Francois D'Hooge

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody–Drug Conjugate Targeting 5T4

Antibody–drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576–87. ©2017 AACR.

ADCT-402, a PBD dimer-containing antibody drug conjugate targeting CD19-expressing malignancies

Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19- cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody–Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody–drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient–derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176–86. ©2018 AACR.

Abstract 3111A: ADCT-401/MEDI3726, a novel pyrrolobenzodiazepine (PBD)-based antibody-drug conjugate (ADC) targeting PSMA-expressing prostate cancers

Prostate-specific membrane antigen (PSMA) is an attractive target for an ADC approach as it is over-expressed by virtually all prostate cancers and its expression is highest in poorly-differentiated, metastatic and castration-resistant cases. PSMA has limited expression in non-prostatic tissues, it is not secreted or cleaved by PSMA-expressing cells, and it is constitutively internalized, a process that may be accelerated by specific antibody binding. ADCT-401/MEDI3726 is an ADC composed of a monoclonal antibody (J591), directed against human PSMA, site-specifically conjugated (drug-to-antibody ratio of 1.8) to a highly cytotoxic DNA cross-linking PBD dimer with a valine-alanine dipeptide linker. In vitro, ADCT-401/MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-expressing prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. In vivo, ADCT-401/MEDI3726 showed strong antitumor activity against CWR22Rv1 and LNCaP human-derived prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with low, heterogeneous PSMA expression, ADCT-401/MEDI3726 achieved dose-dependent antitumor activity when administered as single dose at either 0.33 or 1 mg/kg, which resulted in significant increase in survival compared to the vehicle-treated animals. Moreover, a single dose of ADCT-401/MEDI3726 showed remarkable superior antitumor activity compared to multiple doses of another PSMA-targeted ADC stochastically conjugated to the auristatin payload vcMMAE with a DAR of ~4. In the LNCaP model, ADCT-401/MEDI3726 resulted in dose-dependent antitumor activity when dosed once at 0.11, 0.33 or 1 mg/kg. In the PSMA-negative prostate cancer-derived PC3 xenograft model, ADCT-401/MEDI3726 and an isotope-control ADC showed limited tumor growth inhibition highlighting target-mediated antitumor activity. ADCT-401/MEDI3726 demonstrated potent and specific in vitro and in vivo antitumor activity in prostate cancer-derived models of differing levels of PSMA and this warrants further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Simon Chivers, Karin Havenith, David G. Williams, Lauren Adams, Maria Mellinas-Gomez, Simon Corbett, Peter Tyrer, Francois D9Hooge, Song Cho, Nazzareno Dimasi, Mary Jane Hinrichs, Phil W. Howard, John A. Hartley, Patrick H. van Berkel. ADCT-401/MEDI3726, a novel pyrrolobenzodiazepine (PBD)-based antibody-drug conjugate (ADC) targeting PSMA-expressing prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3111A. doi:10.1158/1538-7445.AM2017-3111A