13 reacted to completion with maleimide, based on the 13 C NMR spectrum of the product.…”

Maleimide spacers as versatile linkers in the synthesis of bioconjugates of anthracyclines

“…The conjugation method of the drug to a carrier should not affect the activity of the drug itself, but it should allow the facile and reproducible incorporation of the drug in the delivery system while avoiding aggregation of the conjugate and the formation of homopolymers of carrier or drug. 8 Finally and in addition to biological and mechanistic considerations, the maleimide spacers are generally easy to prepare in a reproducible manner, and stable. 12 From a synthetic point of view, they result from the initial condensation of the appropriate amine onto maleic anhydride followed by further functionalisation at the distal end of the amine chain for connection to the anthracycline drug at the required position.…”

“…5 In addition, spacers may be attached to the anthracycline molecule in different positions which also affects the potency of the drug. 6 Initial bioconjugation studies included monoclonal antibodies 7, 8 as carriers, and it is also in this early work that most of the spacers were designed and assessed. Synthetic polymers were then used as a means to deliver the drug to malignant tissue.…”