# US 10,871,490 B2 · Assay for detection of chimeric antigen receptor T cells

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- Number: US 10,871,490 B2
- Document ID: US10871490B2
- Status: Granted
- Kind: B2
- Application number: 15/417,292
- Filed: 2017-01-27
- Published: 2017-08-31
- Granted: 2020-12-22
- Earliest claimed: 2016-02-29
- Assignees: Multenyi Biotec, gmbH
- Applicants: Miltenyi Biotec GmbH
- Inventors: Anne Richter; Michaela Niemoller; Volker Nolle; Stefan Miltenyi; Mario Assenmacher
- Web page: https://patentgrep.com/patent/US10871490B2

## CPC

- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- C07K 14/70503: {Immunoglobulin superfamily}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- G01N 33/56972: {White blood cells}
- G01N 2333/70596: Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention is directed to a detection reagent for CAR expressing cells according to general formula (1) Xn—Sm—Do 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.

## Claims

### Claim 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,
Xn—Sm—Do
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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

The method according to claim 1 characterized in that after detection, the detection moiety D is subjected to photo- and/or chemical bleaching.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.