搜索结果: 'easysep direct'

T cell-derived cancers are hallmarked by heterogeneity,aggressiveness,and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here,we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept,we identified an antibody with unique specificity toward a distinct T cell receptor (TCR) and developed antibody-drug conjugates,precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization,associated with induction of cancer cell death with strong signs of apoptosis. Furthermore,cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules’ anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge,this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies. Graphical abstract Harald Kolmar and colleagues report a novel approach for the treatment of the difficult-to-treat T cell lymphoma/leukemia based on targeting the clonally rearranged T cell receptor expressed by the malignant T cell population. The developed antibody-drug conjugates precisely eliminate target T cells while preserving the integrity of the T cell repertoire and cellular immunity. View Publication

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based,and stable mammalian systems have been challenging to establish and apply. To this end,we develop PROTein Evolution Using Selection (PROTEUS),a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS,we alter the doxycycline responsiveness of tetracycline-controlled transactivators,generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution,and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall,PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells. Subject terms: Synthetic biology,Synthetic biology,Molecular evolution,Next-generation sequencing View Publication

Biological mechanisms are inherently dynamic,requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales,making them unsuitable for studying dynamic processes or characterizing essential genes,where chronic depletion can cause cell death. We compare five inducible protein degradation systems—dTAG,HaloPROTAC,IKZF3,and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency,basal degradation,target recovery after ligand washout,and ligand impact. This analysis identifies OsTIR1-based AID 2.0 as the most robust system. However,AID 2.0’s higher degradation efficiency comes with target-specific basal degradation and slower recovery rates. To address these limitations,we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants,including S210A,that significantly enhance the overall degron efficiency. The resulting degron system,named AID 2.1,maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout,enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts. Subject terms: Genetic engineering,CRISPR-Cas9 genome editing,Peptides View Publication

Adoptive cell therapy (ACT) targeting tumor-specific antigens holds promise for solid tumors,but limited neoantigen presentation remains a key barrier to efficacy. Here,we identify and characterize a T cell receptor (TCR),T104,for the KRAS.G12V mutation,a prevalent neoantigen in colorectal,lung,and pancreatic cancers. TCR-T104 selectively recognizes and kills KRAS.G12V-expressing tumor cells. Combining T cell therapy with lymphodepleting chemotherapy significantly enhances tumor cell killing,particularly by TCR-T cells,tumor-infiltrating lymphocytes (TILs),and T cell engager antibodies across multiple cancer types and target antigens. Mechanistically,chemotherapy upregulates immunoproteasome activity and human leukocyte antigen (HLA)-I surface expression. HLA-immunopeptidome analyses reveal that chemotherapy remodels the antigenic landscape across tumor cell lines and in vivo models,increasing peptide abundance and hydrophobicity while altering proteasomal cleavage preferences. These findings establish a synergistic role for chemotherapy in enhancing neoantigen presentation and T cell-mediated tumor recognition and suggest that fine-tuning these regimens could improve ACT efficacy,particularly in tumors with low-abundance neoantigens. View Publication

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations,which is lacking in the field,is also crucial. Here,we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine,a small molecule that activates the SHH pathway,could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+,Irx3+,and Pax7-),with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus,the directed neural differentiation system with small molecules,even without further purification,will facilitate basic and translational studies using human motoneurons at a minimal cost. View Publication

IL-33,a new member of the IL-1 family,has been described as an important inducer of Th2 cytokines and mediator of inflammatory responses. In this study,we demonstrate that murine basophils sorted directly from the bone marrow,without prior exposure to IL-3 or Fc(epsilon)R cross-linking,respond to IL-33 alone by producing substantial amounts of histamine,IL-4,and IL-6. These cells express ST2 constitutively and generate a cytokine profile that differs from their IL-3-induced counterpart by a preferential production of IL-6. In vivo,IL-33 promotes basophil expansion in the bone marrow (BM) through an indirect mechanism of action depending on signaling through the beta(c) chain shared by receptors for IL-3,GM-CSF,and IL-5. IL-3 can still signal through its specific beta(IL-3) chain in these mutant mice,which implies that it is not the unique growth-promoting mediator in this setup,but requires IL-5 and/or GMCSF. Our results support a major role of the latter growth factor,which is readily generated by total BM cells as well as sorted basophils in response to IL-33 along with low amounts of IL-3. Furthermore,GM-CSF amplifies IL-3-induced differentiation of basophils from BM cells,whereas IL-5 that is also generated in vivo,affects neither their functions nor their growth in vitro or in vivo. In conclusion,our data provide the first evidence that IL-33 not only activates unprimed basophils directly,but also promotes their expansion in vivo through induction of GM-CSF and IL-3. View Publication

Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease,therapeutic drug screening,and transplant-based regenerative medicine. However,methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here,we present a novel,small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2),we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC),sporadic FTD,and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid,efficient,and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy,terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient,novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases. View Publication

It is unclear by which receptor cyclic adenosine monophosphate (cAMP) acts to promote neutrophil survival. We found that 8-(4-chlorophenylthio)-2'-O-methyl-cAMP,a specific activator of the recently discovered cAMP receptor,cAMP-regulated guanosine 5'-triphosphate exchange protein directly activated by cAMP,failed to protect human neutrophils from cell death. In contrast,specific activators of cAMP-dependent protein kinase type I (cA-PKI) could protect against death receptor [tumor necrosis factor receptor 1 (TNFR-1),Fas]-mediated apoptosis as well as cycloheximide-accelerated spontaneous" apoptosis. A novel "caged" cA-PK-activating analog� View Publication

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10(9)-10(10) cells,because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage,to allow for at least an eightfold increase in cell number,with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array,and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition,our transcriptome,protein and functional studies,including albumin secretion,drug-induced CYP450 expression and urea production,all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. View Publication