Fig. 3

Screening of asTCRs. a Collected fractions of size-exclusive chromatography-purified soluble pHLAs are presented on 12% non-reducing SDS-PAGE. b TCR-phage output obtained from three rounds of panning against NY-ESO-1_(157–165) pHLA. c Monoclonal ELISA of 66 TCR clones randomly selected to evaluate their binding with NY-ESO-1_(157–165) pHLA. d Monoclonal ELISA of NY-ESO-1_(157–165) pHLA-TCR binders against irrelevant pHLA. asTCRs were identified when they only bound to _(157–165) pHLA. e asTCRs binding variance. All ELISA values at OD450 nm were normalized against signal from KM13 with TCRafp, an AFP_(158–166) asTCR serving as a control standard. f asTCRs phylogenetic family. TCR0 and TCRe represent 1G4 TCR wild-type and its affinity-enhanced TCR derived from the directed evolution of its CDRS. TCR1, TCR2, TCR3, TCR4, TCR5, and TCR6 are asTCRs selected from the naïve library after normalization. Most asTCRs were selected around the threshold of OD450 nm = 0.5. Data are arrayed as mean ± SD and compared making use of one-way ANOVA (Brown-Forsythe and Welch multiple comparison tests) where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001