CCR FOCUS Antibody-Drug Conjugates of Calicheamicin Derivative: Gemtuzumab Ozogamicin and Inotuzumab Ozogamicin Alejandro D. Ricart Abstract Antibody-drug conjugates (ADC) are an attractive approach for the treatment of acute myeloid leukemia and non-Hodgkin lymphomas, which in most cases, are inherently sensitive to cytotoxic agents. CD33 and CD22 are specific markers of myeloid leukemias and B-cell malignancies, respectively. These endocytic receptors are ideal for an ADC strategy because they can effectively carry the cytotoxic payload into the cell. Gemtuzumab ozogamicin (GO, Mylotarg) and inotuzumab ozogamicin consist of a derivative of calichea- micin (a potent DNA-binding cytotoxic antibiotic) linked to a humanized monoclonal IgG4 antibody directed against CD33 or CD22, respectively. Both of these ADCs have a target-mediated pharmacokinetic disposition. GO was the first drug to prove the ADC concept in the clinic, specifically in phase II studies that included substantial proportions of older patients with relapsed acute myeloid leukemia. In contrast, in phase III studies, it has thus far failed to show clinical benefit in first-line treatment in combination with standard chemotherapy. Inotuzumab ozogamicin has shown remarkable clinical activity in relapsed/refractory B-cell non-Hodgkin lymphoma, and it has started phase III evaluation. The safety profile of these ADCs includes reversible myelosuppression (especially neutropenia and thrombocyto- penia), elevated hepatic transaminases, and hyperbilirubinemia. There have been postmarketing reports of hepatotoxicity, especially veno-occlusive disease, associated with GO. The incidence is 2%, but patients who undergo hematopoietic stem cell transplantation have an increased risk. As we steadily move toward the goal of personalized medicine, these kinds of agents will provide a unique opportunity to treat selected patient subpopulations based on the expression of their specific tumor targets. Clin Cancer Res; 17(20); 6417–27. Ó2011 AACR. Introduction The sialic-acid–binding immunoglobulin-like lectins (siglecs) include a family of receptors that are restrictedly It is surprising that few monoclonal antibodies (mAb) expressed on one or a few immune cell types, making them have been approved for the treatment of acute myeloid attractive for targeted therapy (8, 9). CD33 (also known as leukemia (AML) and non-Hodgkin lymphoma (NHL) since siglec-3) and CD22 (siglec-2) were identified as markers of the first approval of rituximab in 1997. These malignancies myeloid leukemias and B-cell lymphomas, respectively, localize in areas that are readily accessible to circulating almost 25 years ago. Siglecs are endocytic receptors and mAbs, such as the circulating blood, lymph nodes, and thus ideal for an ADC strategy because they can effectively bone marrow. The disposition of intact mAbs is also favor- carry the complex with its cytotoxic payload into the cell (8). able for their treatment: Distribution into other tissues is This is a striking difference from other antibody targets, such slow, and volumes of distribution are generally low (1). as CD20, for which human cells are highly heterogeneous in Antibody-drug conjugates (ADC) are an attractive approach their internalization ability (10). for the treatment of these diseases, which in most cases are inherently sensitive to cytotoxic agents. This CCR Focus Gemtuzumab Ozogamicin section describes a significant number of ADCs currently in clinical development (2–7). Description Gemtuzumab ozogamicin (GO, Mylotarg; Fig. 1) consists of a semisynthetic derivative of calicheamicin (N-acetyl-g calicheamicin 1,2-dimethyl hydrazine dichloride), a potent Author's Affiliation: Biotechnology Unit and Oncology Clinical Research, Pfizer Inc., San Diego, California enediyne DNA-binding cytotoxic antibiotic, linked to an engineered humanized monoclonal IgG4 antibody Corresponding Author: Alejandro D. Ricart, Biotechnology Unit and Oncology Clinical Research, La Jolla Laboratories, Pfizer Inc., 10646 (hP67.6) directed against the CD33 antigen present on Science Center Drive (CB1), San Diego, CA 92121. Phone: 858-622- leukemic myeloblasts in most patients with AML 7575; Fax: 877-481-3011; E-mail: alejandro.d.ricart@pfizer.com (80%). IgG4 has interesting properties for a carrier. It has doi: 10.1158/1078-0432.CCR-11-0486 the longest circulating half-life of all isotypes, with limited Ó2011 American Association for Cancer Research. ability for complement fixation and antibody-dependent www.aacrjournals.org 6417 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. CCR FOCUS A CH3 Figure 1. A, immunoconjugate binding and internalization. B, immunoconjugate intracellular trafficking from endosome to lysosome, with linker cleavage. Acid-labile AcBut hydrazone linker is cleaved in the acid environment of lysosome. C, calicheamicin derivative is released intracellularly. The reduction to the active enediyne form requires glutathione. D, the active enediyne form binds to the minor groove in DNA and causes double-strand breaks, resulting in cell death. E, Pgp- mediated efflux may be a mechanism of drug resistance in leukemic cells. The immunoconjugate structure on the upper right is modified from Advani et al. (52). Reprinted with permission. Ó 2008 American Society of Clinical Oncology. D cellular toxicity. The unconjugated antibody hP67.6 is not cells in vitro. This effective intracellular hydrolytic release of known to be cytotoxic per se (11). Calicheamicin was the calicheamicin derivative was important for its intracel- isolated from the actinomycete Micromonospora echinospora lular trafficking and the subsequent access to its DNA target. calichensis (12). The cytotoxic drug is attached to the anti- This indicates the importance of designing linkers that are body through a covalent linkage (condensation) of a specific for the individual target cell type (11). bifunctional linker, 4-(4-acetylphenoxy)butanoic acid (AcBut linker), which allows stability in physiologic Pharmacodynamics buffers (pH 7.4) and efficient calicheamicin release inside Saturation and internalization were analyzed in a phase II lysosomes (pH 4; ref. 11). The average loading of cali- study (13). Within 3 to 6 hours after a 9 mg/m2 infusion of cheamicin on the antibody is 2.5 mol/mol (drug-loading GO, nearly complete saturation of CD33 antigens was range of 2–3 mol of calicheamicin per mole of antibody; reached for leukemic and normal myeloid cells in blood. ref. 11). Calicheamicin binds to the minor groove in the Although the mean maximal binding of GO to myeloid DNA and causes double-strand DNA breaks, resulting in cell blast cells was comparable to monocytes, the maximal death (please refer to Fig. 1 to follow this sequence; ref. 12). binding to granulocytes was significantly lower, with no The custom-made, well-controlled, hydrolysable bond with binding to lymphocytes. These data are in accordance with the AcBut linker showed significantly more potent and the known expression levels of the CD33 antigen on these selective calicheamicin conjugates of P67.6 against HL-60 cells. On the other hand, this target is not (considerably) 6418 Clin Cancer Res; 17(20) October 15, 2011 Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. Antibody-Drug Conjugates of Calicheamicin Derivative þ expressed on normal CD34 pluripotent hematopoietic (RP2D) was determined to be 9 mg/m2 infused i.v. over 2 stem cells or on nonhematopoietic tissues (see "Safety" hours, repeated on day 14 (16). Increased serum concentra- section for gemtuzumab ozogamicin below; ref. 14). Satu- tions were observed after the second dose and were believed ration levels before the second infusion were increased to be due to a decrease in clearance by CD33-positive blast compared with baseline levels (before the first infusion) in cells, a result of the lower peripheral leukemic burden after that phase II study, apparently due to remaining GO from the first dose (21, 22). The concentration profiles of cali- the first infusion. Upon binding to CD33, GO was rapidly cheamicin derivative followed the same time course as internalized, as determined by the decrease in maximal hP67.6, evidence that the payload remained conjugated to membrane binding (13). In vitro studies using pulse label- the antibody and was delivered to the leukemic cells. No ing with GO showed a continuous renewal of CD33, which relationship was found between plasma concentration and could increase the internalization and thereby the intracel- response at the RP2D (21). Despite high interpatient vari- lular accumulation of the cytotoxic agent (13). Using a ability, the pharmacokinetic parameters of the components lentivirus-mediated gene transfer to manipulate CD33 (hP67.6, total and unconjugated calicheamicin derivative) expression in cell lines that normally lack CD33 or have were not different based on gender or age, including a very low levels of CD33, Walter and colleagues (15) showed comparison between pediatric patients and adults (22, a quantitative relationship between CD33 expression and 23). Weight and body surface area did not affect the phar- GO-induced cytotoxicity. The CD33 cytoplasmic immunor- macokinetics of GO (23). Although most mAbs in oncology eceptor tyrosine-based inhibitory motifs controlled the are administered on the basis of body weight or surface area, internalization process, and point mutations that disrupted