# US 10,370,414 B2 · Binding proteins comprising at least two repeat domains against HER2

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- Number: US 10,370,414 B2
- Document ID: US10370414B2
- Status: Granted
- Kind: B2
- Application number: 14/648,611
- Filed: 2013-12-02
- Published: 2015-10-22
- Granted: 2019-08-06
- Earliest claimed: 2012-11-30
- Assignees: Molecular Partners AG
- Applicants: Molecular Partners AG
- Inventors: Ulrike Fiedler; Ignacio Dolado; Heike Strobel
- Web page: https://patentgrep.com/patent/US10370414B2

## CPC

- C07K 14/001: {by chemical synthesis}
- A61P 35/00: Antineoplastic agents
- C07K 14/47: from mammals
- C07K 16/32: against translation products of oncogenes
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/31: multispecific
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/35: Valency
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2318/20: Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
- C07K 2319/21: containing a His-tag
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to a recombinant binding protein comprising at least a first and a second repeat domain, wherein each of said two repeat domains binds the extracellular region of HER2 and wherein said repeat domains are covalently linked.

## Claims

### Claim 1

A recombinant protein comprising at least a first and a second ankyrin repeat domain, wherein each of said first and second ankyrin repeat domains binds the extracellular region of human epidermal growth factor receptor 2 (HER2), wherein said first ankyrin repeat domain binds domain II of HER2 and said second ankyrin repeat domain binds domain IV of HER2, wherein said first and second ankyrin repeat domains are located on the same polypeptide and wherein said first ankyrin repeat domain is located N-terminally to said second ankyrin repeat domain, and wherein
a) said first ankyrin repeat domain comprises an amino acid sequence that has at least 91% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs:62 to 68, 72, and 114 to 121, and
b) said second ankyrin repeat domain comprises an amino acid sequence that has at least 91% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs:74 to 82.

### Claim 2 (depends on 1)

The recombinant protein of claim 1, wherein said first ankyrin repeat domain does not compete for binding to HER2 with pertuzumab.

### Claim 3 (depends on 1)

The recombinant protein of claim 1, wherein said second ankyrin repeat domain does not compete for binding to HER2 with trastuzumab.

### Claim 4 (depends on 1)

The recombinant protein of claim 1, wherein
a) said first ankyrin repeat domain comprises an amino acid sequence that has at least 95% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs:62 to 68, 72, and 114 to 121, and
b) said second ankyrin repeat domain comprises an amino acid sequence that has at least 95% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs:74 to 82.

### Claim 5 (depends on 1)

The recombinant protein of claim 1, wherein said first ankyrin repeat domain binds the extracellular region of HER2 in PBS with a Kd below 10−7M and said second ankyrin repeat domain binds the extracellular region of HER2 in PBS with a Kd below 10−7M.

### Claim 6 (depends on 1)

The recombinant protein of claim 1, wherein said protein inhibits stimulated proliferation of BT474 cells with an IC50 value below 100 nM.

### Claim 7 (depends on 1)

The recombinant protein of claim 1, wherein said protein induces apoptosis in BT474 cells with an EC50 value below 100 nM.

### Claim 8 (depends on 1)

The recombinant protein of claim 1, wherein said first and second ankyrin repeat domains are connected by a polypeptide linker.

### Claim 9 (depends on 1)

The recombinant protein of claim 1, wherein
a) said first ankyrin repeat domain comprises an amino acid sequence that has at least 98% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs:62 to 68, 72, and 114 to 121, and
b) said second ankyrin repeat domain comprises an amino acid sequence that has at least 98% amino acid sequence identity with an ankyrin repeat domain selected from the group consisting of SEQ ID NOs:74 to 82.

### Claim 10 (depends on 1)

The recombinant protein of claim 1, wherein
a) said first ankyrin repeat domain is selected from the group consisting of SEQ ID NOs:62 to 68, 72, and 114 to 121, and
b) said second ankyrin repeat domain is selected from the group consisting of SEQ ID NOs:74 to 82,
and wherein further
a) G at position 1 and/or S at position 2 of said ankyrin repeat domains are optionally missing; and
b) L at the second last position and/or N at the last position of said ankyrin repeat domains are optionally substituted with A.

### Claim 11

A recombinant protein comprising at least a first and a second ankyrin repeat domain, wherein each of said two repeat domains binds the extracellular region of human epidermal growth factor receptor 2 (HER2), wherein said repeat domains are covalently linked, and wherein said recombinant protein comprises a polypeptide, wherein said polypeptide has at least 90% amino acid sequence identity with a polypeptide selected from the group consisting of SEQ ID NO:83 to 98, 102, 103, 122, 123, and 136 to 141.

### Claim 12 (depends on 1)

A pharmaceutical formulation comprising a recombinant protein of claim 1 and a pharmaceutically acceptable carrier.

### Claim 13 (depends on 11)

The recombinant protein of claim 11, wherein said first repeat domain binds domain II of HER2 and said second repeat domain binds domain IV of HER2, wherein said first and second repeat domains are located on the same polypeptide, and wherein said first repeat domain is located N-terminally to said second repeat domain.

### Claim 14 (depends on 11)

The recombinant protein of claim 11, wherein said protein induces apoptosis in BT474 cells with an EC50 value below 100 nM.

### Claim 15 (depends on 11)

The recombinant protein of claim 11, wherein said polypeptide has at least 95% amino acid sequence identity with a polypeptide selected from the group consisting of SEQ ID NO:83 to 98, 102, 103, 122, 123, and 136 to 141.

### Claim 16 (depends on 11)

The recombinant protein of claim 11, wherein said polypeptide has at least 98% amino acid sequence identity with a polypeptide selected from the group consisting of SEQ ID NO:83 to 98, 102, 103, 122, 123, and 136 to 141.

### Claim 17 (depends on 11)

A pharmaceutical formulation comprising the recombinant protein of claim 11 and a pharmaceutically acceptable carrier.

### Claim 18

A recombinant protein comprising at least a first and a second ankyrin repeat domain, wherein each of said two repeat domains binds the extracellular region of human epidermal growth factor receptor 2 (HER2), wherein said repeat domains are covalently linked, and wherein said recombinant protein comprises a polypeptide, wherein said polypeptide comprises said first and second ankyrin repeat domains and has at least 90% amino acid sequence identity with SEQ ID NO: 87.

### Claim 19 (depends on 18)

The recombinant protein of claim 18, wherein said polypeptide has at least 95% amino acid sequence identity with SEQ ID NO: 87.

### Claim 20 (depends on 18)

The recombinant protein of claim 18, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 87.

### Claim 21 (depends on 18)

The recombinant protein of claim 18, wherein said protein induces apoptosis in BT474 cells with an EC50 value below 100 nM.

### Claim 22 (depends on 18)

A pharmaceutical formulation comprising the recombinant protein of claim 18 and a pharmaceutically acceptable carrier.

### Claim 23 (depends on 1)

A nucleic acid encoding the recombinant protein of claim 1.