Med. 19, 465–472 (2013). Article81. Fu, X. et al. A simple and sensitive method for measuring tumor-specific T cell cytotoxicity. PLoS ONE 5, e11867 (2010). Acknowledgements Research reported in this publication was performed in the Columbia University Department of Microbiology and Immunology Flow Cytometry core facility, the Columbia Center for Translational Immunology (CCTI) and the Herbert Irving Comprehensive Cancer Center (HICCC) Flow Cytometry core facility. Grant numbers NIH/NCI R01CA249160, NIH/NCI R01CA259634, NIH/NCI U01CA247573, NIH/NIGMS T32GM145766, NIH S10OD030282, Searle Scholars Program SSP-2017-2179, Cancer Research Institute CRI 3446, Pershing Square Foundation PSSCRA CU20-0730 and a Roy and Diana Vagelos Precision Medicine Pilot grant supported the study. Author contributions A.R., T.D. and N.A. conceived the study. A.R. designed the system and experiments, conducted sequencing analysis and neoantigen selection, constructed all therapeutics and performed most experiments. A.R. and J.I. designed microbial genetic knockouts. A.R. and B.R. designed and performed all in vitro and ex vivo T cell assays, and flow-cytometric immunophenotyping. A.R., J.I., S.H., M.K., Y.J., W.S. and A.V. performed cloning. J.I., S.H., M.K. and Y.J. performed knockouts. M.R., F.L. and J.I. assisted with sample processing for immunophenotyping. J.I., W.S., Z.S., C.R.G., S.H., J.H., E.R.B., R.L.V., M.K. and A.V. assisted with some in vitro, in vivo and/or ex vivo experiments. J.I., Z.S., S.H., M.K. and Y.J. performed blood bactericidal assays and qPCR. A.R. and M.R. performed immunofluorescence studies. A.R., T.D. and N.A. analysed data and wrote the paper with input from all authors. Competing interests A.R., J.I., T.D. and N.A. have filed a provisional patent application with the US Patent and Trademark Office related to this work. The other authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41586-024-08033-4 . Correspondence and requests for materials should be addressed to Tal Danino or Nicholas Arpaia. Peer review information Nature thanks Paul Robbins and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at http://www.nature.com/reprints. ab c de f G8S2 CsL-25kDa10- -20 75- DnaKNeomE1Neo1Neo2 -kDa pTac pTac pTacpTacpTac NeoAgp (CsL)NeoAgp (G8S2)NeomE1Neo1Promoter:020406080100 Neo2ng/OD bacteriapMHC IC50≤500nM MHC-I MHC-IIShared Neither 7% 37% 9%47% Total = 406Pcdhgc3 Snx5 Ubqln 1Aldh18a1 Lin7cDdx27 Nap1l 4Tdg Cad Steap2 Trip12Gpc1 Smarcd1 pTacLO-pTacLO+pHelpGS-linker NeoAg LP Stop NeoaNeobNeocNeodNeoeNeonRBS NeoAgp NeoAgp(G4S1)051015Relative expression (intensity) NeoAgp(G4S1)0.00.51.01.52.02.5Relative expression (intensity)pTacLO-pTacLO+pHelpEcNMHCI/IIv MHCIIakDa 15-20- 20-25- 75-MHCIa NeoAgpBL21 EcN BL21 EcN BL21 EcN BL21pHelpp TacLO+pTacLO-NeoAgp pHelpp TacLO-pTacLO+NeoAgp(GS) 15-kDaNeoAg 75- DnaK - pHelp pTacLO+ NeoAgpNeoAgp(G4S1)Promoter:ng per OD bacteria pTacLO-(His6) Prototype Neoantigen Construct NeoAg LP Stop NeoaNeobNeocNeodNeonRBS kDapTacLO+pTacLO-pHelp NeoAgNeoAgp 15- 75- DnaK(His6) EcN BL2105101520Relative expression (intensity)MHCI/IIv MHCIIa MHCIaNeoAgp(G4S1) BL210123Expression relative to prototyp e (intensity )MHCI/IIv MHCIIa MHCIa020406080100 pHelp pTacLO+ pTacLO- DnaK Extended Data Fig. 1 | Neoantigen prediction and synthetic construct design. 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.