A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.

A novel sequence-selective pyrrolobenzodiazepine (PBD) dimer 5 (SJG-136) has been developed that comprises two C2-exo-methylene-substituted DC-81 (3) subunits tethered through their C8 positions via an inert propanedioxy linker. This symmetric molecule is a highly efficient minor groove interstrand DNA cross-linking agent (XL(50) = 0.045 microM) that is 440-fold more potent than melphalan. Thermal denaturation studies show that, after 18 h incubation with calf thymus DNA at a 5:1 DNA/ligand ratio, it increases the T(m) value by 33.6 degrees C, the highest value so far recorded in this assay. The analogous dimer 4 (DSB-120) that lacks substitution/unsaturation at the C2 position elevates melting by only 15.1 degrees C under the same conditions, illustrating the effect of introducing C2-exo-unsaturation which serves to flatten the C-rings and achieve a superior isohelical fit within the DNA minor groove. This behavior is supported by molecular modeling studies which indicate that (i) the PBD units are covalently bonded to guanines on opposite strands to form a cross-link, (ii) 5 has a greater binding energy compared to 4, and (iii) 4 and 5 have equivalent binding sites that span six base pairs. Dimer 5 is significantly more cytotoxic than 4 in a number of human ovarian cancer cell lines (e.g., IC(50) values of 0.0225 nM vs 7.2 nM, respectively, in A2780 cells). Furthermore, it retains full potency in the cisplatin-resistant cell line A2780cisR (0.024 nM), whereas 4 loses activity (0.21 microM) with a resistance factor of 29.2. This may be due to a lower level of inactivation of 5 by intracellular thiol-containing molecules. A dilactam analogue (21) of 5 that lacks the electrophilic N10-C11/N10'-C11' imine moieties has also been synthesized and evaluated. Although unable to interact covalently with DNA, 21 still stabilizes the helix (Delta T(m) = 0.78 degrees C) and has significant cytotoxicity in some cell lines (i.e., IC(50) = 0.57 microM in CH1 cells), presumably exerting its effect through noncovalent interaction with DNA.

The pyrrolo[2,1‐c][1,4]benzodiazepines (PBDs) are a family of sequence‐selective DNA minor‐groove binding agents that form a covalent aminal bond between their C11‐position and the C2‐NH2 groups of guanine bases. The first example of a PBD monomer, the natural product anthramycin, was discovered in the 1960s, and the best known PBD dimer, SJG‐136 (also known as SG2000, NSC 694501 or BN2629), was synthesized in the 1990s and has recently completed Phase II clinical trials in patients with leukaemia and ovarian cancer. More recently, PBD dimer analogues are being attached to tumor‐targeting antibodies to create antibody–drug conjugates (ADCs), a number of which are now in clinical trials, with many others in pre‐clinical development. This Review maps the development from anthramycin to the first PBD dimers, and then to PBD‐containing ADCs, and explores both structure–activity relationships (SARs) and the biology of PBDs, and the strategies for their use as payloads for ADCs.

Both traditional drug discovery approaches and some more recently developed innovative strategies have already provided valuable tools for the discovery of PPI modulators, and a number of successful examples have highlighted the potential of targeting PPIs for therapeutic intervention, especially in the oncology area.

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The structure of the interstrand cross-linked adduct formed between a C8-C8'-linked pyrrolobenzodiazepine (PBD) dimer (DSB-120; 1,1'-(propane-1,3-diyldioxy)bis[(11aS)-7-methoxy-1,2,3,11a-t etrahydro-5H- pyrrolo[2,1-c][1,4]benzodiazepin-5-one]) and a self-complementary d(CICGATCICG)2 duplex has been determined from high-field 1D- and 2D-NMR data using a simulated annealing procedure. The refined structure supports earlier observations from solution NMR experiments and indicates that the covalently bound molecule spans six DNA base pairs in the minor groove, forming a symmetric cross-link between the spatially separated internal guanines and with active recognition of an embedded 5'-GATC bonding site. This result confirms that template-directed approaches are useful for the design of linked DNA-interactive PBD dimers with viable DNA cross-linking potential. Further, head-to-head connection of the PBD moieties results in an overall retention of 5'-GA bonding site preference for each alkylating PBD subunit. Structural analysis indicates that cross-link formation results in a localized perturbation of the DNA duplex, attributable in part to a mutual reduction in dynamic mobility or "covalent clamping" within the Gua4-Cyt7 base tract. However, ligand-induced distortion is confined to the Cyt7 and Ino8 residues on each strand. The Gua(N2)-Gua(N2) cross-link is stabilized by two directed H-bonds from the formed animal residues to N3 acceptor atoms of adenine bases on the 3'-side of each covalently modified guanine. Evidence for sequence-specific cross-linking with DSB-120 is provided by extended modeling studies which suggest that recognition of the favored d(.GATC.) motif is dominated by van der Waals steric factors, although electrostatic and H-bonded interaction terms also play a key role. This conclusion supports recent covalent footprinting studies revealing that this PBD dimer shows a selectivity for embedded base sequences of the type 5'-(pu/py)GATC(py/pu).

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials. Using a HPLC/MS-based methodology developed to evaluate the binding of DNA-interactive agents to oligonucleotides of varying length and sequence, we have demonstrated that, in addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, 1 can form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences, and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra- or interstrand cross-links are feasible because of the unavailability of two appropriately positioned guanines. Crucially, we have demonstrated a preference for the extended intrastrand cross-link with Pu-GAATG-Py, which forms more rapidly than the other cross-links (rank order: Pu-GAATG-Py > Pu-GATC-Py >> Pu-GATG-Py and Pu-GAATC-Py). However, thermal denaturation studies suggest that the originally reported Pu-GATC-Py interstrand cross-link is more stable, consistent with the covalent joining of both strands of the duplex and a lower overall distortion of the helix according to modeling studies. These observations impact on the proposed mechanism of action of SJG-136 (1) both in vitro and in vivo, the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials.