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Cell Chemical Biology Oct 2023Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer...
Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.
Topics: Humans; Proliferating Cell Nuclear Antigen; Chromatin; Protein Binding; Neoplasms; DNA; DNA Replication
PubMed: 37531956
DOI: 10.1016/j.chembiol.2023.07.001 -
Nature Jan 2024Emerging data have shown that previously defined noncoding genomes might encode peptides that bind human leukocyte antigen (HLA) as cryptic antigens to stimulate...
Emerging data have shown that previously defined noncoding genomes might encode peptides that bind human leukocyte antigen (HLA) as cryptic antigens to stimulate adaptive immunity. However, the significance and mechanisms of action of cryptic antigens in anti-tumour immunity remain unclear. Here mass spectrometry of the HLA class I (HLA-I) peptidome coupled with ribosome sequencing of human breast cancer samples identified HLA-I-binding cryptic antigenic peptides that were noncanonically translated by a tumour-specific circular RNA (circRNA): circFAM53B. The cryptic peptides efficiently primed naive CD4 and CD8 T cells in an antigen-specific manner and induced anti-tumour immunity. Clinically, the expression of circFAM53B and its encoded peptides was associated with substantial infiltration of antigen-specific CD8 T cells and better survival in patients with breast cancer and patients with melanoma. Mechanistically, circFAM53B-encoded peptides had strong binding affinity to both HLA-I and HLA-II molecules. In vivo, administration of vaccines consisting of tumour-specific circRNA or its encoded peptides in mice bearing breast cancer tumours or melanoma induced enhanced infiltration of tumour-antigen-specific cytotoxic T cells, which led to effective tumour control. Overall, our findings reveal that noncanonical translation of circRNAs can drive efficient anti-tumour immunity, which suggests that vaccination exploiting tumour-specific circRNAs may serve as an immunotherapeutic strategy against malignant tumours.
Topics: Animals; Female; Humans; Mice; Antigens, Neoplasm; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Mass Spectrometry; Melanoma; Peptides; Protein Biosynthesis; Ribosome Profiling; RNA, Circular; Survival Analysis
PubMed: 38093017
DOI: 10.1038/s41586-023-06834-7 -
Nature Jun 2024The introduction of AlphaFold 2 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in...
The introduction of AlphaFold 2 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein-ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein-nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody-antigen prediction accuracy compared with AlphaFold-Multimer v.2.3. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.
Topics: Humans; Antibodies; Antigens; Deep Learning; Ions; Ligands; Models, Molecular; Molecular Docking Simulation; Nucleic Acids; Protein Binding; Protein Conformation; Proteins; Reproducibility of Results; Software
PubMed: 38718835
DOI: 10.1038/s41586-024-07487-w -
Journal of Hematology & Oncology Jul 2023Immune cell engagers are engineered antibodies with at least one arm binding a tumor-associated antigen and at least another one directed against an activating receptor... (Review)
Review
Immune cell engagers are engineered antibodies with at least one arm binding a tumor-associated antigen and at least another one directed against an activating receptor in immune effector cells: CD3 for recruitment of T cells and CD16a for NK cells. The first T cell engager (the anti-CD19 blinatumomab) was approved by the FDA in 2014, but no other one hit the market until 2022. Now the field is gaining momentum, with three approvals in 2022 and 2023 (as of May): the anti-CD20 × anti-CD3 mosunetuzumab and epcoritamab and the anti-B cell maturation antigen (BCMA) × anti-CD3 teclistamab, and another three molecules in regulatory review. T cell engagers will likely revolutionize the treatment of hematological malignancies in the short term, as they are considerably more potent than conventional monoclonal antibodies recognizing the same tumor antigens. The field is thriving, with a plethora of different formats and targets, and around 100 bispecific T cell engagers more are already in clinical trials. Bispecific NK cell engagers are also in early-stage clinical studies and may offer similar efficacy with milder side effects. Trispecific antibodies (engaging either T cell or NK cell receptors) raise the game even further with a third binding moiety, which allows either the targeting of an additional tumor-associated antigen to increase specificity and avoid immune escape or the targeting of additional costimulatory receptors on the immune cell to improve its effector functions. Altogether, these engineered molecules may change the paradigm of treatment for relapsed or refractory hematological malignancies.
Topics: Humans; T-Lymphocytes; Antibodies, Bispecific; Immunotherapy; Hematologic Neoplasms; Killer Cells, Natural; Antigens, CD19; Antineoplastic Agents; Antigens, Neoplasm
PubMed: 37501154
DOI: 10.1186/s13045-023-01482-w -
Cell Oct 2023The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid...
The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells.
Topics: Humans; Antigen Presentation; Antigens, CD1; Lipidomics; Lipids; T-Lymphocytes; Amino Acid Motifs
PubMed: 37725977
DOI: 10.1016/j.cell.2023.08.022 -
Current Protocols Aug 2023Visualizing fluorescence-tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is...
Visualizing fluorescence-tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence-labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine-tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free-swimming Tetrahymena cells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super-resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such as Tetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly-l-lysine-coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super-resolution images with SRRF microscopy.
Topics: Microscopy, Fluorescence; Fluorescent Antibody Technique; Microtubules; Cytoskeleton; Fibroblasts; Antibodies; Antigens
PubMed: 37540554
DOI: 10.1002/cpz1.842 -
Immunity Nov 2023The accurate selection of neoantigens that bind to class I human leukocyte antigen (HLA) and are recognized by autologous T cells is a crucial step in many cancer...
The accurate selection of neoantigens that bind to class I human leukocyte antigen (HLA) and are recognized by autologous T cells is a crucial step in many cancer immunotherapy pipelines. We reprocessed whole-exome sequencing and RNA sequencing (RNA-seq) data from 120 cancer patients from two external large-scale neoantigen immunogenicity screening assays combined with an in-house dataset of 11 patients and identified 46,017 somatic single-nucleotide variant mutations and 1,781,445 neo-peptides, of which 212 mutations and 178 neo-peptides were immunogenic. Beyond features commonly used for neoantigen prioritization, factors such as the location of neo-peptides within protein HLA presentation hotspots, binding promiscuity, and the role of the mutated gene in oncogenicity were predictive for immunogenicity. The classifiers accurately predicted neoantigen immunogenicity across datasets and improved their ranking by up to 30%. Besides insights into machine learning methods for neoantigen ranking, we have provided homogenized datasets valuable for developing and benchmarking companion algorithms for neoantigen-based immunotherapies.
Topics: Humans; Antigens, Neoplasm; Neoplasms; Histocompatibility Antigens Class I; Machine Learning; Peptides; Immunotherapy
PubMed: 37816353
DOI: 10.1016/j.immuni.2023.09.002 -
Proceedings of the National Academy of... Jul 2023A common event upon receptor-ligand engagement is the formation of receptor clusters on the cell surface, in which signaling molecules are specifically recruited or...
A common event upon receptor-ligand engagement is the formation of receptor clusters on the cell surface, in which signaling molecules are specifically recruited or excluded to form signaling hubs to regulate cellular events. These clusters are often transient and can be disassembled to terminate signaling. Despite the general relevance of dynamic receptor clustering in cell signaling, the regulatory mechanism underlying the dynamics is still poorly understood. As a major antigen receptor in the immune system, T cell receptors (TCR) form spatiotemporally dynamic clusters to mediate robust yet temporal signaling to induce adaptive immune responses. Here we identify a phase separation mechanism controlling dynamic TCR clustering and signaling. The TCR signaling component CD3ε chain can condensate with Lck kinase through phase separation to form TCR signalosomes for active antigen signaling. Lck-mediated CD3ε phosphorylation, however, switched its binding preference to Csk, a functional suppressor of Lck, to cause the dissolvement of TCR signalosomes. Modulating TCR/Lck condensation by targeting CD3ε interactions with Lck or Csk directly affects T cell activation and function, highlighting the importance of the phase separation mechanism. The self-programmed condensation and dissolvement is thus a built-in mechanism of TCR signaling and might be relevant to other receptors.
Topics: Receptors, Antigen, T-Cell; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Signal Transduction; Phosphorylation; Antigens
PubMed: 37399423
DOI: 10.1073/pnas.2217301120 -
Immunity Oct 2023Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous...
Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.
Topics: Mice; Humans; Animals; Peyer's Patches; B-Lymphocytes; Antigens; Receptors, Antigen, B-Cell; Immunoglobulin A; Intestinal Mucosa
PubMed: 37714151
DOI: 10.1016/j.immuni.2023.08.018 -
Immunity Jul 2023CD4+ T cell responses are exquisitely antigen specific and directed toward peptide epitopes displayed by human leukocyte antigen class II (HLA-II) on antigen-presenting...
CD4+ T cell responses are exquisitely antigen specific and directed toward peptide epitopes displayed by human leukocyte antigen class II (HLA-II) on antigen-presenting cells. Underrepresentation of diverse alleles in ligand databases and an incomplete understanding of factors affecting antigen presentation in vivo have limited progress in defining principles of peptide immunogenicity. Here, we employed monoallelic immunopeptidomics to identify 358,024 HLA-II binders, with a particular focus on HLA-DQ and HLA-DP. We uncovered peptide-binding patterns across a spectrum of binding affinities and enrichment of structural antigen features. These aspects underpinned the development of context-aware predictor of T cell antigens (CAPTAn), a deep learning model that predicts peptide antigens based on their affinity to HLA-II and full sequence of their source proteins. CAPTAn was instrumental in discovering prevalent T cell epitopes from bacteria in the human microbiome and a pan-variant epitope from SARS-CoV-2. Together CAPTAn and associated datasets present a resource for antigen discovery and the unraveling genetic associations of HLA alleles with immunopathologies.
Topics: Humans; Captan; Deep Learning; COVID-19; SARS-CoV-2; HLA Antigens; Epitopes, T-Lymphocyte; Peptides
PubMed: 37301199
DOI: 10.1016/j.immuni.2023.05.009