# US 9,518,131 B2 · Generating metal ion binding proteins

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- Number: US 9,518,131 B2
- Document ID: US9518131B2
- Status: Granted
- Kind: B2
- Application number: 14/242,725
- Filed: 2014-04-01
- Published: 2014-10-02
- Granted: 2016-12-13
- Earliest claimed: 2013-04-02
- Assignees: Miltenyi Biotech GmbH
- Applicants: Miltenyi Biotec GmbH
- Inventors: Holger Thie; Kristina Reck; Volker Nolle
- Web page: https://patentgrep.com/patent/US9518131B2

## CPC

- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- C07K 16/2815: {against CD8}
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- 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 present invention provides for a method for generating a metal ion binding protein, the method comprising a) integrating the amino acid sequence DDD into CDR1 of a light chain variable region of an antibody or fragment thereof; and b) combining the sequence of step a) with a heavy chain variable region of an antibody or fragment thereof; and c) isolating the protein. Also provided is the use of metal ion binding proteins generated by the method of the present invention for isolation and purification of proteins and for the reversible labeling of a target molecule. Also provided is a metal ion binding anti-CD8 protein.

## Claims

### Claim 1

A method for the targeted generation of antibodies or fragments thereof that bind antigen in a Ca2+ or Mg2+ dependent manner, the method comprising: a) combining SEQ ID No: 1 or a fragment comprising the CDRs of SEQ ID NO: 1 with antibody heavy chain variable regions; b) screening for antibodies that bind antigen in a Ca2+ or Mg2+ dependent manner; and c) identifying individual antibodies or antigen binding fragments thereof that bind antigen in a Ca2+ or Mg2+ dependent manner.

### Claim 2 (depends on 1)

The method of claim 1, wherein said step b) is performed by a method selected from the group consisting of high-throughput ELISA screening, high-throughput cell-based screening, phage display selection, and yeast display selection.

### Claim 3 (depends on 1)

The method of claim 1, wherein said step c) is performed by ELISA or flow cytometry.