Identification of patient-specific CD4+ and CD8+ T cell neoantigens through HLA-unbiased genetic screens

Abstract

Cancer neoantigens that arise from tumor mutations are drivers of tumor-specific T cell responses, but identification of T cell-recognized neoantigens in individual patients is challenging. Previous methods have restricted antigen discovery to selected HLA alleles, thereby limiting the breadth of neoantigen repertoires that can be uncovered. Here, we develop a genetic neoantigen screening system that allows sensitive identification of CD4⁺ and CD8⁺ T cell-recognized neoantigens across patients' complete HLA genotypes.

Fig. 1. Overview and validation of neoantigen discovery technology.

a, Schematic overview of the methodology. b, Antigen discovery screen of CD8⁺ TCR #53 T cells against immortalized HLA-A*02:01⁺ B cells transduced with a model antigen library of n = 4,764 minigenes. Dots represent individual minigenes. Fold change, defined as the relative abundance of minigenes in the presence of TCR #53 T cells compared with mock T cells, and mean normalized read counts are plotted for each individual minigene. Minigenes encoding the model CDK4 mutant and WT epitopes are highlighted. CDK4_R24L minigenes: P = 9.4 × 10⁻⁴³, P = 1.5 × 10⁻⁹. P values were generated using the DESeq2 Wald test (one-sided) and adjusted for several comparisons. c, Antigen screen using CD8⁺ T cells expressing either the DMF4 (left panel) or DMF5 (right panel) TCR against model library-expressing HLA-A*02:01⁺ B cells. Data are plotted as in b with fold change showing relative minigene abundance when exposed to either DMF4 or DMF5 TCR T cells as compared with mock T cells. DMF4 MART1-ELA minigenes: P = 1.9 × 10⁻¹², P = 1.7 × 10⁻⁹; DMF5 MART1-ELA minigenes: P = 4.1 × 10⁻¹⁴, P = 8.8 × 10⁻⁷. P values were generated as in b. d, Antigen screen using CD4⁺ T cells expressing patient-derived TCRs specific for the MHC class II-restricted SNORD73A_R>W and MANSC1_D>H against patient-matched immortalized B cells transduced with the CD74 signal-fused model library. Data are plotted as in b with fold change showing relative minigene abundance in the presence of CD4⁺ SNORD73A TCR or MANSC1 TCR T cells relative to mock T cells. SNORD73A_R>W: P = 2.1 × 10⁻⁵³; P = 2.5 × 10⁻⁴⁴; MANSC1_D>H: P = 2.1 × 10⁻²⁶, P = 3.2 × 10⁻⁸. P values were generated as in b.

Fig. 2. Personalized and HLA-agnostic neoantigen screening of patient-derived CD4⁺ and CD8⁺ T cells.

a,b, Nonsynonymous tumor mutations of patient NKIRTIL063 were identified by exome and RNA sequencing and used to design a personalized mutanome minigene library consisting of n = 2,762 unique minigenes. Patient B cells were immortalized, transduced with the mutanome library and screened with in vitro-expanded tumor-infiltrating CD8⁺ (a) and CD4⁺ (b) T cells. Fold change represents relative minigene abundance in cultures with or without patient T cells. Screen hits were defined as outlined in Methods and are marked by colored dots. c,d, Validation of neoantigen hits identified in a and b by incubating patient CD8⁺ (c) or CD4⁺ (d) with autologous B cells expressing either neoantigen hits (mut) or respective WT sequences as single minigenes. T cell activation was assessed by measuring IFNγ levels in supernatants. Dots represent technical replicates. e, NKIRTIL063 CD8⁺ and CD4⁺ T cells were incubated with patient B cells expressing indicated TMG constructs, followed by measuring IFNγ concentrations in culture supernatants. Asterisks indicate TMG constructs that encode a neoantigen identified using the antigen screens in a–d. Dots represent technical replicates. f, Summary of NKIRTIL063 neoantigens identified using the HANSolo screens and TMG approach. g,h, Patient NKIRTIL027 immortalized B cells were transduced with the patient mutanome library (n = 2,586 minigenes) and screened using in vitro-expanded NKIRTIL027 CD8⁺ (g) and CD4⁺ (h) tumor-infiltrating T cells. Fold change depicts relative minigene abundance in cultures with or without patient T cells. Screen hits are marked by colored dots. i,j, NKIRTIL027 CD8⁺ TIL screen hits were validated by incubating patient T cells with matched B cells expressing the single mutant or corresponding WT sequences, and measuring IFNγ levels in supernatants (i) or killing of transduced B cells after exposure to patient T cells at indicated effector:target (E:T) ratios (j). Dots represent technical replicates. N/A, not available. k, The NKIRTIL027 CD4⁺ T cell screen hit was validated as in i. l,m, Neoantigen specificities of patient ITO34 CD8⁺ TIL were screened against the patient mutatome library (n = 952 minigenes) and validated as in g and i.