Objective: Integrins are heterodimeric receptors that convey cell-to-cell and cell-to-matrix interactions. Integrin α v β 3 is expressed in several tumour entities including melanoma, glioblastoma, breast, pancreatic and prostate cancer, where it promotes tumour cell survival and metastasis. Here, we generated α v β 3 -specific chimeric antigen receptor (CAR) T-cells and analysed their antitumour function in pre-clinical models in vitro and in vivo ., Methods: α v β 3 -CARs comprising a super-humanised hLM609 targeting domain with either high or low affinity (hLM609v7, K d = 3 nM vs. hLM609v11, K d = 160 nM) and equipped with either a long or a short IgG4-Fc extracellular spacer (229 vs. 12 amino acids) were expressed in CD8 + and CD4 + T-cells through lentiviral transduction., Results: α v β 3 -CAR T-cells eliminated α v β 3 -positive tumour cells rapidly and specifically, produced IFN-γ and IL-2 (CD4 + > CD8 + ) and exhibited productive proliferation. In vitro , we observed the strongest reactivity with the higher-affinity hLM609v7 α v β 3 -CAR in the short spacer configuration, consistent with the tumour membrane-distal localization of the hLM609 epitope. In a murine xenograft model of metastatic A-375 melanoma, the strongest antitumour effect was mediated by the lower-affinity hLM609v11 α v β 3 -CAR. Notably, a single administration of hLM609v11 α v β 3 -CAR T-cells was able to induce complete elimination of melanoma lesions, leading to long-term tumour-free survival., Conclusions: These data establish α v β 3 integrin as a novel target for CAR T-cell immunotherapy, and affirm our previous notion that binding domain affinity and spacer length can be calibrated to augment CAR reactivity., Clinical Implications: α v β 3 -CAR T-cells have therapeutic potential in several prevalent solid tumours, including melanoma and triple-negative breast cancer., Competing Interests: Conflict of Interest Statement M.H. is co-inventor on a patent application (PCT/US2013/055862) related to CAR spacer design that has been filed by the Fred Hutchinson Cancer Research Center (Seattle, WA) and licensed by JUNO Therapeutics, Inc.. C.R. is co-inventor of U.S. Patent 7,087,409 that claims the humanised LM609 variants and is owned by The Scripps Research Institute (La Jolla, CA).