CD33 is expressed by acute myeloid leukemia (AML) cells in >80% of patients but not by normal hematopoietic stem cells, suggesting that elimination of CD33(+) cells may be therapeutically beneficial. A conjugate of a calicheamicin hydrazide derivative attached via hydrazone formation to the oxidized carbohydrates of the anti-CD33 murine antibody P67.6 had been chosen for use in AML prior to humanization of this antibody. However, the CDR-grafted humanized P67.6 could not be used to make the carbohydrate conjugate because of the unexpected sensitivity of this antibody to periodate oxidation. Exploration of a series of bifunctional linkers resulted in a new class of calicheamicin conjugates, termed the hybrid conjugates, that allows for the attachment of the calicheamicin to lysines but incorporates the site of hydrolytic release, a hydrazone, previously shown to be required for activity. The optimized conjugate chosen for clinical trials, gemtuzumab ozogamicin ("gem-ozo", Mylotarg, formerly designated CMA-676), was significantly more potent and selective than the carbohydrate conjugate it replaced. It was selectively cytotoxic to HL-60 leukemia cells in tissue culture with an IC(50) in the low to sub-pg cal/mL range (cal = calicheamicin equivalents). Doses of gem-ozo as low as 50 microg cal/kg given three times to mice bearing HL-60 xenografts routinely resulted in long-term, tumor-free survivors, while a nonbinding control conjugate was relatively inactive. Gem-ozo at a concentration of 2 to 10 ng cal/mL selectively inhibited leukemia colony formation by marrow cells from a significant proportion of AML patients. Gem-ozo has also shown significant activity against AML in Phase II trials and is the first antibody-targeted chemotherapeutic agent approved by the FDA.

5 g tumor mass). In contrast, unconjugated CalichDMH, unconjugated G5/44, and an isotype-matched control conjugate, CMA-676, were ineffective against these BCL xenografts. Thus, CD22-targeted delivery of CalichDMH is a potent and effective preclinical therapeutic strategy for BCLs. The strong antitumor profile of CMC-544 supports its clinical evaluation as a treatment option for B-lymphoid malignan-

The anti-CD33 antibody, P67.6, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to acute myeloid leukemia (AML) due to the presence of CD33 on >80% of patient samples and its lack of expression outside the myeloid cell lineages, especially its lack of expression on pluripotent stem cells. Previous calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This results in a "carbohydrate conjugate" capable of releasing active drug by hydrolysis of a hydrazone bond in the lysozomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This results in an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the likely site of drug release from the conjugate. In this article, these two classes of calicheamicin-antibody conjugates are compared for potential use in AML with the anti-CD33 antibody P67.6. Conjugates of P67.6 are shown to require the site of hydrolytic release afforded by the carbohydrate conjugates in order to retain good potency and selectivity in vitro, in vivo, and ex vivo. The P67.6 carbohydrate conjugate of calicheamicin is selectively cytotoxic at <0.006 ng/mL of calicheamicin equivalents (cal equiv) toward HL-60 promyelocytic leukemia cells in tissue culture. Long-term, tumor-free survivors are seen in xenograft models when mice bearing HL-60 subcutaneous tumors are treated with the P67.6 carbohydrate conjugate at a dose of 300 microg/kg cal equiv given three times. This conjugate also selectively inhibits the formation of colonies from AML marrow samples at 2 ng/mL cal equiv. The P67.6 carbohydrate conjugate of calicheamicin therefore appears to have promise as an antibody-targeted chemotherapeutic agent for CD33-positive diseases such as AML.

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy. Cancer Res; 71(12); 4236-46. Ó2011 AACR.