An Anti-CD33 Antibody−Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia. Choice of Linker

Abstract

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.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…UV/vis spectroscopy is the most commonly implemented technique to determine DAR. This molar ratio can be derived from the Beer-Lambert law by comparing the IgG absorbance maxima at 280 nm to that of a drug molecule in simultaneous equations (34,67,(71)(72)(73)(74). ADCs with a variety of payloads have been evaluated using this method, including DM1, methotrexate, calicheamicin analogues, and auristatins (11,75,76).…”

Antibody Drug Conjugates: Preclinical Considerations

“…1). These agents bind to the minor groove of DNA and are extremely cytotoxic, with picomolar potency across human tumor cell lines (38)(39)(40). Unlike tubulin inhibitors, DNA damaging payloads impact both proliferating and non-proliferating cells.…”

“…Clinical demonstration of this payload is the antibody drug conjugate gemtuzumab ozogamicin (Mylotarg®; Pfizer), a humanized anti-CD33 IgG4 conjugated to calicheamicin (39,40,48). Mylotarg® was approved in 2000 by the FDA for the treatment of acute myeloid leukemia but was withdrawn in 2010 due to limited clinical activity and safety concerns owing to the rate of fatal toxicity observed in patients.…”

In Vitroandin VivoActivity of the Maytansinoid Immunoconjugate huN901-N2′-Deacetyl-N2′-(3-Mercapto-1-Oxopropyl)-Maytansine against CD56+ Multiple Myeloma Cells

“…11), a humanized anti-CD33 mAb linked to a semisynthetic, highly cytotoxic calicheamicin ␥ 1 derivative. Gemtuzumab ozogamicin has demonstrated potent antitumor activity in tumor xenograft models of myeloid leukemia in athymic mice (12) and in patients with CD33…”