Introduction: A method to study biodistribution of adduct forming antibody drug conjugates (ADCs) by LC-MS Description: ADCs are increasingly used to deliver cytotoxic payloads to tumor cells. While the targeted approach has vastly improved the toxicity profiles of these drugs, it is still important to understand the bio-distribution and the enhanced tumor localization this approach offers. Radio-labeled methods are still the most reliable approach to study the bio-distribution, but may not be able to differentiate between the drug that is present in the tissue from those that have formed covalent adducts upon non-specific internalization. Here, we describe a mass-spectrometric methods to identify these covalent adducts these cytotoxic agents form and how such methods could be utilized to map the bio-distribution in xenograft models. Data: The antibody drug conjugate is highly localized in the tumor with a %ID/gm value of almost 40 within 24 hours of dosing and no non-specific accumulation in other organs observed. Conclusions: A simple and robust LC-MS based method has been developed to understand the bio-distribution of antibody-drug conjugates by detecting and quantifying the actual and final chemical state of the toxin in the targeted cell. This method could be used to evaluate the (1) PK of antibody-drug conjugates (2) make lead selections at advanced preclinical stages of antibody-drug development (3) understand the ADME properties of the chosen antibody-drug conjugate Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2446.

2202 CTLA-4 is a negative costimulatory molecule expressed by multiple T cell subsets. Inhibition CTLA-4 activity by monoclonal antibodies has been shown to enhance anti-tumor responses in a variety of murine models. Ipilimumab (MDX-010; BMS-734016), a human antibody against CTLA-4, has shown activity against multiple f tumor types in clinical trials. Anti-cancer responses have been frequently associated with inflammatory adverse events that are suggestive of T cell activation, such as colitis, rash, and hypopituitarism. Serious adverse events are managed by cessation of antibody and the use immunosuppressive doses of corticosteroids, while durable anti-tumor responses have been maintained. Using a murine Sa1N fibrosarcoma model sensitive to anti-CTLA-4 monotherapy, the effect of dexamethasone (Dex) on anti-tumor activity was investigated. Sa1N cells were implanted in A/J mice and treated at ~100 mm3 with an anti-CTLA-4 antibody (9D9, 10 mg/kg), Q3Dx4. Dex (1 or 10 mg/kg; Q2Dx7) was initiated 7 days after anti-CTLA-4 treatment. In two separate experiments (example below), anti-CTLA-4 resulted in 85% and 97% mean tumor growth inhibition (TGI). Delayed treatment with Dex at 1 mg/kg did not effect TGI mediated by anti-CTLA-4 (83% and 95% TGI), while Dex at 10 mg/kg resulted in a limited abrogation of the anti-tumor effect (78% and 91% TGI). When Dex (1 and 10 mg/kg) was given concurrently with anti-CTLA4 (5 mg/kg), the anti-tumor effect by anti-CTLA-4 was still retained at early time points (day 18) but ultimately resulted in a reversal of TGI and tumor outgrowth at 24 days. Dex treatment alone did not significantly effect tumor volume in any study. These data support the use of corticosteroids in the treatment of adverse events following anti-CTLA-4 immunotherapy.