Background: The Lung Cancer Master Protocol (Lung-MAP) is designed to evaluate novel targeted therapies in patients with advanced squamous lung carcinoma. In the S1400F sub-study (non-match), we tested whether combined CTLA-4 and PD-1 inhibition with durvalumab plus tremelimumab (D+T) could overcome primary or acquired resistance to anti-PD-(L)1 therapy. Response, progression-free (PFS) and overall survival, and safety in the acquired resistance cohort are reported herein. Methods: Patients with previously treated squamous lung carcinoma, performance status (PS) 0-1, and adequate organ function that developed disease progression after ≥24 weeks of anti-PD-(L)1 monotherapy were eligible. Prior severe immune-related toxicities, intervening systemic therapy and combination chemo-immunotherapy were not permitted. Patients received D1500 mg + T75 mg IV q28 days for 4 cycles then D maintenance until disease progression. The primary endpoint was best objective response (RECIST 1.1). Interim analysis for futility was planned after 20 patients evaluable for response were enrolled. If no responses were observed, the cohort would stop enrolment. Results: 30 eligible patients were accrued to the acquired resistance cohort. Median age was 68 years, 60% of patients were male, 33% PS 0 and had received a median of 2 prior lines of therapy (maximum 4). Best response to prior anti-PD-(L)1 therapy was CR/PR/SD in 3/7/20 patients, with a median duration of anti-PD-(L)1 therapy of 8.6 months (5.2-30.4). No objective responses were seen with D+T; 47% had SD as best response. Median PFS was 2.0 months (95% CI 1.6-2.9) and survival 7.5 months (95% CI 5.3-8.7). Among the 14 patients with SD as best response, the median PFS calculated from first disease assessment is 2.8 months (95% CI: 1.4-3.9). Grade≥3 adverse events at least possibly related to protocol therapy were seen in 10/30 patients. These include 1 treatment-related death due to pneumonitis and 1 death not otherwise specified. Other adverse events include grade 3 confusion (1), dehydration (2), diarrhea (3), encephalopathy (1), weakness (1), hyperglycemia (1), hypoxia (1), lymphopenia (1), nausea, (1), neutropenia (1), thrombocytopenia (1), rash (1), vomiting (1), grade 4 dyspnea (1), leucopenia (1) and lymphopenia (1). Conclusions: D+T did not demonstrate activity in patients with acquired resistance to PD-1 checkpoint inhibitors and pretreated advanced squamous lung carcinoma. Clinical trial information: NCT03373760.