title: Engineering improved T cell receptors using an alanine-scan guided T cell display selection system.
abstract: T cell receptors (TCRs) on T cells recognize peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells and this interaction determines the T cell immune response. Due to negative selection, naturally occurring TCRs bind self (tumor) peptides with low affinity and have a much higher affinity for foreign antigens. This complicates isolation of naturally occurring, high affinity TCRs that mediate more effective tumor rejection for therapeutic purposes. An attractive approach to resolve this issue is to engineer high affinity TCRs in vitro using phage, yeast or mammalian TCR display systems. A caveat of these systems is that they rely on a large library by random mutagenesis due to the lack of knowledge regarding the specific interactions between the TCR and pMHC. We have focused on the mammalian retroviral display system because it uniquely allows for direct comparison of TCR-pMHC-binding properties with T-cell activation outcomes. Through an alanine-scanning approach, we are able to quickly map the key amino acid residues directly involved in TCR-pMHC interactions thereby significantly reducing the library size. Using this method, we demonstrate that for a self-antigen-specific human TCR (R6C12) the key residues for pMHC binding are located in the CDR3β region. This information was used as a basis for designing an efficacious TCR CDR3α library that allowed for selection of TCRs with higher avidity than the wild-type as evaluated through binding and activation experiments. This is a direct approach to target specific TCR residues in TCR library design to efficiently engineer high avidity TCRs that may potentially be used to enhance adoptive immunotherapy treatments.
authors.affiliation: NYU Cancer institute, New York University School of Medicine, NewYork, NY 10016, USA.
authors.forename: Karolina
authors.initials: K
authors.lastname: Malecek
authors.forename: Shi
authors.initials: S
authors.lastname: Zhong
authors.forename: Katelyn
authors.initials: K
authors.lastname: McGary
authors.forename: Connie
authors.initials: C
authors.lastname: Yu
authors.forename: Kevin
authors.initials: K
authors.lastname: Huang
authors.forename: Laura A
authors.initials: LA
authors.lastname: Johnson
authors.forename: Steven A
authors.initials: SA
authors.lastname: Rosenberg
authors.forename: Michelle
authors.initials: M
authors.lastname: Krogsgaard
mesh_terms: D000409:Alanine / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000941:Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000235:genetics / Q000276:immunology; D016296:Mutagenesis / Q000235:genetics / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
chemical_list: D000941:Antigens; D016207:Cytokines; D015789:HLA-A2 Antigen; D011948:Receptors, Antigen, T-Cell; D000409:Alanine