Journal: BMC cancer

Article Title: First molecules to reactivate RAS G12V GTPase activity.

doi: 10.1186/s12885-025-13580-8

Figure Lengend Snippet: Fig. 1 The best- scored flexible docking poses of PER154A (A), PER160A (B), PER170A (C), and PER176A (D) docked to KRASG12V (PDB ID: 8AFD). Experi mental V12 and Q61 conformations have been shown in light gray, and results from flexible docking are shown in green. Hydrogen bonds are repre sented by dashed cyan lines

Article Snippet: Briefly, 500 ng of KRAS-WT or KRASG12V protein (both from SignalChem), 500 ng of GAP RT 01001-PER, molecules at selected concentrations – 10 μM, 1 μM DTT, 5 μM GDP and GTPase/GAP Buffer were applied to white non-transparent 96-well plates to a final volume of 25 μl per well.

Techniques:

Journal: BMC cancer

Article Title: First molecules to reactivate RAS G12V GTPase activity.

doi: 10.1186/s12885-025-13580-8

Figure Lengend Snippet: Fig. 3 Analysis of the GTPase activity of the KRASG12V protein in the presence of selected molecules. (A) Verification of the action of GAP family proteins – a commercially available and self-obtained GAP RT 01001-PER on KRASWT and KRASG12V proteins. No functionality was observed for the commercial pro tein, while GAP RT 01001-PER showed GTPase activity for KRASWT and had no effect on KRASG12V. The GTPase activity of the KRASG12V protein was partially restored by selected molecules. These effects were observed both in the absence (B) and presence (C) of the GAP RT 01001-PER protein

Article Snippet: Briefly, 500 ng of KRAS-WT or KRASG12V protein (both from SignalChem), 500 ng of GAP RT 01001-PER, molecules at selected concentrations – 10 μM, 1 μM DTT, 5 μM GDP and GTPase/GAP Buffer were applied to white non-transparent 96-well plates to a final volume of 25 μl per well.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) NetMHCPan 4.1 summary of the top 10 peptide sequences that are most likely to be presented by HLA-A2 from the first 30 amino acids of KRAS G12V . Peptides containing the G12V mutation (including mutations in red text) are in black text; others are in gray. (b) Log 10 (EC 50 (M)) comparison of recognition of synthetic KRAS G12V 5-14 peptide (KLVVVGAVGV) presented by HLA-A2 by an initially generated set of MHC class I-restricted TCRs isolated to be specific for this epitope. Lower values indicate higher functional avidity. (c) % IFNγ + T cells, transduced with TCR 2 , after exposure to B-LCL cell lines expressing different Class I HLA alleles found on CFPAC1 cells without KRAS G12V 5-14 peptide loaded. Cell lines are color coded by allele. (d) MS results of RAS family peptide fragments detected after tryptic digestion of immunoprecipitated whole KRAS protein from cell lysate of DAN-G and CFPAC1 pancreatic adenocarcinoma cell lines. Lysine side chain trimethylation is indicated in red with an asterisk. (e) Reconstitution of properly folded HLA-A2 surface expression on T2 cells after exogeneous loading by wild type or G12V/D mutant KRAS 5-14 peptides with or without lysine-5 side chain methylations. (f) Additional views of structural models of HLA-A2-presented KRAS G12V 5-14 peptide including possible lysine-5 side chain methylation, supplementing by highlighting the protrusion of the lysine-5 side chain (circled in red) out of the HLA-A2 groove. (g) Overview of structural prediction of a TCR-pMHC complex using a combined Alphafold-Rosetta pipeline. Alphafold allows for deep learning-based prediction of proteins and protein complexes, which we applied to predict TCR-pMHC complex structure. However, this does not yet contain PTMs, which Alphafold cannot incorporate reliably. Thus, we used the resulting structure as input into Rosetta, which used physics-based energy functions to introduce PTMs and reoptimize the structure to accommodate them.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Mutagenesis, Comparison, Generated, Isolation, Functional Assay, Transduction, Expressing, Immunoprecipitation, Methylation, Structural Proteomics, Introduce

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Log 10 (EC 50 (M)) comparison of recognition of different concentrations of a synthetic KRAS G12V 5-14 peptide (KLVVVGAVGV) presented by HLA-A2 by different MHC class I-restricted TCRs specific for the epitope. Lower values indicate higher functional avidity. (b) Killing of live CFPAC1 and DAN-G HLA-A2 + KRAS G12V+ pancreatic adenocarcinoma cells cocultured with CD8 + T cells expressing the TCRs shown in (a) using a 4:1 T cell:tumor cell ratio. Cocultures were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR: red fluorescent protein; NG: green fluorescent protein. (c) ARTEMIS MS data of peptides eluted from HLA-A2. Data is from the 293F cell line, as commonly used for proteomics assays requiring high protein expression. All cell lines were transduced with an HLA-A2 single chain secreted dimer as well as a KRAS G12V constitutive expression construct to increase presentation and the likelihood of detecting a presented peptide by MS, of KRAS G12V -related epitopes. The first 100 amino acids of KRAS G12V are shown, with the G12V mutation highlighted in yellow. Epitopes predicted by NetMHCPan 4.1 are shown in purple; epitopes detected from ARTEMIS are shown in blue. ARTEMIS detection of KRAS G12V 5-14 is outlined in red. (d) Heatmap of – log 10 (EC 50 (μg/ml)) of CD8 + T cells expressing TCR 2 or TCR 19 against candidate KRAS G12V epitopes, with and without methylation of the lysine-5 side chain of the epitope, presented by HLA-A2. Higher values indicate higher functional avidity. EC 50 values were calculated from T cell exposure to peptide concentrations ranging from 1 µg/ml to 10 - µg/ml. Gray squares indicate EC 50 calculations that lacked a stable fit, e.g., due to lack of response to peptide even at high doses. (e) Rosetta structural modeling of HLA-A2 presenting KRAS G12V 5-14 and its methylated variants. Red circles indicate the amine group of the lysine-5 side chain; arrows indicate its movement with different methylation states. A reference point is drawn at the same position in each image to help illustrate positional changes. (f) Alanine scan of the KRAS G12V epitope and resulting response by CD8 + T cells expressing TCR 2 and TCR 19 . The epitope with each individual residue substituted with alanine (or, for alanine-11, substituted with either glycine or threonine) was presented by antigen-presenting cells to stimulate primary human CD8 + T cells expressing a TCR, and subsequent IFNγ expression was measured. Higher IFNγ induction indicates greater tolerance for amino acid substitution at that position. X-axis numbers indicate the residue number on the KRAS protein, and letters indicate the amino acid substitution. A: alanine; G: glycine; T: threonine. Last column is unmodified KRAS G12V 5-14.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Comparison, Functional Assay, Expressing, Imaging, Transduction, Construct, Mutagenesis, Methylation, Residue

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Structural model, as predicted by a combined Alphafold-Rosetta pipeline, of a ‘core’ CDR3α sequence (sequence EDNT) of an existing TCR recognizing KRAS G12V 5-14 and its relation to the lysine-5 side chain (circled in red) of the epitope as presented by HLA-A2. (b) Generation of TCR mutagenesis libraries by mutagenesis of the core CDR3α sequence followed by transduction of the TCRs into Nur77-GFP reporter Jurkat cells and subsequent clonal expansions. Tetramer binding patterns of HLA-A2-KRAS G12V 5-14 peptide with unmodified or methylated lysine-5 side chain at the end of cell stimulation are shown. Each circle indicates a population with a specific pattern of unmethylated vs. methylated peptide tetramer binding that was sorted and sequenced. (c) Examples of mutated CDR3α ‘core’ sequences resulting from sorted clones of the mutagenesis library that exhibit the noted different patterns of PTM peptide recognition. (d) Heatmap of – log 10 (EC 50 (μg/ml)) of TCRs derived from the mutagenesis library and the original TCR 2 responding to different degrees of lysine-5 methylation of KRAS G12V 5-14 presented by HLA-A2, as calculated from dose response assays. Gray squares indicate EC 50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. TCR 4UM is the 4 th TCR clonotype identified from an initial sequencing dataset from sorted Jurkat cells binding to both unmethylated (“U”) and methylated (“M”) tetramers; other TCRs are labeled by changes in the ‘core’ CDR3α sequence (e.g., EDST). (e) Killing of HLA-A2 + KRAS + CFPAC1 and DAN-G pancreatic adenocarcinoma cells by CD8 + T cells expressing selected mutagenized TCRs. Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Sequencing, Mutagenesis, Transduction, Binding Assay, Methylation, Cell Stimulation, Clone Assay, Derivative Assay, Labeling, Expressing, Imaging

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: Structural predictions of unmethylated and methylated variants of KRAS G12V 5-14, as well as unmethylated wild type or G12D peptide, presented by HLA-A2 to interface with (a) TCR 2 and (b) mutagenized TCR EDST TCRs. The CDR3α ‘core’ sequence is shown as individual amino acids within the TCRs. In (b), the TCR 2 -pMHC interfaces from (a) are overlaid with transparency to facilitate visual comparison. With TCR EDST , residues in the core sequence of CDR3α and the lysine-5 side chain of each variant of KRAS G12V epitope appear closer to each other than with TCR 2 .

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Methylation, Sequencing, Comparison, Variant Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Dose response curves of CD8 + T cells expressing selected TCRs generated from the HLA-A2 KRAS G12V TCR mutagenesis library after exposure to wild type or G12D KRAS 5-14 peptides presented by HLA-A2. (b) Failure of CD8 + T cell killing and progressive growth of human HLA-A2 + tumor cell lines that express wild type KRAS (HeLa cervical carcinoma cell line; left) or KRAS G12D (Panc1 pancreatic adenocarcinoma; right) and no KRAS G12V . Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein. (c) Alanine scan of KRAS G12V 5-14 epitope tested against TCRs selected from the mutagenesis library. Each cell indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine) and with or without lysine-5 side chain dimethylation. (d) IFNγ secretion by CD8 + T cells expressing mutagenized TCRs after exposure to synthetic peptides from the human proteome fitting the K-x-x-V-V-x-A-x-x-x tolerance pattern identified from the alanine scan shown in (c), with a concentration of 0.1 μg/ml for each peptide. (e) Dose response curves of CD8 + T cells expressing mutagenized TCRs after exposure to the potential human off-target peptides demonstrated above to generate the three largest responses at 0.1 μg/ml: CFA61 23-32/607-616/667-676 , RSLBB 35-44 , and TRXR1 342-351 , compared to the KRAS G12V epitope.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Expressing, Generated, Mutagenesis, Imaging, Residue, Concentration Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Experimental workflow to enrich and characterize T cell clones with TCRs recognizing a defined peptide epitope. (b) Tetramer binding patterns of primary human T cells stimulated with unmethylated and methylated KRAS G12V peptide presented by HLA-A2. Plots also indicate gates used to sort cells with different affinities for unmethylated and methylated epitopes. (c) Dose response curves of TCRs against wild type and KRAS G12D 5-14 peptides presented by HLA-A2. (d) Tumor cell growth during coculture of CD8 + T cells expressing selected TCRs with HLA-A2 + , wild type KRAS HeLa and KRAS G12D Panc1 pancreatic adenocarcinoma cell lines. Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein. (e) Alanine scan of KRAS G12V 5-14 epitope tested against TCR A2UoM1-1 . Each box indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine). (f) Dose response curve of CD8 + Nur77-GFP Jurkat cells expressing TCR A2UoM1-1 after exposure to EPIPL 1948-1957 , the potential human off-target peptide as determined from alanine scan results.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Clone Assay, Binding Assay, Methylation, Expressing, Imaging, Residue

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Heatmap of –log 10 (EC 50 (μg/ml)) of selected TCRs responding to the unmethylated and methylated epitopes presented by HLA-A2, as calculated from dose response assays. Higher values indicate higher functional avidity. Gray squares indicate EC 50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. (b) Killing of HLA-A2 + KRAS G12V+ CFPAC1 and DAN-G pancreatic adenocarcinoma cells by CD8 + T cells expressing selected TCRs. Cocultures of fluorescently labeled tumor cells and TCR-T cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR: red fluorescent protein; NG: green fluorescent protein. (c) Alphafold-Rosetta visualizations of TCR:pMHC complexes for TCR A2UoM1-1 , isolated from expansion of responding primary human CD8 + T cells from normal repertoires. Structures were generated for unmethylated and mono/di/trimethylated versions of the epitope, presented by HLA-A2. The four CDR3α amino acids in closest proximity to the lysine-5 side chain of KRAS G12V are shown with individual molecular bonds.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Methylation, Functional Assay, Expressing, Labeling, Imaging, Isolation, Generated

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Overall workflow: DAN-G pancreatic adenocarcinoma cells were transduced with a CRISPR knockout library targeting methylation-related genes, and then cocultured with CD8 + T cells expressing either TCR 2 or mutagenized TCR EDST , which respectively have weak or strong recognition of methylated KRAS G12V epitopes. Tumor cells surviving at the end of coculture were sequenced to identify gRNA enrichment or depletion associated with immune evasion. (b) Volcano plot summarizing log 2 (fold change) of gRNA read frequencies targeting individual genes recovered after coculture of DAN-G cells with T cells expressing TCR EDST versus TCR 2 . The threshold of significance was set at log 10 ( p ) < 1.2 ( p < 0.05) and is denoted by the horizontal dashed line. Mean counts of all gRNAs per gene were used to calculate fold change values. The data point for gRNAs targeting SUPT6H is specifically labeled. The knockouts depleted or enriched after cocultures using TCR EDST versus TCR 2 are colored blue and red, respectively. (c) Killing of DAN-G cells with or without SUPT6H knockout in coculture with selected TCR-T cells (untransduced or expressing TCR 2 , TCR EDST , or TCR A2UoM1-1 ). Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed green nuclear fluorescent protein (“NG”). (d) Kaplan-Meier survival plots for individuals who have tumors with KRAS mutations, using public TCGA data. Left: only tumors with the KRAS G12V mutant, with or without concurrent SUPT6H mutation. Right: tumors with any KRAS mutant, with or without a concurrent SUPT6H mutation.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Transduction, CRISPR, Knock-Out, Methylation, Expressing, Labeling, Imaging, Mutagenesis