# US 9,752,190 B2 · Composition for detecting undifferentiated human pluripotent stem cell, monoclonal antibody 6-1 and use thereof

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- Number: US 9,752,190 B2
- Document ID: US9752190B2
- Status: Granted
- Kind: B2
- Application number: 14/806,514
- Filed: 2015-07-22
- Published: 2016-04-07
- Granted: 2017-09-05
- Earliest claimed: 2014-08-12
- Assignees: Korea Research Institute of Bioscience and Biotechnology
- Applicants: Korea Research Institute of Bioscience and Biotechnology
- Inventors: Jeong Ki Min; Jongjin Park; Kwang-Hee Bae; Sang Chul Lee; Yee Sook Cho
- Web page: https://patentgrep.com/patent/US9752190B2

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- G01N 33/56966: {Animal cells}
- C12Q 2600/158: Expression markers
- G01N 2333/705: Assays involving receptors, cell surface antigens or cell surface determinants
- G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)
- 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)

## Abstract

The present invention relates to a composition for detecting the undifferentiated human pluripotent stem cells comprising an agent useful for measuring the level of Desmoglein 2 (Dsg 2) mRNA or the protein thereof, a kit for detecting the undifferentiated human pluripotent stem cells comprising the said composition, a method for detecting the undifferentiated human pluripotent stem cells containing the step of measuring the level of Desmoglein 2 mRNA or the protein thereof, a method for evaluating the differentiation of human pluripotent stem cells and thereafter for separating the undifferentiated human pluripotent stem cells, a method for reducing the undifferentiated status of human pluripotent stem cells by inhibiting the expression or activation of Desmoglein 2, and a monoclonal antibody binding specifically to human Desmoglein 2.

## Claims

### Claim 1

A method for detecting undifferentiated human pluripotent stem cells comprising the step of measuring levels of Desmoglein 2 protein in separated human pluripotent stem cells,
wherein the measurement of Desmoglein 2 protein levels comprises reacting human pluripotent stem cells with a Dsg2 protein specific antibody to form an antigen-antibody complex and measuring the antigen-antibody complex,
wherein the antibody comprises a heavy chain variable region (VH) comprising the heavy chain CDR1 represented by SEQ. ID. NO: 60, the heavy chain CDR2 represented by SEQ. ID. NO: 61, and the heavy chain CDR3 represented by SEQ. ID. NO: 62; and a light chain variable region (VL) comprising the light chain CDR1 represented by SEQ. ID. NO: 64, the light chain CDR2 represented by SEQ. ID. NO: 65, and the light chain CDR3 represented by SEQ. ID. NO: 66.

### Claim 2

A method for separating undifferentiated human pluripotent stem cells comprising the following steps:
(a) reacting human pluripotent stem cells with an agent specifically binding to Desmoglein 2 protein; and
(b) separating the human pluripotent stem cells that have been conjugated with said agent,
wherein the agent specifically binding to Desmoglein 2 protein in step (a) is a Dsg2 specific antibody comprising a heavy chain variable region (VH) comprising the heavy chain CDR1 represented by SEQ. ID. NO: 60, the heavy chain CDR2 represented by SEQ. ID. NO: 61, and the heavy chain CDR3 represented by SEQ. ID. NO: 62; and a light chain variable region (VL) comprising the light chain CDR1 represented by SEQ. ID. NO: 64, the light chain CDR2 represented by SEQ. ID. NO: 65, and the light chain CDR3 represented by SEQ. ID. NO: 66.

### Claim 3

A monoclonal antibody specifically binding to Desmoglein 2 having a heavy chain variable region (VH) comprising the heavy chain CDR1 represented by SEQ. ID. NO: 60, the heavy chain CDR2 represented by SEQ. ID. NO: 61, and the heavy chain CDR3 represented by SEQ. ID. NO: 62; and a light chain variable region (VL) comprising the light chain CDR1 represented by SEQ. ID. NO: 64, the light chain CDR2 represented by SEQ. ID. NO: 65, and the light chain CDR3 represented by SEQ. ID. NO: 66.

### Claim 4 (depends on 1)

The method of claim 1 wherein measuring the antigen-antibody complex is performed using a western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay, complement fixation assay, radio-immuno assay (RIA), immunodiffusion assay, protein chip assay, immunohisto staining, flow cytometry, or combinations thereof.

### Claim 5 (depends on 1)

The method of claim 1 wherein the separating step is performed using an ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay, complement fixation assay, radio-immuno assay (RIA), immunodiffusion assay, protein chip assay, flow cytometry, or combinations thereof.