A single point mutation on FLT3L-Fc protein increases the risk of Immunogenicity
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ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Qui Phung
PROVIDER: MSV000096859 | MassIVE | Wed Jan 15 15:07:00 GMT 2025
REPOSITORIES: MassIVE
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Project description:IntroductionAs a crucial asset for human health and modern medicine, an increasing number of biotherapeutics are entering the clinic. However, due to their complexity, these drugs have a higher potential to be immunogenic, leading to the generation of anti-drug antibodies (ADAs). Clinically significant ADAs have an impact on pharmacokinetics (PK), pharmacodynamics (PD), effectiveness, and/or safety. Thus, it is crucial to understand, manage and minimize the immunogenicity potential during drug development, ideally starting from the molecule design stage.MethodsIn this study, we utilized various immunogenicity risk assessment methods, including in silico prediction, dendritic cell internalization, MHC-associated peptide proteomics, in vitro HLA peptide binding, and in vitro T cell proliferation, to assess the immunogenicity risk of FLT3L-Fc variants.ResultsWe identified a single point mutation in the human FLT3L-Fc protein that introduced highly immunogenic T cell epitopes, leading to the induction of T cell responses and thereby increasing the immunogenicity risk in clinical settings. Consequently, the variant with this point mutation was removed from further consideration as a clinical candidate.DiscussionThis finding underscores the necessity for careful evaluation of mutations during the engineering of protein therapeutics. The integration of multiple immunogenicity risk assessment tools offers critical insights for informed decision-making in candidate sequence design and therapeutic lead selection.
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Project description:MotivationMutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling.ResultsWe developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon point mutations.Availability and implementationhttp://zhanglab.ccmb.med.umich.edu/STRUM/ CONTACT: qiang@suda.edu.cn and zhng@umich.eduSupplementary informationSupplementary data are available at Bioinformatics online.
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Project description:We investigated the RNAPII and γH2AX occupancy genome wide by ChIP-Seq in MLL2 F/F and FC/FC80 MEF cells. We found that a week after MLL2 excision (FC/FC cells), a group of genes present higher levels of γH2AX and RNAPII near the TSS, as compared to the control (F/F cells). H3K4Me1, H3K4M2 and H3K4Me3 levels near the TSS were also studied. There is a total of 52 samples. 3 independent replicates for each experiment were performed. H3, H2AX and IgG ChIPs were used for normalisation or as controls.The experiments were performed using immortalised mouse embryonic fibroblasts (MEF) in which both MLL2 alleles were targeted by the loxp system (F/F cells). Tamoxifen treatment of the F/F cells for 24 hours results in the excision of both MLL2 alleles (FC/FC cells).
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