Journal: bioRxiv

Article Title: A novel HLA Class II presentation prediction algorithm deciphers immunogenic CD4 epitopes specific to KRAS G12C

doi: 10.1101/2024.12.06.627073

Figure Lengend Snippet: A) Schematic showing patient S2 tumor profile and therapies prior to enrollment and vaccination in study NCT03953235 B) T cell responses to KRAS G12C peptide pool assessed by ex vivo IFNγ ELISpot. Bar graphs (mean ± SD of replicate wells) show SFU/10 6 cells for timepoints prior to (baseline; week 0), and after vaccination (weeks 2, 4, 5, 6, 8, 12, 16, 20, and 24). Assay LOD (30 SFU/10 6 cells) is indicated by dotted line. Vaccination conditions are indicated by adenovirus and self-amplifying mRNA (samRNA) symbols. C) & D) T cell responses to KRAS G12C peptide pool and individual peptides or controls as assessed by ex vivo IFNγ ELISpot for PBMCs (C), CD4 depl PBMCs, and CD8 depl PBMCs (D) are shown. Bar graphs (mean ± SD of replicate wells) indicate SFU/10 6 cells at post-vaccination timepoints. Assay LOD (30 SFU/10 6 cells) is indicated by dotted line.

Article Snippet: Custom-made, recombinant, lyophilized peptides specific for G12C mutation were produced by Genscript (Piscataway, NJ, USA) and reconstituted at 2 or 5mg/ml/peptide in sterile DMSO (VWR International, Pittsburgh, PA, USA), aliquoted, and stored at - 80°C.

Techniques: Ex Vivo, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: A novel HLA Class II presentation prediction algorithm deciphers immunogenic CD4 epitopes specific to KRAS G12C

doi: 10.1101/2024.12.06.627073

Figure Lengend Snippet: A) Maximal presentation probability among all 15-mers containing the G12C mutation against the HLA genotype from a patient that received a vaccine containing KRAS G12C antigen. Presentation probability was scored per-allele. B) Saliency map of the first 26 amino acids of KRAS G12C computed by extracting the gradient of each 15-mer among G12C containing epitopes, over Patient S2’s HLA class II genotype (Methods). Larger saliency indicates greater importance to prediction of HLA class II presentation. C) Left: UMAP representation of all unique HLA alleles for which pseudosequences are available, colored by gene family. HLA alleles were embedded using pre-trained ESM2 (not EDGE-II). Right: Contour plots show EDGE-II scores as a function of the embedded HLA sequence-space, modeled as a 2D Gaussian Process (Methods).

Article Snippet: Custom-made, recombinant, lyophilized peptides specific for G12C mutation were produced by Genscript (Piscataway, NJ, USA) and reconstituted at 2 or 5mg/ml/peptide in sterile DMSO (VWR International, Pittsburgh, PA, USA), aliquoted, and stored at - 80°C.

Techniques: Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: A novel HLA Class II presentation prediction algorithm deciphers immunogenic CD4 epitopes specific to KRAS G12C

doi: 10.1101/2024.12.06.627073

Figure Lengend Snippet: A) Post-immunotherapy T cell responses to KRAS G12C peptide pool (Supplementary Table S5) and controls assessed by post-IVS IFNγ ELISpot for Patient S2. Bar graphs (mean ± SD of replicate wells) show SFU/10 6 cells. Assay limit of detection (LOD; 180 SFU/10 6 cells) is indicated by dotted line. Box and arrow indicate post-ELISpot cells collected for 10x emulsions and single cell sequencing. B) Schematic outlining single cell sequencing approach for TCR seq and digital gene expression (DGE) analyses.

Article Snippet: Custom-made, recombinant, lyophilized peptides specific for G12C mutation were produced by Genscript (Piscataway, NJ, USA) and reconstituted at 2 or 5mg/ml/peptide in sterile DMSO (VWR International, Pittsburgh, PA, USA), aliquoted, and stored at - 80°C.

Techniques: Enzyme-linked Immunospot, Sequencing, Expressing

Journal: bioRxiv

Article Title: A novel HLA Class II presentation prediction algorithm deciphers immunogenic CD4 epitopes specific to KRAS G12C

doi: 10.1101/2024.12.06.627073

Figure Lengend Snippet: A) Activation of Jurkat cells transduced with recombinant TCRs (rTCRs) from patient S2 co-cultured with antigen presenting cells pulsed with KRAS G12C pools and peptides or controls are shown. Bar graphs (median of replicate experiments ± SEM) show surface expression of activation markers CD25 (yellow) and CD69 (blue) as percent (%) positive populations. B) T cells from timepoint Dose-4 Day-1 visualized in two dimensions using UMAP and colored by clonal expansion. Top: Stimulation with CEF peptides. Bottom: Stimulation with G12C peptides. C) Approximately 23K T cells from multiple experimental conditions were visualized in two dimensions using UMAP (Methods). Left: annotation of different cell types from transcription profiles. Right: Same visualization with clonal abundance as the colormap. The T cells colored in red represent the clonotypes that are hyperexpanded (each unique clonotype is represented by 100 to 500 cells) and the T cells colored in blue represent the clonotypes that are singletons (each unique clonotype is represented by a single cell). D) UMI counts (expression level) of genes (x-axis) associated with T cell types and functional markers from single cell sequencing data. TCR969 (blue, n = 5 cells) and TCR995 (yellow, n = 7 cells) were experimentally validated for binding to a G12C peptide.

Article Snippet: Custom-made, recombinant, lyophilized peptides specific for G12C mutation were produced by Genscript (Piscataway, NJ, USA) and reconstituted at 2 or 5mg/ml/peptide in sterile DMSO (VWR International, Pittsburgh, PA, USA), aliquoted, and stored at - 80°C.

Techniques: Activation Assay, Transduction, Recombinant, Cell Culture, Expressing, Functional Assay, Sequencing, Binding Assay