Philip Wilson Howard

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.

Abstract 52: Mechanistic and benchmarking studies of ADCT-502, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting HER2-expressing solid tumors

ADCT-502 is an ADC composed of an engineered version of humanized IgG1 trastuzumab, directed against human HER2, site-specifically conjugated to the highly cytotoxic PBD-based linker-drug tesirine (drug-antibody ratio of 1.7). In vitro, ADCT-502 has highly potent and targeted cytotoxicity against various solid cancer cell lines. In vivo, ADCT-502 demonstrates strong and durable antitumor activity in mouse xenografts with various levels of HER2, but is inactive in a HER2-negative xenograft. ADCT-502 is stable, well tolerated and has a favorable PK profile both in rat and cynomolgus monkey. The current study aimed to define further the mechanism of action of ADCT-502 and to benchmark its activity in xenograft models against ado-trastuzumab emtansine (T-DM1), the ADC currently approved for the treatment of HER2+ metastatic breast cancers. ADCT-502 bound and internalized efficiently in JIMT-1 cells (HER2+) and co-localized with lysosomes within 2 hours. PBD dimers bind in the DNA minor groove and exert cytotoxicity via the formation of DNA interstrand cross-links. Following a 2-hour exposure to ADCT-502, DNA interstrand cross-linking peaked between 12 and 24 hours, after which cross-links persisted at least 36 hours. In contrast, cross-link formation by an equimolar concentration of warhead alone, peaked immediately following drug exposure and a non-targeted ADC did not produce DNA crosslinks in these cells. Moreover, ADCT-502 showed indirect bystander killing activity in HER2-negative MDA-MB-468 cells incubated with conditioned medium from ADCT-502-treated HER2+ SK-BR-3 cells. In vivo, antitumor activity of ADCT-502 was compared to T-DM1 in both cell line- and patient-derived-xenograft (PDX) models. For example, in a HER2 1+, FISH- breast cancer PDX, ADCT-502 showed dose-dependent antitumor activity resulting in 1/8 and 8/8 TFS after a single dose at 0.1 and 0.2 mg/kg, respectively. Conversely, a single dose of T-DM1 at 30 mg/kg showed only marginal activity compared to the control. Similarly, in a HER2 1+, FISH- esophageal cancer PDX, while a single dose of ADCT-502 at 0.44 mg/kg resulted in strong and durable antitumor activity, single doses of T-DM1 at either 10 or 30 mg/kg showed no activity compared to the control. These data confirm that the mechanism of cell killing of ADCT-502 is via target-specific internalization and subsequent cross-linking of DNA. They also show superior in vivo antitumor activity of ADCT-502 compared to T-DM1 in various tumor xenografts, including those with low HER2 levels. Taken together, these results support the development of ADCT-502 not only in patients that have become resistant/refractory to T-DM1, but also in patients whose tumors express low levels of HER2, and are not eligible for treatment with T-DM1. Citation Format: Francesca Zammarchi, Halla W. Reinert, Narinder Janghra, Simon Corbett, Maria Mellinas-Gomez, Sajidah Chowdhury, Neha Arora, Peter Tyrer, Francois Bertelli, David G. Williams, Philip W. Howard, John A. Hartley, Patrick H. van Berkel. Mechanistic and benchmarking studies of ADCT-502, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting HER2-expressing solid tumors [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 52. doi:10.1158/1538-7445.AM2017-52

Abstract 3601: Antibody-drug conjugates (ADCs) with tubulysin and PBD warheads, maintain potent in vitro cytotoxicity against multidrug-resistant tumor cells expressing P-glycoprotein (P-gp)

Development of resistance to initially-effective therapies remains a major challenge in the treatment of cancer. Resistance to antibody-drug conjugates (ADCs) can arise due to many factors including down-regulation of the target, modified internalization or trafficking of the internalized ADC/antigen complexes, decreased sensitivity to the warhead conjugated to the ADC, or combinations of these. One of the common mechanisms of resistance to ADCs is increased expression of efflux drug pumps such as P-glycoprotein (P-gp) which can then transport small molecule warheads out of the cell leading to multi-drug resistance (MDR). In fact, the warheads used in the two clinically-approved ADCs, Adcetris® and Kadcyla™, have been reported to be P-gp substrates and P-gp overexpression can lead to acquired resistance against these ADCs. Studies were conducted to determine if pyrrolobenzodiazepine (PBD) dimer or tubulysin warheads, and/or ADCs conjugated with these warheads were susceptible to P-gp-mediated resistance. The P-gp susceptibility of these warheads and associated ADCs was evaluated by comparing the relative cytotoxicity of warheads/ADCs in parental tumor cell lines compared to the same cell lines that have been manipulated to overexpress P-gp. Verapamil, an inhibitor of P-gp-mediated efflux, was used to confirm whether decreased sensitivity was due to P-gp activity. Unlike known P-gp substrates MMAE, paclitaxel, and vinblastine, the majority of PBD and tubulysin warheads tested were not significant P-gp substrates. These warheads and ADC9s conjugated with these warheads potently induced cytotoxicity of the parental cancer cell lines, and maintained this potency in the same cell lines overexpressing P-gp. Interestingly, certain tubulysins had differential susceptibility to P-gp depending on whether they were conjugated or depending on the linker that was used. For example, one tubulysin warhead showed moderate susceptibility to P-gp-mediated efflux as a naked warhead, but ADCs conjugated with this warhead had equivocal efficacy in parental and P-gp-overexpressing cell lines. An alternative tubulysin warhead was equipotent as an unconjugated small molecule in either parental or P-gp-overexpressing cell lines, however an ADC conjugated with this warhead using a cleavable linker had no activity in the resistant cells; an effect that was reversible with verapamil treatment, confirming the role of P-gp. These data suggest that ADCs conjugated with either PBD or tubulysin warheads may be active in MDR settings where resistance is mediated by P-gp expression, however activity against MDR cancers may be dependent on the particular warhead that was used and/or the particular linker that is used to conjugate the warhead to the antibody. Citation Format: Shenlan Mao, Ryan Fleming, Binyam Bezabeh, Nazzareno Dimasi, Dorin Toader, Thais Cailleau, Philip Howard, Changshou Gao, Bob Hollingsworth, Adeela Kamal, Jay Harper. Antibody-drug conjugates (ADCs) with tubulysin and PBD warheads, maintain potent in vitro cytotoxicity against multidrug-resistant tumor cells expressing P-glycoprotein (P-gp). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3601. doi:10.1158/1538-7445.AM2015-3601