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Achieving transplant tolerance with regulatory T cell (Treg) adoptive immunotherapy is currently under investigation as a therapy to reduce graft rejection and improve long-term outcomes. Traditional approaches involve the of polyclonal Tregs, which are known to be less potent than antigen-specific cells, or antigen-expanded Tregs, which have several technical limitations. We and others have developed an alternate approach to generate antigen-specific Tregs by expressing a chimeric antigen receptor specific for HLA-A*02:01 (A2-CAR). In our initial studies the antigen-binding region (scFV) of the A2-CAR was derived from the mouse BB7.2 hybridoma, which, due to the high degree of homology between HLA molecules, has been reported to bind to HLA-A alleles in addition to *02:01. Here we sought to systematically define the antigen-specificity of the A2-CAR as well as humanize the sequence to minimize the risk of immunogenicity.We designed 20 humanized versions of the A2-CAR and systematically tested them to determine which were highly expressed on the surface of human Tregs and capable of mediating A2-stimulated activation, expansion, and suppression. We also developed a novel method to systematically, and comprehensively test HLA-allele specificity.Of the 20 humanized A2-CARs, 10 were expressed on Tregs and retained A*02:01 binding capacity. We used a series of functional screens to define which of these 10 A2-CARs most effectively stimulated Treg activation, proliferation and proliferation. We then took advantage of the Panel Reactive Antibody (PRA) assay (One Lambda) and created a new method to test CAR-expressing Tregs to bind to specific HLA-alleles. We found that the majority of the humanized A2-CARs had a significantly reduced reactivity to binding to alleles other than A*02:01. We also tested the biological relevance of HLA cross reactivity by stimulating A2-CAR expressing Tregs with cell lines expressing HLA alleles that were or were not found to be cross reactive using the PRA assay. Ultimately, six humanized anti-A2 CARs showed the desired properties, with an ability to activate Tregs, bind to HLA-A2 but not to a comprehensive panel of other common A or B alleles. The potent ability of one of these variants to suppress rejection was confirmed in a humanized model of xenogeneic graft-versus-host disease.We successfully developed a series of humanized A2-CARs which were comprehensively screened for desirable properties to generate antigen-specific Tregs. This body of pre-clinical data will support the development of a first-in-human clinical trial of A2-CAR-engineered Tregs to prevent organ allograft rejection.