Abstract Background: Probody® therapeutics (Pb-Tx) are masked antibodies designed to be selectively activated in the tumor microenvironment by tumor-associated proteases, while remaining largely inactive in normal tissue and in circulation. This allows Pb-Tx to address previously undruggable targets that are highly expressed in both tumor and normal tissue, such as the adhesion molecule CD166 (ALCAM). CX-2009, an investigational PDC consisting of an anti-CD166 monoclonal antibody conjugated to the microtubule inhibitor DM4, is being evaluated in a Phase 1/2 clinical trial for patients with advanced solid tumors (NCT03149549). Clinical data have been previously presented (Boni V, et al. J Clin Oncol. 2020; 38 [15 suppl]: abstract 526); here we present translation data assessing tumoral CX-2009 activation and DM4 accumulation in patients receiving ≥4 mg/kg every 2 or 3 weeks (Q2W or Q3W), alongside clinical results from breast cancer (BC) patients. Methods: Eligible patients with metastatic cancer after ≥2 prior standard treatments were required to submit tumor tissue for CD166 IHC analysis. CX-2009 was administered at escalating doses Q3W (0.25-10 mg/kg) or Q2W (4 or 6 mg/kg) until discontinuation criteria were met. Tumor response was assessed Q8W. On-treatment tumor biopsies in consenting patients were collected 3-5 days after the first dose. Levels of CD166 and of intact and activated CX-2009 in patient biopsies were measured by capillary immunoelectrophoresis; levels of DM4 were evaluated by a competitive ELISA, following depletion of the majority of CX-2009 from the sample. Results: 99 patients, including 45 with HR+/Her2- BC, HER2+ BC, or TNBC, were enrolled from 27 sites. The median number of prior therapies was 5.5 (range, 1-10) in all patients and 7 (3-16) in BC patients. Patients received a median of 2 (range, 1-16) CX-2009 doses. 92 patients discontinued treatment; the majority for disease progression/symptomatic deterioration (n=65) or adverse events (n=11). In 26 evaluable BC patients treated at ≥4 mg/kg, ORR was 11% (HR+/HER2-; n=18) and 38% (TNBC; n=8) including confirmed and unconfirmed responses, with a Clinical Benefit Rate at 24 weeks (CBR24) of 35%. CX-2009 remained predominantly (median >95%) in the inactive form in circulation (Stroh M, et al. J Clin Oncol. 2020; 38 [15 suppl]: abstract 3599). Over the dose range of 4-10 mg/kg, activated CX-2009 was quantifiable in 18 out of 22 biopsy samples, including 6 of 7 samples from the BC subset. The intratumoral concentrations of activated CX-2009 and CD166 were significantly correlated (r2=0.59, p=0.004). DM4 was measured in 12 evaluable biopsy samples. Levels of normalized intratumoral DM4 were significantly correlated with the relative change in target lesions (r2=0.34, p=0.045). Conclusions: CX-2009 behaved as designed, with activated drug in tumor and predominantly intact drug in plasma, leading to clinically meaningful disease control in patients with advanced BC. The relationship between normalized intratumoral DM4 and tumor volume changes support the premise that accumulation of payload toxin is related to anti-tumor activity. The correlation between activated CX-2009 and target expression suggests a role for CD166 in intratumoral accumulation of activated CX-2009. Taken together, the data support continued development of CX-2009 in patients with HR+/HER2- BC and TNBC. Email address for questions or comments: joyce_liu@dfci.harvard.edu Citation Format: Joyce F. Liu, Ivan A. Zein, Tam Dang, Susan K. Lyman, Alexander Spira, Nataliya Uboha, Patricia LoRusso, Mary J. Fidler, Funda Meric-Bernstam, Tobias Arkenau, Misako Nagasaka, Luc R. Desnoyers, William M. Kavanaugh, Virginia Paton, Alison Hannah, Valentina Boni. Intratumoral activation and phase 1/2 clinical activity of CX-2009, a probody drug conjugate (PDC) targeting CD166 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-07.