Prodrug applications for targeted cancer therapy

Abstract

Prodrugs are widely used in the targeted delivery of cytotoxic compounds to cancer cells. To date, targeted prodrugs for cancer therapy have achieved great diversity in terms of target selection, activation chemistry, as well as size and physicochemical nature of the prodrug. Macromolecular prodrugs such as antibody-drug conjugates, targeted polymer-drug conjugates and other conjugates that self-assemble to form liposomal and micellar nanoparticles currently represent a major trend in prodrug development for cancer therapy. In this review, we explore a unified view of cancer-targeted prodrugs and highlight several examples from recombinant technology that exemplify the prodrug concept but are not identified as such. Recombinant "prodrugs" such as engineered anthrax toxin show promise in biological specificity through the conditionally targeting of multiple cellular markers. Conditional targeting is achieved by structural complementation, the spontaneous assembly of engineered inactive subunits or fragments to reconstitute functional activity. These complementing systems can be readily adapted to achieve conditionally bispecific targeting of enzymes that are used to activate low-molecular weight prodrugs. By leveraging strengths from medicinal chemistry, polymer science, and recombinant technology, prodrugs are poised to remain a core component of highly focused and tailored strategies aimed at conditionally attacking complex molecular phenotypes in clinically relevant cancer.

Fig. 1

Passive and active conversion of prodrugs. Shown are illustrative examples of prodrugs that are activated by endogenous (passively) or exogenous (actively) enzymes, proteins, or conditions. In the case of conjugates, the active drug moiety is colored in red. a Examples of prodrugs that are substrates for endogenous proteases (① prostate-specific antigen, PSA) (8), membrane transporters (② PEPT1 oligopeptide transporter in pancreatic carcinomas) (14), or intracellular reductases (③ DT-diaphorase and ④ NADPH:cytochrome P450 reductase). b Prodrugs requiring exogenously administered enzymes or energy for activation. Activation of 5-fluorocytosine (⑤ 5-FC) and gemcitabine (⑥ dFdC) by engineered chimeric enzymes to their first cytotoxic antimetabolites. “Designer” conjugates of cytotoxic compounds as substrates for specific exogenous enzymes: ⑦ a recombinant carboxylesterase for dipiperinyl-VP-16 (40) and ⑧ β-lactamase for cephalosporinyl-5-FU (41). ⑨ A conjugate of the photosensitizer chlorin e6 with a single-stranded DNA aptamer that targets epithelial cancers presenting hypo-glycosylated MUC1 antigens (20). Irradiation at 664 nm generates cytotoxic singlet oxygen

Fig. 2

Diversity of targeted macromolecular prodrug conjugates. Shown are illustrative examples of cytotoxic agents (drawn in red) that are covalently attached to targeting moieties (blue) to form macromolecular prodrug conjugates. In some cases, a specifically cleavable linker (or spacer; green) connects the drug and targeting moiety. Cleavage sites are marked with a dashed line; enzyme-mediated cleavage is denoted with a scissors symbol. a Antibody-drug conjugates (ADCs), such as trastuzumab emtansine (T-DM1), in which the anti-tubular agent DM1 is conjugated to trastuzumab that targets HER2-positive metastatic breast cancer (16). In T-DM1, there are on average n = 3.5 equivalents of DM1 per antibody. b Targeted polymer-drug conjugates. HPMA-based copolymers are frequently used as a biocompatible polymeric scaffold to form polymeric nanoparticles. ① A pH-sensitive HPMA-doxorubicin conjugate in which the drug and anti-thymocyte globulin are linked at different HPMA units (94). Release of doxorubicin is triggered by hydrolysis of a hydrazone linker at endosomal pH (5 to 6). ② In HPMA-JHPD, L12ADT (an alkylated thapsigargin analog) targets prostate cancer cells via a sequence-specific peptide linker that is cleaved by prostate-specific antigen (95). ③ Carbon nanotube as a novel macromolecular carrier for Pt(IV)-based prodrugs. The targeting moiety (folic acid) and “longboat” carrier are anchored to the two axial positions present in Pt(IV), which are eliminated when the metal center is reduced to Pt(II), generating cisplatin, under intracellular conditions (53). c Redirected toxins, exemplified by the immunotoxin moxetumomab pasudotox, which is a recombinant conjugate of an anti-CD22 single-chain variable fragment (scFv) to residues 251 to 613 of Pseudomonas exotoxin A. Cytotoxicity encoded in domain III (PE3) is conditionally activated by furin-mediated cleavage between residues 279 and 280 in domain II

Fig. 3

Off-target effects of transductionally bispecific toxin conjugates. A cartoon showing the various cellular interactions of bispecific toxin conjugates harboring two receptor-targeting ligands (blue and red). Since binding by each ligand to its targeted receptor is independent, and each receptor is capable is endocytosis, intoxication ensues in all cells harboring one or both of the targeted cell-surface receptors. Activity in normal cells harboring only one of the targeted receptor leads to off-target, dose-limiting toxicity

Fig. 4

Conditionally bispecific prodrug conjugates. Shown are illustrative examples of cytotoxic agents (red) that are covalently linked to a promoiety that targets two independent markers in a sequential, conditional manner. Receptor-targeting ligands are colored in blue and substrates for target-specific cleavage in green. a “Low-MW” prodrug conjugates targeting cell-surface integrins with cyclic RGD motifs. ① A prodrug conjugate of SN38 (the active form of irinotecan) linked by a nitroquinone trigger. Specific two-electron reduction of the indole nitrogen by intracellular DT-diaphorase (DTD) leads to fragmentation of the linker and drug release (96). ② A doxorubicin conjugate linked by a substrate for plasmin (97). Plasmin cleavage conditionally triggers 1,6-elimination of the adjacent p-aminobenzyl alcohol (PABOH), releasing the free drug. b Brentuximab vedotin is a conjugate of monomethyl auristatin E (MMAE), an anti-mitotic agent, with the anti-CD30 antibody brentuximab (98). Upon endocytosis, cleavage of the valine-citrulline linker by lysosomal cathepsin B, followed by decomposition of the adjacent PABOH moiety, releases MMAE (99). This ADC (Adcetris®) is currently approved for use or in clinical trials for several lymphomas. c A targeted HPMA-doxorubicin (termed PK1) in which DOX and an antibody targeting the surface antigen OA3 on ovarian cancer cells are attached to polymeric HPMA via a peptide substrate (GFLG) for lysosomal cathepsin B (100)

Fig. 5

Conditionally bispecific intoxication of cells by engineered anthrax toxin targeted at two ECM proteases: uPA and MMP. Shown is the implementation as reported by Phillips et al. (84). (1) Anthrax protective antigen (PA) recognizes the two receptors ANTXR1 and ANTXR2. Wildtype PA undergoes proteolytic cleavage by furin to a 63-kDa truncated form (PA₆₃) that self-associates in the receptor-bound state. Two variants of protective antigen (PA) were engineered to redirect wildtype PA’s specificity for furin to uPA (2) and MMP (3). (4) Oligomeric PA₆₃ (pre-pore) binds lethal factor (LF) or engineered fusions of its N-terminal domain (LF _N) with a cargo effector. (5) The complex is internalized into endosomes. (6) Acidification within the endosomes triggers a transition of the pre-pore to a pore that translocates LF or the LF_N-based fusion into the cytosol. Wildtype LF leads to cell death by activating the mitogen-activated protein kinase kinase (MAP2K) pathway. An alternative cytotoxin is a LF_N conjugate with Pseudomonas exotoxin A (FP59) that induces apoptosis through inhibition of protein synthesis (72)

Fig. 6

Two schemes for conditionally bispecific enzyme activators for low-MW prodrugs. a Enzymes can be constructed as fusions with the N-terminus of LF to be translocated by conditional bispecific anthrax toxins (ATx). Proteolytic activation of PA and translocation of LF_N-based cargos are as described in Fig. 5. In the cytosol of cells expressing both markers, the enzyme would catalyze the bioactivation of a low-MW prodrug. b Alternatively, inactive fragments or subunits of an enzyme can be targeted transcriptionally as transgenes. By placing each transgene under the control of a cell- or disease-specific different promoter (P1 and P2), functional reconstitution becomes conditional within target cells in which both promoters are active. For natively monomeric enzymes, this requires splitting of the primary sequence and likely fusion to a high-affinity oligomerization domain to drive re-assembly. Complementing split enzymes have been demonstrated for a number of systems, including β-lactamase (93), which can be used to bioactivate cephalosporin conjugates of 5-FU (41)