Conditionally Active Chimeric Antigen Receptors for Modified T-Cells
a technology of chimeric antigen receptors and t-cells, which is applied in the field of protein evolution to achieve the effect of enhancing the wellness of a human
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example 1
escription of a Multiwall Assay (for Example, 96-Well Assay) for Temperature Mutants
[0344]Fluorescent substrate is added to each well of a multiwall plate, at both wild-type and new, lower reaction temperatures (for example, either 37° C. or 25° C. as mentioned above) for an appropriate time period. Fluorescence is detected by measuring fluorescence in a fluorescent plate reader at appropriate excitation and emission spectra (for example, 320 nm excitation / 405 nm emission). Relative fluorescence units (RFU) are determined. Supernatant from wild type molecule and plasmid / vector transformed cells are used as positive and negative controls. Duplicate reactions are performed for each sample, reaction temperature, and positive and negative control.
[0345]Mutants that are active at the lower temperature (for example, the mutants active at 25° C.) and that have a decrease in activity at the wild type temperature (for example, a 10%, 20%, 30%, 40% or more decrease in activity at 37° C.), thu...
example 2
escription of a Different Assay Format for Confirmation of Activity (for Example, a 14-mL Assay) for Temperature Mutants
[0346]Mutants that are identified as temperature sensitive primary hits are expressed in 14 ml culture tubes and their enzymatic activity is measured at wild type (for example, 37° C.) and the lower temperature (for example, 25° C.). Protein is expressed and purified as described above for the multiwall format, with the exception that the expression is performed in different format (14 ml tubes) rather than the multiwall (96-well plate) format.
[0347]Each mutant supernatant is transferred to a multiwall plate, for example a 96-well microplate. Fluorescent substrate is added to each tube at the indicated reaction temperatures (wild-type, lower temperature) for a required period of time. Wild-type molecules are used as a positive control and supernatant from cells transformed with only vector is used as a negative control. Fluorescence is detected by measuring fluores...
example 3
escription of Further Evolution of Hits Discovered
[0350]If desired, a new, combinatorial variant library is generated from all or selected mutant hits previously identified. The new library can be designed to contain every possible combination of amino acid variants for each of the selected mutants, and rescreened as described for new hits.
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