Upon binding to cell-surface antigens, many mAbs are internalized through a process known as receptor mediated endocytosis, which carries the mAb into lysosomes that are both acidic and rich in proteolytic enzymes (7). Considerable attention has been directed at developing linkers that are relatively stable at neutral pH, but undergo hydrolysis under the mildly acidic (circa pH 5) conditions within the lysosomes. The cleavable linker system that has been most extensively exploited contains the hydrazone functionality.
There are two general methods for producing mAb-drug conjugates through hydrazone bond formation. Treatment of mAbs with sodium periodate generates aldehydes and ketones through carbohydrate oxidation. Addition of hydrazido drug derivatives leads to the formation of hydrazones that are hydrolyzed under acidic conditions (10-12). One advantage of this methodology is that the mAbs are modified regiospecifically, since the carbohydrates on mAbs are largely restricted to the Fc region. However, the oxidation method leads to a variety of reactive species, and the resulting hydrazones are poorly defined. In addition, the oxidative conditions used for hydrazone formation can also lead to methionine oxidation, and this can be highly detrimental to mAb binding activity (13). An alternative approach to forming mAb-hydrazone linked conjugates is to attach an aldehyde, ketone, or preformed hydrazone to the surface of an mAb by acylation of lysine amino groups. This approach allows for much greater control over the relative hydrolysis rates of the hydrazone bond. Aromatic ketones generally appear to have the most promising characteristics since they are stable for several days under neutral pH conditions, but are much less stable at pH 5 (10,13,14). However, there are some examples of aliphatic hydrazones that are conditionally labile under mildly acidic conditions.
BR96-doxorubicin - A maleimido derivative of doxorubicin was conjugated to the anticarcinoma chimeric mAb cBR96 through a hydrazone that was selected for stability at pH 7 and drug release at pH 5 (15). Conjugates, having structures represented by !, were formed by reduction of interchain disulfides, then adding the maleimido drug derivative containing the hydrazone linkage. As many as eight drugs could be attached to each mAb with complete retention of binding activity. This methodology was later extended to include branched linkers, allowing for more drug to be attached to each thiol group in the mAb (16).
Preclinical studies demonstrated immunologically specific cures at well-tolerated doses in both mice and rats (15). However, the amount of conjugate needed to achieve these effects was very high (>100 mg conjugate/kg), reflecting the relatively low potency of the targeted drug. Pharmacokinetic studies indicated that the intratumoral drug concentration was much higher in animals treated with conjugate than in animals treated with the maximum tolerated dose (MTD) of unconjugated doxorubicin (17).
In a Phase I clinical trial, the MTD of BR96-doxorubicin was found to be 600700 mg/m2 with gastrointestinal dose-limiting toxicities (18). The half-life of drug release from circulating conjugate was approximately 43 hours, which is suboptimal, given that the half-life of the mAb in circulation was approximately 12 days. The conjugate was marginally active in this trial. A subsequent Phase II trial confirmed that the response rate was low, with gastrointestinal dose limiting toxicities (19). This study showed that unconjugated BR96 mAb elicited the same toxicities as the conjugate, suggesting that normal tissue cross-reactivity and mAb-mediated activities might have contributed to the toxicity. One of the noteworthy findings in this study was that active drug was detected within tumor masses, providing support for the concept of using mAbs for anticancer drug delivery.
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