Development of GO

The class of toxin selected were the calicheamicins, highly potent and reactive antitumor antibiotics of the enediyne family that were originally isolated from fermentations of the soil microorganism Micromonospora echinospora ssp. calichensis in a screen for potent DNA damaging agents (78-81). The parent compound, calicheamicin-g₁^I, has been shown to interact with double-stranded DNA in the minor groove in a relatively sequence-specific manner in vitro (82). Following reduction by cellular thiols, the enediyne moiety undergoes rearrangement to form a 1,4-benzenoid diradical that abstracts hydrogens from the phosphodiester backbone of DNA, resulting in single- and double-strand lesions (82, 83) (Figure 3); the latter involve direct double-strand breaks and, as a major lesion, bistranded damage that consists of an abasic site on one strand and a direct strand break on the other (84). This DNA damage elicits a strong cellular response with cell cycle arrest in the G₂/M phase followed by either DNA repair or, if damage is overwhelming, apoptosis and cell death. While the response to the initial DNA damage remains incompletely understood, calicheamicin-induced double-strand breaks activate DNA repair through activation of ATM/ATR and DNA-dependent protein kinase (DNA-PK) (85, 86). In turn, ATM activation leads to activation of Chk1/2 and G₂/M cell cycle arrest (85, 87). DNA-PK phosphorylates H2AX in rapid response to DSBs, a step that is required for subsequent recruitment of DNA damage repair proteins (88). Consistently, cells defective in ATM or DNA-PK are hypersensitive to calicheamicins (83, 89), as are cells deficient in the ERCC2/XRD gene, which is involved in the nucleotide excision repair pathway (90), supporting the notion that the extent of DNA damage and damage repair is central for the toxic effects of calicheamicins. Some experimental studies have suggested that calicheamicin-induced cytotoxicity could involve non-apoptotic (i.e. necrotic) pathways, e.g. through activation of poly(ADP-ribose) polymerase 1 (PARP1) and exhaustion of NAD⁺ levels (91). However, the mitochondrial pathway of apoptosis appears to be predominantly utilized during calicheamicin-induced cell death, which may be triggered in a p53-independent and death receptor/FADD-independent manner via activation of mitochondrial permeability transition, cytochrome c release, involvement of pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak), and activation of caspases (92, 93). In line with this cytotoxic mechanism, microarray studies in yeast indicate that calicheamicin-g₁^I alters the expression of genes involved in chromatin arrangement, DNA repair and/or oxidative damage, DNA synthesis and cell cycle checkpoint control but also a variety of metabolic, biosynthetic, and stress response genes, as well as ribosomal proteins (94).

Experiments with free and antibody-bound calicheamicin analogues determined the structure-activity

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profile of a series of these toxins (95). For conjugation via periodate oxidized carbohydrates contained on the anti-MUC1 antibody, CTM01, thiol hydrazide derivatives were prepared by displacement at the methyltrisulfide moiety of the parent analogues. This process results in a “carbohydrate conjugate” capable of releasing active drug both by hydrolysis of the hydrazone bond at low pH as well as by reduction of the disulfide bond. Compared with calicheamicin-g₁^I, analogues that were missing the rhamnose at the end of the DNA binding region were found ineffective as conjugates in vivo; in contrast, 2 analogues (calicheamicin-a3I and N-acetyl-calicheamicin-g1I) in which the DNA binding region was intact yet the amino sugar was either eliminated or modified showed a clear therapeutic advantage over calicheamicin-g1I. Addition of methyl groups as steric bulk adjacent to the disulfide in the linker resulted in enhanced anti-tumor activity and an improved therapeutic window, likely because of increased stability of the linker in the serum. Together, these early studies identified the N-acetyl-calicheamicin-g₁^I dimethyl hydrazide derivative as having an optimal therapeutic window when conjugated to an antibody (95). Of note, although the potency of this hydrazide is 2-8 fold less than that of the corresponding parent compounds, it remains 100-1,000-fold more potent than clinically used anti-cancer agents. While not suited as free drug due to a narrow therapeutic window, this potency renders a calicheamicin a good candidate as toxin for antibody-based therapeutics (78).

During early development, a murine antibody (P67.6) recognizing the V-set Ig-like domain of CD33 (96) was conjugated to the N-acetyl-calicheamicin-g₁^I dimethyl hydrazide derivative (“carbohydrate conjugate”) as well as to a N-acetyl-calicheamicin-g₁^I dimethyl acid, N-hydroxysuccinimide ester; conjugation of the latter occurred via lysine residues of the antibody, resulting in an “amide conjugate” stable to hydrolysis (97). While inclusion of the hydrazone was not necessary for anti-tumor activity of the anti-MUC1 antibody (98), only the carbohydrate conjugate of P67.6 showed good potency and selectivity against CD33⁺ human AML cells in vitro and in xenograft models, demonstrating the importance of rapid release of the toxic moiety from its conjugated state under acidic conditions such as those in lysosomal vesicles for anti-AML activity (97). Subsequently, P67.6 was humanized by grafting complementarity-determining regions into a human IgG₄ kappa framework (hP67.6) to minimize immunogenicity, and then conjugated with the calicheamicin derivative via an acid-labile hybrid 4-(4’-

Table 1. Cellular parameters implicated in GO efficacy

Factor	Comment
Uptake of CD33/GO complexes
Receptor-mediated uptake
·   CD33 expression levels	Quantitative relationship between CD33 expression and GO efficacy in engineered cell lines; expression levels associated with cytogenetic risk of AML and CD33 SNPs
·   CD33 saturation	In vitro evidence linking reduced CD33 saturation to reduced GO cytotoxicity
·   CD33 internalization	Relatively slow process, controlled by intracellular tyrosine motifs and likely tyrosine phosphorylation and ubiquitylation status of CD33
·   Re-expression of CD33 binding sites	Surface CD33 levels return to pretreatment levels within 72 hours after CD33 antibody administration; could contribute to amount of internalized GO, in particular if given in fractionated doses.
Non-receptor mediated uptake	Suggested by experimental studies with CD33- cell lines (clinical role unknown)
Intracellular trafficking of GO	Hypothetical
Activation of GO	Low pH in lysosomes required for release of calicheamicin-g1I moiety from antibody
Extrusion of GO
·   ABC family of drug transporters	Established role of P-glycoprotein and MRP1; role of other transporters unknown
Induction of cytotoxicity
·   Generation of SS- and DS-DNA breaks	Hypersensitivity of cell lines with defects in DNA repair to calicheamicins
·   Mitochondrial pathways of apoptosis	Good experimental evidence for role of pro- and anti-apoptotic Bcl-2 protein family members
·   Other downstream pro- or anti-apoptotic signaling pathways	Not examined in detail but MEK1/2 and AKT signaling may confer relative resistance
·   Cell cycle status	Limited in vitro data suggesting that resting cells are relatively less susceptible to GO

Abbreviations: DS, double-stranded; GO, gemtuzumab ozogamicin; SNP, single-nucleotide polymorphism; SS, single-stranded Modified from a table that was initially published in Blood (58). Reproduced with permission from The American Society of Hematology

acetylphenoxy)butanoic acid linker to yield GO (99) (Figure 4). Of note, hP67.6 contains an IgG₄ core-hinge mutation that protects the therapeutic from Fab-arm exchange with endogenous human IgG₄ and thereby provides stabilization of the drug (100). However, only about 50% of the antibody is linked to calicheamicin-g₁ moieties, with an average loading of 4-6 molecules of the calicheamicin-g₁ derivate per antibody, while the remaining antibody is unconjugated (101).