Assay for detection of chimeric antigen receptor T cells
The invention is directed to a detection reagent for CAR expressing cells according to general formula (1) X n —S m —D o wherein X is a polypeptide comprising at least 20 amino acids of which at least two amino acids are connected by at least one disulfide group S is a spacer comprising at least 15 carbon atoms D is a detection moiety m, o are integers independently between 1 and 200 n is an integer between 4 and 200 with the provisio that X binds to the antigen binding domain of a chimeric antigen receptor of a cell. The detection agent can be used in a method to detect cells having an antigen binding domain of a chimeric antigen receptor (CAR) binding the detection reagent and preferable to remove the detection moiety D from the detected cell.
1. A method for detecting cells expressing an antigen binding domain of a chimeric antigen receptor (CAR), comprising:
providing a detection reagent according to the general formula (1) to a sample of cells,
X n —S m —D o
wherein
X is a polypeptide comprising at least 20 amino acids of which at least two amino acids are connected by at least one disulfide group having an extracellular sequence of CD20
S is a spacer comprising a polysaccharide having at least 15 carbon atoms selected from the group consisting of dextrans, pullulans, inulins, amylose, cellulose, hemicelluloses, xylan, glucomannan, pectin, chitosan and chitin
D is a fluorescent moiety as detection moiety
m, o are integers independently between 1 and 200
n is an integer between 4 and 200
with the proviso that X binds to the antigen binding domain of a chimeric antigen receptor of a cell
wherein the cells have an antigen binding domain of a chimeric antigen receptor (CAR) binding the detection reagent;
detecting the cells via the detection moiety of the detection reagent; and after detecting the cell via the detection moiety,
enzymatically digesting the spacer S thereby removing the detection moiety D from the detected cell.
2. The method according to claim 1 characterized in that the cells recognizing the conjugate are detected by fluorescence emission, applying a magnetic field or by chemical reaction of the chemically detectable moiety.
3. The method according to claim 1 characterized in that the cells detected via the detection moiety D of the detection reagent are separated from the not detected cells.
4. The method according to claim 1 characterized in that after detection, the detection moiety D is subjected to photo- and/or chemical bleaching.
5. The method according to claim 1 , characterized in that polypeptide X has a sequence of amino adds of CEPANPSEKNSPSTQYC (SEQ ID NO. 1), wherein the amino adds C are connected by a disulfide group.
6. The method according to claim 1 , characterized in that polypeptide X has the sequence of amino adds of NIYNCEPANPSEKNSPSTQYCYSIQ, (SEQ ID NO. 2), wherein the amino adds Care connected by a disulfide group.