TCR-like antibodies represent a unique type of engineered antibodies with specificity towards peptide:Human Leukocyte Antigen (pHLA), a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed Complementarity Determining Region (CDR) engineering in combination with framework engineering for affinity and thermostability optimization. The strategy allowed the identification of HLA-DQ2.5 gluten peptide specific T-cell receptor (TCR)-like antibodies with low picomolar affinity. Our method outlines an efficient and general method for development of this promising class of antibodies, which should facilitate their utility including translation to human therapy.

Antibodies specific for antigenic peptides bound to major histocompatibility complex (MHC) molecules are valuable tools for studies of antigen presentation. Such T-cell receptor (TCR)-like antibodies may also have therapeutic potential in human disease due to their ability to target disease-associated antigens with high specificity. We previously generated celiac disease (CeD) relevant TCR-like antibodies that recognize the prevalent gluten epitope DQ2.5-glia-α1a in complex with HLA-DQ2.5. Here, we report on second-generation high-affinity antibodies towards this epitope as well as a panel of novel TCR-like antibodies to another immunodominant gliadin epitope, DQ2.5-glia-α2. The strategy for affinity engineering was based on Rosetta modeling combined with pIX phage display and is applicable to similar protein engineering efforts. We isolated picomolar affinity binders and validated them in Fab and IgG format. Flow cytometry experiments with CeD biopsy material confirm the unique disease specificity of these TCR-like antibodies and reinforce the notion that B cells and plasma cells have a dominant role in gluten antigen presentation in the inflamed CeD gut. Further, the lead candidate 3.C11 potently inhibited CD4 + T-cell activation and proliferation in vitro in an HLA and epitope specific manner, pointing to a potential for targeted disease interception without compromising systemic immunity. Significance StatementConsumption of gluten-containing food drives celiac disease in genetically predisposed individuals. The underlying disease mechanism is not fully understood, but it is strictly dependent on activation of pathogenic T cells. We have engineered high-affinity human antibodies recognizing the T-cell target HLA-DQ2.5 in complex with gluten epitopes and studied cell-specific antigen presentation in patients, which shows that plasma cells and not dendritic cells dominate the inflamed tissue. The only available treatment is lifelong adherence to a gluten-free diet, which is difficult and not effective in all cases. We show that at least one of our antibodies can specifically inhibit activation of pathogenic T-cells in vitro and therefore shows promise for therapy.