Peptidelinked Conjugates

The inherent instability of hydrazone and disulfide linkers has prompted several studies towards the identification of peptide sequences that can be used to attach drugs to mAbs. Such peptides are designed for rapid lysosomal hydrolysis and high serum stability. Initial studies indicated that cathepsin B, one of the most abundant of the lysosomal enzymes readily cleaved doxorubicin-peptide derivatives, but only if a self-immolative spacer was inserted between the peptide and the drug (41). Several dipeptides were found to be suitable for drug attachment, based on indefinite stabilities in serum and neutral pH, and rapid cleavage by lysosomal extracts or purified cathepsin B. This work was extended to include other drugs, such as paclitaxel and mitomycin C (42). This approach is particularly attractive, since the proteases that lead to drug release are mainly intracellular, and are less active in the blood, since they have acidic pH optima and are strongly inhibited by serum protease inhibitors.

BR96-peptide-doxorubicin conjugates - From a series of several dipeptide derivatives, BR96-phenylalanine-lyslne-p-aminobenzyl-doxorubicin (BR96-Phe-Lys-Dox) and the corresponding valine-citrulline derivative, BR96-Val-Cit-Dox, were found to be serum stable and to undergo rapid cleavage by cathepsin B, leading to the release of doxorubicin (43). One of the enzymes responsible for drug release was cathepsin B, but kinetic data using crude lysosomal preparations indicated that other enzymes might also play a role. The stability characteristics of these conjugates were evident from in vitro assays, in which high levels of specificity were observed even upon prolonged treatment.

While it appears that the peptide-linked doxorubicin conjugates present significant advantages over the corresponding hydrazone conjugates described earlier, low drug potency remains an issue. For that reason, the investigators explored bivalent linkage systems that allowed for increased levels of drug substitution (44). Although the conjugates had enhanced potency, the substitution with as many as 16 drugs/mAb led to non-covalent dimerization. To circumvent this, hydrophilic ethyleneglycol hydrazides were appended in a reversible manner to the free carbonyl group of doxorubicin, and the resulting highly substituted conjugates were mostly monomelic (45). This approach, while interesting, suffers from significant complexity, since two Independent events, proteolysis, followed by acid catalyzed hydrazone hydrolysis, must take place for intracellular drug activation.

mAb-auristatin conjugates - The auristatins are structurally related to dolastatin 10, a pentapeptlde natural product that has been the subject of several human clinical trials for cancer therapy (46). Molecules in this family exert potent antitumor activities through the inhibition of tubulin polymerization, and may also lead to intratumoral vascular damage. The activities are generally 100-1,000 times more potent than doxorubicin. Auristatins can be prepared in large quantities through total synthesis, and unlike calicheamicin and the CC1065-like minor groove binders described earlier, the drugs tend to be exceedingly stable.

Peptide derivatives of the synthetic auristatin MMAE were prepared using methodologies similar to those described for the peptlde-llnked doxorubicin conjugates (47). mAb-Val-Clt-MMAE and mAb-Phe-Lys-MMAE conjugates (4) were prepared using the mAb interchain mAb disulfides, producing conjugates with approximately 8 drugs/mAb. Unlike the doxorubicin conjugates, mAb-peptide-MMAE conjugates effected immunologically specific cell kill at concentrations well below that required for antigen saturation. The conjugates were highly stable in human serum, with projected half-lives of greater than 80 days. By comparison, the hydrazone-linked mAb-AEVB conjugate released free drug a ty2 of only 2-3 days in plasma. Thus, the peptide linker is much more stable than an optimized hydrazone linker.

In vivo studies in both carcinoma and hematologic tumor xenograft models demonstrated that the mAb-Val-Cit-MMAE conjugate was highly effective, immunologically specific, and well-tolerated. Studies were reported with cAC10-Val-Cit-MMAE, a conjugate that bound to the CD30 antigen on hematologic malignancies, in which cures of established tumors were obtained at doses as little as 1/60lh the MTD. Therapeutic windows this high have not been reported in the drug conjugate field, and the results underscore the importance of drug potency, linker design, and mAb trafficking in developing conjugates with optimal chances for therapeutic efficacy.

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