Engineered cells and methods of use
A cell and engineering technology, applied in the medical field, can solve problems that limit the practical application of vaccination methods
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0131]Cell engineers are continually striving to grow cells that are active therapeutics. The past few years have witnessed amazing results in patients treated with adoptive cell transfer (ACT). Most notably, recent chimeric antigen receptor T-cell (CAR-T) therapy (tisagenlecleucel) (registered trademark of Novartis) approved as the first cell-based gene therapy in the United States. Genetic engineering is a common and reliable method for designing engineered cells with new functions. However, genetic engineering has shown limited success due to several technical challenges and safety concerns such as resistance to viral transduction of primary cells, heterogeneous expression levels, and potential for disruption of endogenous genes. To break through these limitations, engineering cell surfaces from the "outside" by biochemical, biophysical or enzymatic methods has become a general and generally applicable approach. Modification sites for "external" non-genetic approaches c...
Embodiment 2
[0138] figure 1 One embodiment of a two-step one-pot reaction and a one-step one-pot reaction is illustrated.
[0139] Two-step one-pot reaction : The reaction is usually carried out in a 1.5 mL Eppendorf tube containing 1.0 mL buffer (pH 7.5) containing L-fucose or its C-5 substituted analog (final concentration 10 mM), ATP (10 mM ), GTP (10mM), MgSO4 (10mM), inorganic pyrophosphatase (90 units, no endotoxin) and FKP (9 units, no endotoxin). The reaction mixture was incubated at 37°C with shaking (225 rpm) for 5 to 6 hours. The progress of the reaction was monitored by TLC using 10 mM tetrabutylammonium hydroxide in 80% acetonitrile in water as the developing solvent (aldose staining with p-anisole). After the disappearance of fucose or fucose analogues, the crude product can be directly used for cell surface fucosylation. If fucose-alkynes or azides are used, the crude product can be further modified by CuAAC reactions to generate GDP-fucose conjugates. For example, re...
Embodiment 3
[0142] like figure 2 As shown, in one embodiment, a one-pot fucosylation reaction is performed on cultured CHO cells. After fucosylation, conversion of cell surface sLacNAc to sLe X , which can be detected by an APC-conjugated anti-CLA antibody ( figure 2 A). Live CHO cells were washed 3 times with PBS and resuspended in 100 μL of fucosylation buffer (containing Helicobacter pylori α-1,3-FucT) containing purified GDP-fucose (in Fig. Indicated as a general reaction) or a one-pot method of GDP-fucose. After 20 min incubation at 37°C, cells were washed, blocked, and stained with APC-anti-CLA. After washing, samples were analyzed by flow cytometry. like figure 2 B and figure 2 As shown in C, the fucosylation reaction works well with the one-pot GDP-fucose. Concentration titrations showed that the reaction could be saturated with 100 μM one-pot GDP-fucose, which is the same as purified GDP-fucose according to the results disclosed herein. Control experiments were also ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More