The results from these clinical studies prompted further investigation into the use of conjugates in combination with other drugs. One such study demonstrated that paclitaxel strongly synergized with BR96-doxorubicin (20). It was found that this combination was highly effective in lung, colon, and breast tumor xenograft models. Synergistic activities between BR96-doxorubicin and docetaxel were also reported, and a mechanism for synergy was proposed based on conjugate-mediated G2 cell-cycle arrest, leading to enhanced sensitization of the cells to taxanes (21). The promising in vitro and in vivo synergy studies provide the basis for ongoing clinical trials in which BR96-doxorubicin is combined with chemotherapeutic agents (22).

Anti-CD33-calicheamicin conjugates - Mylotarg, the first mAb-drug conjugate to be approved for human use, is a radical departure from previously described conjugates. The cytotoxic element in Mylotarg is N-acetyl-gamma calicheamicin, a minor groove binder that produces DNA double strand breaks (23) with an IC5o in the low ng/mL range (24). The linkage between the mAb and the drug incorporates two labile bonds as shown in structure 2: a hydrazone and a sterically hindered disulfide. It is believed that the hydrazone is cleaved before the disulfide is reduced.

The mAb moiety in Mylotarg is a humanized form of P67.6, a murine mAb that binds to the CD33 antigen present on myeloid cells, notably transformed cells of acute myeloid leukemia (AML) patients (25). CD33 has been shown to internalize and is not present on pluripotent stem cells (26). Any non-transformed myeloid cells eliminated by the conjugate can therefore be replaced. Greater than 80% of AML patients express the CD33 antigen (27).

Mylotarg consists of a 1:1 mixture of hP67.6, a humanized lgG4, with hP67.6 conjugated to 4-6 moles N-acetyl-gamma calicheamicin, providing an average drug loading ratio of 2-3 drugs/mAb (28). In conjugates prepared with murine P67.6, the hydrazone linkage was generated through periodate oxidation of the mAb carbohydrate, followed by condensation of the resulting putative aldehydes with an acyl hydrazide derivative of N-acetyl-gamma calicheamicin. However, the humanized form of the mAb, hP67.6, lost binding activity on periodate treatment, possibly because of a sensitive methionine residue in the antigen binding region (13). Consequently, the conjugate was formed by reacting the drug-hydrazone-linker complex with mAb lysine residues. The hydrazone in Mylotarg is a derivative of p-hydroxyacetophenone, which underwent 6% hydrolysis in 24 hr at pH 7.4 and was cleaved quantitatively under the same conditions at pH 4.5 (13). The role that conjugation technology can play in efficacy was further underscored by the finding that humanized lysine-linked conjugate was substantially more potent (45 fold on HL-60 cells) and more selective (70 fold on HL-60 cells) in vitro and somewhat more active in vivo than the murine carbohydrate linked version.

A Phase I clinical trial of Mylotarg in refractory or relapsed AML patients demonstrated tolerable levels of toxicity at doses up to 9 mg/m2 (29). Responses were documented in 8 of 40 patients. Toxicities included fever and chills, reversible elevation of liver enzymes, myelosupression with severe neutropenia, and veno-occlusive disease (29, 30). The CD33 antigen was saturated in treated patients within 30 min at the 9 mg/m2 dose. Response correlated with a combination of antigen saturation and low tumor cell efflux activity. The serum half-life of the conjugate was found to be 38 ± 21 hours. Immune response to the calicheamicin was detected in 2 of 40 patients, but no anti-hP67.7 response was observed. In the Phase II trial, patients were given up to 3 doses at 9 mg/m2 at intervals of 14 to 28 days apart, and the overall response rate was 30%, with a relapse-free survival time of 6.8 months (31). In May, 2000, Mylotarg was clinically approved for the treatment of AML. Being the first such mAb-drug conjugate, this represents a significant breakthrough in the field.

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