Stephen J. Gregson

A Potent Anti-CD70 Antibody-Drug Conjugate Combining a Dimeric Pyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology

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.

SJG-136 (NSC 694501), a Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 1: Cellular Pharmacology, In vitro and Initial In vivo Antitumor Activity

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.

SJG-136 (NSC 694501), A Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 2: Efficacy Evaluations

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250–400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd×5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd×5 schedule, the maximum-tolerated dose was ∼120 μg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was ∼16 μg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.

SG2285, a Novel C2-Aryl-Substituted Pyrrolobenzodiazepine Dimer Prodrug That Cross-links DNA and Exerts Highly Potent Antitumor Activity

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

Synthesis of a novel C2/C2′-exo unsaturated pyrrolobenzodiazepine cross-linking agent with remarkable DNA binding affinity and cytotoxicity

A C2/C2′-exo unsaturated pyrrolobenzodiazepine dimer 1 has been synthesised which is cytotoxic at the picomolar level and has remarkable covalent DNA binding affinity, raising the melting temperature of duplex-form calf thymus DNA by 34 °C after 18 h incubation.

Antistaphylococcal activity of DNA-interactive pyrrolobenzodiazepine (PBD) dimers and PBD-biaryl conjugates

Objectives Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. Methods DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. Results Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. Conclusions PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates.

Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody–drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871–8. ©2017 AACR.

Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates: Synthesis and Evaluation of Non-Cleavable Drug-Linkers.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.