# US 12,181,475 B2 · Reagents for detection of chimeric antigen receptor cells

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- Number: US 12,181,475 B2
- Document ID: US12181475B2
- Status: Granted
- Kind: B2
- Application number: 17/226,202
- Filed: 2021-04-09
- Published: 2021-12-02
- Granted: 2024-12-31
- Earliest claimed: 2020-05-27
- Assignees: Miltenyi Biotec B.V. &Co.KG
- Applicants: Miltenyi Biotec B.V. & Co. KG
- Inventors: Michaela Niemöller; Jonathan Fauerbach; Christian Dose; Anne Richter; Tina Borke
- Web page: https://patentgrep.com/patent/US12181475B2

## CPC

- G01N 33/56966: {Animal cells}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- 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 2319/03: containing a transmembrane segment
- 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 method for preparing a detection reagent for CAR expressing cells according to general formula (I) (X-S)m-D (I) wherein D is a detection moiety, S a spacer unit and X a polypeptide comprising 10 to 80 amino acids and a N3 group, which binds to the antigen binding domain of a chimeric antigen receptor of a cell and m=4-200, characterized in reacting p detection moieties D with a spacer S according to general formula (II) wherein R is a leaving group, q=1 to 5, and n=4 to 200 and p is 4-250 thereby forming modified detection moiety Sp-D and reacting the modified detection moiety Sp-D with a plurality of polypeptides X to yield (X-S)m-D.

## Claims

### Claim 1

A method for preparing a detection reagent for CAR expressing cells according to general formula (I)
(X-S)m-D (I)
wherein D is a detection moiety, S a spacer unit, m=4-200 and X is a polypeptide comprising 10 to 80 amino acids and a N3 group wherein X binds to the antigen binding domain of a chimeric antigen receptor of a cell, characterized in reacting p detection moieties D with a spacer S according to general formula (II)
wherein R is a leaving group, q=1 to 5, and n=4 to 200 and p is 4-250;
thereby forming modified detection moiety Sp-D and reacting the modified detection moiety Sp-D with a plurality of polypeptides X to yield (X-S) m-D.

### Claim 2 (depends on 1)

The method according to claim 1, characterized in that the leaving group R is selected from the group consisting of succinimidyl ester, tertafluorophenyl ester, sulfodicholorphenol ester, hydroxybenzotriazole ester and acyl chloride.

### Claim 3 (depends on 1)

The method according to claim 1, characterized in that the polypeptide X is an extracellular sequence of CD20.

### Claim 4 (depends on 1)

The method according to claim 1, characterized in that polypeptide X has a sequence of amino acids of CEPANPSEKNSPSTQYC (SEQ ID NO: 1.

### Claim 5 (depends on 1)

The method according to claim 1, characterized in that polypeptide amino acids of X has the sequence of NIYNCEPANPSEKNSPSTQYCYSIQ (SEQ ID NO: 2.

### Claim 6 (depends on 1)

The method according to claim 1, characterized in that polypeptide X comprises at least two amino acids which are connected by at least one disulfide group.

### Claim 7 (depends on 1)

The method according to claim 1, characterized in that the detection moiety D is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, chemically detectable moieties and magnetic particles.

### Claim 8 (depends on 1)

The method for detecting cells expressing an antigen binding domain of a chimeric antigen receptor (CAR), characterized in that a detection reagent obtained with the method according to claim 1 is provided to a sample of cells and the cells having an antigen binding domain of a chimeric antigen receptor (CAR) binding the detection reagent are detected via the detection moiety of the detection reagent.