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ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development. In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301. We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours. Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987). In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples. These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. Disclosures Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.