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AXL, a member of the tyrosine kinase receptor family TAM, is a transmembrane receptor overexpressed in many solid (e.g. lung, breast, pancreas, glioma and esophageal) and hematological malignancies (acute myeloid and chronic lymphocytic leukemia) and its overexpression is maintained in both primary tumors and metastasis. Moreover, expression and activation of AXL is associated with poor clinical prognosis and several studies suggest that overexpression of AXL results in resistance to both conventional chemotherapy and targeted therapies. All these features make AXL an attractive target for the development of an ADC to treat AXL-expressing cancers. ADCT-601 is an ADC composed of a humanized IgG1 antibody against human AXL, site-specifically conjugated using GlycoconnectTM technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-601 in human cancer cell lines and xenograft models and to determine its safety and tolerability in the rat. In vitro, ADCT-601 demonstrated potent cytotoxicity in a panel of cell lines of different origin and levels of AXL, while its potency was strongly reduced in AXL-negative cell lines. In vivo, ADCT-601 showed potent anti-tumor activity in the human triple-negative breast cancer-derived MDA-MB-231 xenograft, expressing moderate levels of AXL (≈36,000 copies/cell). In this model, a single dose of ADCT-601 at 1 mg/kg resulted in strong and sustained anti-tumor activity and resulted in 5/10 partial responders (PR) and 4/10 tumor-free survivors (TFS) at the end of the study on day 60. In the SN12C model, a human renal cell carcinoma-derived xenograft with high level of AXL expression (≈88,000 copies/cell), single doses of ADCT-601 at 0.3, 0.6 or 1 mg/kg showed dose-dependent anti-tumor activity compared to the vehicle- and isotype control ADC-treated mice. At the highest dose, ADCT-601 resulted in 7/8 and 6/8 complete responder (CR) and TFS, respectively, at the end of the study on day 60. Moreover, ADCT-601 showed potent and durable anti-tumor activity in a pancreatic cancer patient-derived xenograft model expressing heterogeneous levels of AXL where each single dose of ADCT-601 tested (0.3, 0.6 and 1 mg/kg) resulted in complete eradication of the tumors at the end of the study on day 43. Conversely, none of the mice in the vehicle and isotype control ADC groups had any PR, CR or TFS. ADCT-601 was stable, well tolerated and showed a favorable PK profile in the rat with a half-life of 9 days and a MTD of 6 mg/kg. In conclusion, ADCT-601 demonstrated potent and specific in vitro and in vivo anti-tumor activity in various cancer-derived models with different levels of membranous AXL. ADCT-601 was stable and well tolerated in the rat, warranting further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Simon Chivers, Paul W. Hogg, Charlie Britten, Sandamali Dissanayake, Peter Tyrer, Francois Bertelli, Ian Hutchinson, Luke Masterson, Phil Howard, John A. Hartley, Patrick H. van Berkel. Preclinical activity of ADCT-601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting AXL-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2792A.

Delta-like 1 homolog protein (DLK-1) is an EGF-like membrane bound protein consisting of six tandem EGF-like repeats, a juxtamembrane region with a TACE (ADAM17)-mediated cleavage site, a transmembrane domain, and a short intracellular tail. DLK-1 is strongly expressed during fetal development, while its expression is highly restricted in adults. Conversely, DLK-1 gets re-expressed in several tumors, such as neuroblastoma, hepatocellular carcinoma (HCC), rhabdomyosarcoma, small cell lung cancer, myelodysplastic syndrome and acute myeloid leukemia. Interestingly, in HCC DLK-1 has been shown to be a marker of cancer stem cells, a subpopulation of cells responsible for tumor initiation, growth, metastasis, and recurrence. Altogether, DLK-1 represents an attractive target for an antibody-drug conjugate (ADC) approach based on its selective expression in a wide range of malignancies and restricted expression in healthy organs, as well as its expression on HCC cancer stem cells. ADCT-701 is an ADC composed of a humanized IgG1 antibody against human DLK-1, site-specifically conjugated using GlycoconnectTM technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-701 in human cancer cell lines and patient-derived xenograft (PDX) models and to determine its safety and tolerability in the rat. In vitro, ADCT-701 demonstrated potent cytotoxicity in a panel of human cancer cell lines of different origin and levels of DLK-1, while its potency was strongly reduced in DLK-1-negative cell lines. In vivo, ADCT-701 showed potent anti-tumor activity in the DLK1-expressing neuroblastoma-derived SK-N-FI xenograft in which a single dose of ADCT-701 at 0.5 or 1 mg/kg showed dose-dependent anti-tumor activity compared to the vehicle- and isotype control ADC-treated mice. At 1 mg/kg, ADCT-701 resulted in 1/9 partial responders (PR) and 4/9 complete responder (CR), one of which was tumor-frees survivors (TFS) at the end of the study on day 60. Moreover, ADCT-701 showed dose-dependent anti-tumor activity in a HCC PDX model when tested as a single dose at 0.1, 0.3 or 1 mg/kg. At the highest dose tested, ADCT-701 resulted in 3/8 PR and 2/8 CR, while none of the mice treated with the vehicle or the isotype-control ADC (1 mg/kg, single dose) showed any activity. ADCT-701 was stable, well tolerated and showed a favorable pharmacokinetic profile in the rat with a half-life of 9 days and a maximum tolerated dose of at least 5 mg/kg. In conclusion, ADCT-701 demonstrated potent and specific in vitro and in vivo anti-tumor activity in DLK-1-expressing cancer-derived models and it was stable and well tolerated in the rat, warranting further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Simon Chivers, Paul W. Hogg, Charlie Britten, Sandamali Dissanayake, Peter Tyrer, Francois Bertelli, Ian Hutchinson, Luke Masterson, Phil Howard, John A. Hartley, Patrick H. van Berkel. ADCT-701, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting DLK1-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 744.

ADCT-402, currently in Phase I clinical trials for B-cell hematological malignancies, is an ADC composed of a recombinant humanized IgG1 against human CD19, stochastically conjugated via a cleavable linker to a PBD dimer cytotoxin (DAR of 2.3). PBD dimers, DNA minor groove interstrand cross-linking agents, are gaining increasing attention and are currently being tested as the ADC warheads in several clinical trials. ADCT-402 has potent and targeted cytotoxicity against a panel of human lymphoma and leukemia cell lines in vitro. In vivo, ADCT-402 demonstrates dose-dependent antitumor activity against Burkitt’s lymphoma xenograft models. Moreover, ADCT-402 is markedly superior to maytansinoid- and auristatin-based CD19-targeting ADCs in the Ramos xenograft model. In a rat toxicology study, a single dose of ADCT-402 at 2 mg/kg is well tolerated with a favorable PK profile and excellent stability in vivo. The current study aimed to further define the mechanism of action (MOA) of ADCT-402 and validate its pharmacology and preclinical safety for clinical development. CD19 is a clinically validated target with restricted normal tissue expression and a widespread expression in the majority of B-cell malignancies. Importantly, we show here the consistent expression of CD19 in matched samples (initial diagnosis and relapsed/refractory) from panels of lymphoma patients, indicating that relapsed/refractory patients are appropriate for treatment with ADCT-402. ADCT-402 was shown to be efficiently internalized by CD19+ cells in vitro. Moreover, in line with the PBD dimer MOA, following a 2 hour exposure to ADCT-402, DNA interstrand cross-links reached a peak between 8 - 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of cross-link formation for the PBD dimer warhead alone was observed immediately after 2 hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA cross-links. In SCID mice s.c. implanted with Ramos cells, a single dose of ADCT-402 was administered at 0.33 or 1 mg/kg. Twenty-four hours after treatment, excised tumors showed a dose proportional increase in intensity of staining by an anti-PBD payload antibody, as well as in DNA cross-linking and in γ-H2AX formation. In contrast, no DNA cross-linking was observed in matched lymphocyte samples. The toxicity of ADCT-402 was further evaluated in a repeat dose cynomolgus monkey study. ADCT-402 was clinically well tolerated with an acceptable off-target safety profile. The PK of the ADC was consistent with normal antibody clearance with a half-life of about 12 to 17 days. These data confirm the MOA of ADCT-402 and provide relevant pharmacodynamic assays and preclinical safety assessment to guide the clinical development of this promising ADC in B-cell malignancies. Citation Format: Francesca Zammarchi, Simon Corbett, Karin Havenith, Narinder Janghra, Konstantinos Kiakos, Teresa Marafioti, David G. Williams, Simon Chivers, Phil W. Howard, John A. Hartley, Patrick H. van Berkel. Characterization of the mechanism of action, pharmacodynamics and preclinical safety of ADCT-402, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting CD19-expressing hematological malignancies [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 51. doi:10.1158/1538-7445.AM2017-51