Extended Data Fig. 1: Neoantigen prediction and synthetic construct design.
From: Probiotic neoantigen delivery vectors for precision cancer immunotherapy
a, Percentage of predicted CT26 neoantigens containing mutant-epitope(s) with ≤500 nM MHC-I affinity (MHC-I), MHC-II affinity (MHC-II), both MHC-I and MHC-II affinity (Shared), or no epitope meeting affinity criteria (Neither). Previously validated neoantigens within the set are labeled. b, Upper: prototype neoantigen construct design, Lower: immunoblot of EcN expressing prototype neoantigen constructs. c, Upper: immunoblot of EcN expressing prototype neoantigen constructs with or without GS-linkers. Lower: ELISA quantification of neoantigen construct in soluble fraction with or without GS-linkers in DH5α (n = 3 biological replicates per group). NeoAgp = prototype neoantigen construct, G4S1 = 5-mer GS-linker, pTacLO− = pTac without Lac operator; pTacLO+ = with Lac operator. d, Upper: neoantigen construct design with GS-linkers, Lower-left: Relative immunoblot chemiluminescent intensity for prototype construct with or without interspersing glycine-serine linkers (n = 6 biological replicates per group). Lower-right: relative expression of prototype neoantigen construct with GS-linkers under selected promoters (n = 12 biologically independent samples). e, Upper: immunoblot of EcN expressing alternate prototype neoantigen constructs. Lower: ELISA quantification of alternative neoantigen construct in soluble fraction in DH5α (n = 3 biological replicates per group). NeomE1 = minimal epitope, Neo1 = 1 neoantigen LP in construct, Neo2 = 2 neoantigen LP in construct, G8S2 = 10-mer GS-linker, CsL = immunoprotease sensitive linker. f, Upper: Immunoblot of neoantigen constructs (NeoAgp, MHCIa, MHCIIa, MHCI/IIv), expressed in BL21 or EcN. Lower-left: relative immunoblot chemiluminescent intensity for neoantigen construct expression in EcN vs. BL21 (n = 4 biological replicates per group), Lower-right: relative immunoblot chemiluminescent intensity of predicted neoantigen constructs vs. prototype in BL21 (n = 3 biologically independent samples). c–f, Data are mean ± s.e.m. Gel source data in Supplementary Fig. 1.
