Swiss-PO is a new web tool to map gene mutations on the 3D structure of corresponding proteins and to intuitively assess the structural implications of protein variants for precision oncology. ...Swiss-PO is constructed around a manually curated database of 3D structures, variant annotations, and sequence alignments, for a list of 50 genes taken from the Ion AmpliSeqTM Custom Cancer Hotspot Panel. The website was designed to guide users in the choice of the most appropriate structure to analyze regarding the mutated residue, the role of the protein domain it belongs to, or the drug that could be selected to treat the patient. The importance of the mutated residue for the structure and activity of the protein can be assessed based on the molecular interactions exchanged with neighbor residues in 3D within the same protein or between different biomacromolecules, its conservation in orthologs, or the known effect of reported mutations in its 3D or sequence-based vicinity. Swiss-PO is available free of charge or login at https://www.swiss-po.ch.
We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N ...terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.
The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, ...and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC
50
values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design.
Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such ...interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.
The molecular cause of the majority of rare autosomal recessive disorders remains unknown. Consanguinity due to extensive homozygosity unravels many recessive phenotypes and facilitates the detection ...of novel gene-disease links. Here, we report two siblings with phenotypic signs, including intellectual disability (ID), developmental delay and microcephaly from a Pakistani consanguineous family in which we have identified homozygosity for p(Tyr103His) in the PSMB1 gene (Genbank NM_002793) that segregated with the disease phenotype. PSMB1 encodes a β-type proteasome subunit (i.e. β6). Modeling of the p(Tyr103His) variant indicates that this variant weakens the interactions between PSMB1/β6 and PSMA5/α5 proteasome subunits and thus destabilizes the 20S proteasome complex. Biochemical experiments in human SHSY5Y cells revealed that the p(Tyr103His) variant affects both the processing of PSMB1/β6 and its incorporation into proteasome, thus impairing proteasome activity. CRISPR/Cas9 mutagenesis or morpholino knock-down of the single psmb1 zebrafish orthologue resulted in microcephaly, microphthalmia and reduced brain size. Genetic evidence in the family and functional experiments in human cells and zebrafish indicates that PSMB1/β6 pathogenic variants are the cause of a recessive disease with ID, microcephaly and developmental delay due to abnormal proteasome assembly.
BackgroundThe adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid ...tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101.MethodsPrimary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups.ResultsIn line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1+ Ki67- and LAG-3+ Ki67- CAR-T cells in the TME.ConclusionsThis study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function.
The mutant IDH1 inhibitor ivosidenib improves outcomes for patients with IDH1-mutated cholangiocarcinoma, but resistance inevitably develops. Mechanisms of resistance and strategies to overcome ...resistance are poorly understood. Here we describe two patients with IDH1 R132C-mutated metastatic cholangiocarcinoma who developed acquired resistance to ivosidenib. After disease progression, one patient developed an oncogenic IDH2 mutation, and the second patient acquired a secondary IDH1 D279N mutation. To characterize the putative IDH1 resistance mutation, cells expressing the double-mutant were generated. In vitro, IDH1 R132H/D279N produces (R)-2HG less efficiently than IDH1 R132H. However, its binding to ivosidenib is impaired and it retains the ability to produce (R)-2HG and promote cellular transformation in the presence of ivosidenib. The irreversible mutant IDH1 inhibitor LY3410738 binds and blocks (R)-2HG production and cellular transformation by IDH1 R132H/D279N. These resistance mechanisms suggest that IDH1-mutated cholangiocarcinomas remain dependent on (R)-2HG even after prolonged ivosidenib treatment. Sequential mutant IDH inhibitor therapy should be explored as a strategy to overcome acquired resistance to mutant IDH inhibitors.
We report a case of an uveal melanoma patient with
p.Gly48Leu who responded to MEK inhibition. At the time of the molecular analysis, the pathogenicity of the mutation was unknown. A tridimensional ...structural analysis showed that Gα
can adopt active and inactive conformations that lead to substantial changes, involving three important switch regions. Our molecular modelling study predicted that
p.Gly48Leu introduces new favorable interactions in its active conformation, whereas little or no impact is expected in its inactive form. This strongly suggests that
p.Gly48Leu is a possible tumor-activating driver mutation, consequently triggering the MEK pathway. In addition, we also found an
p.Cys172Gly mutation, which was predicted by molecular modelling analysis to lead to a gain of function by impacting the Ig-like domain 2 folding, which is involved in FGF binding and increases the stability of the homodimer. Based on these analyses, the patient received the MEK inhibitor trametinib with a lasting clinical benefit. This work highlights the importance of molecular modelling for personalized oncology.
The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from ...human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.
Recent clinical developments in antitumor immunotherapy involving T-cell related therapeutics have led to a renewed interest for
uman
eukocyte
ntigen class
(HLA-I) binding peptides, given their ...potential use as peptide vaccines. Databases of HLA-I binding peptides hold therefore information on therapeutic targets essential for understanding immunity. In this work, we use in depth and accurate HLA-I peptidomics datasets determined by mass-spectrometry (MS) and analyze properties of the HLA-I binding peptides with structure-based computational approaches. HLA-I binding peptides are studied grouping all alleles together or in allotype-specific contexts. We capitalize on the increasing number of structurally determined proteins to (1) map the 3D structure of HLA-I binding peptides into the source proteins for analyzing their secondary structure and solvent accessibility in the protein context, and (2) search for potential differences between these properties in HLA-I binding peptides and in a reference dataset of HLA-I motif-like peptides. This is performed by an
developed heuristic search that considers peptides across all the human proteome and converges to a collection of peptides that exhibit exactly the same motif as the HLA-I peptides. Our results, based on 9-mers matched to protein 3D structures, clearly show enriched sampling for HLA-I presentation of helical fragments in the source proteins. This enrichment is significant, as compared to 9-mer HLA-I motif-like peptides, and is not entirely explained by the helical propensity of the preferred residues in the HLA-I motifs. We give possible hypothesis for the secondary structure biases observed in HLA-I peptides. This contribution is of potential interest for researchers working in the field of antigen presentation and proteolysis. This knowledge refines the understanding of the rules governing antigen presentation and could be added to the parameters of the current peptide-MHC class I binding predictors to increase their antigen predictive ability.
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