# US 2014/0155326 A1 · Egfr and c-met fibronectin type iii domain binding molecules

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- Number: US 2014/0155326 A1
- Document ID: US20140155326A1
- Status: Published application
- Kind: A1
- Application number: 14/086,250
- Filed: 2013-11-21
- Published: 2014-06-05
- Earliest claimed: 2012-11-21
- Assignees: Janssen Biotech, Inc.
- Applicants: Janssen Biotech, Inc.
- Inventors: Anderson Mark; Ricardo Attar; Michael Diem; Linus Hyun; Steven Jacobs; Alastair King; Donna Klein; Sheri Moores; Karyn O'Neil; Kristen Picha
- Web page: https://patentgrep.com/patent/US20140155326A1

## CPC

- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- A61P 35/00: Antineoplastic agents
- C07K 14/47: from mammals
- C07K 14/71: for growth factors; for growth regulators
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- 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)
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 2039/505: {comprising antibodies}
- C07K 2317/31: multispecific
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- 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 2319/70: containing domain for protein-protein interaction
- 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

Monospecific and bispecific EGFR and/or c-Met FN3 domain containing molecules, isolated nucleotides encoding the molecules, vectors, host cells, and methods of making thereof are useful in the generation of therapeutic molecules and treatment and diagnosis of diseases and disorders.

## Claims

### Claim 1

1) An isolated bispecific FN3 domain containing molecule comprising a first fibronectin type III (FN3) domain and a second FN3 domain, wherein the first FN3 domain specifically binds epidermal growth factor receptor (EGFR) and blocks binding of epidermal growth factor (EGF) to EGFR, and the second FN3 domain specifically binds hepatocyte growth factor receptor (c-Met), and blocks binding of hepatocyte growth factor (HGF) to c-Met.

### Claim 2 (depends on 1)

2) The bispecific molecule of claim 1, wherein
a) the first FN3 domain inhibits EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with an IC50 value of less than about 2.5×10−6 M when measured in A431 cells using 50 ng/mL human EGF, and the second FN3 domain inhibits HGF-induced c-Met phosphorylation at c-Met residue Tyrosine 1349 with an IC50 value of less than about 1.5×10−6 M when measured in NCI-H441 cells using 100 ng/mL human HGF;
b) the first FN3 domain inhibits EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with an IC50 value between about 1.8×10−8 M and about 2.5×10−6 M when measured in A431 cells using 50 ng/mL human EGF, and the second FN3 domain inhibits HGF-induced c-Met phosphorylation at c-Met residue Tyrosine 1349 with an IC50 value between about 4×10−9 M and about 1.5×10−6 M when measured in NCI-H441 cells using 100 ng/mL human HGF;
c) the first FN3 domain binds human EGFR with a dissociation constant (KD) of less than about 1×10−8 M, and the second FN3 domain binds human c-Met with a KD of less than about 5×10−8 M, wherein the KD is measured using surface plasmon resonance as described in Example 3; or
d) the first FN3 domain binds human EGFR with a KD of between about 2×10−10 to about 1×10−8 M, and the second FN3 domain binds human c-Met with a KD of between about 3×10−10 to about 5×10−8 M, wherein the KD is measured using surface plasmon resonance as described in Example 3.

### Claim 3 (depends on 1)

3) The bispecific molecule of claim 1, wherein the bispecific molecule inhibits NCI-H292 cell proliferation with an IC50 value that is at least 30-fold less when compared to the IC50 value of inhibition of NCI-H292 cell growth with a mixture of the first FN3 domain and the second FN3 domain, wherein the cell proliferation is induced with 10% FBS containing 7.5 ng/mL HGF.

### Claim 4 (depends on 1)

4) The bispecific molecule of claim 1, wherein the bispecific molecule
a) inhibits EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with an IC50 value of less than about 8×10−7 M when measured in NCI-H292 cells using 50 ng/mL human EGF;
b) inhibits HGF-induced c-Met phosphorylation at c-Met residue Tyrosine 1349 with an IC50 value of less than about 8.4×10−7 M when measured in NCI-H441 cells using 100 ng/mL human HGF;
c) inhibits HGF-induced NCI-H292 cell proliferation with an IC50 value of less than about 9.5×10−6M, wherein the cell proliferation is induced with 10% FBS containing 7.5 ng HGF;
d) binds EGFR with a KD of less than about 2.0×10−8 M; or
e) binds c-Met with a KD of less than about 2.0×10−8 M, wherein the KD is measured using surface plasmon resonance as described in Example 3.

### Claim 5 (depends on 1)

5) The bispecific molecule of claim 1, wherein the first FN3 domain comprises an amino acid sequence at least 87% identical to the amino acid sequence of SEQ ID NO: 27, and the second FN3 domain comprises an amino acid sequence at least 83% identical to the amino acid sequence of SEQ ID NO: 41.

### Claim 6 (depends on 5)

6) The bispecific molecule of claim 5, wherein
a) the first FN3 domain comprises
i) an FG loop comprising the amino acid sequence of SEQ ID NO: 179 or the amino acid sequence of SEQ ID NO: 180; and
ii) a BC loop comprising the amino acid sequence of SEQ ID NO: 181; and
b) the second FN3 domain comprises
i) a C strand and a CD loop comprising the amino acid sequence of SEQ ID NO: 184; and
ii) an F strand and an FG loop comprising the amino acid sequence of SEQ ID NO: 185.

### Claim 7 (depends on 6)

7) The bispecific molecule of claim 6, wherein
a) the first FN3 domain comprises the amino acid sequence of SEQ ID NO: 182 or the amino acid sequence of SEQ ID NO: 183; and
b) the second FN3 domain comprises the amino acid sequence of SEQ ID NO: 186.

### Claim 8 (depends on 7)

8) The bispecific molecule of claim 7, wherein the first FN3 domain and/or the second FN3 domain comprises substitutions at one or more residue positions corresponding to positions 11, 14, 17, 37, 46, 73 and 86 in Tencon (SEQ ID NO: 1).

### Claim 9 (depends on 8)

9) The bispecific molecule of claim 8, wherein the first FN3 domain comprises the amino acid sequence of SEQ ID NO: 187; and
the second FN3 domain comprises the amino acid sequence of SEQ ID NO: 188.

### Claim 10 (depends on 5)

10) The bispecific molecule of claim 5, wherein the first FN3 domain comprises the sequence shown in one of SEQ ID NOs: 18-29, 107-110 or 122-137, and the second FN3 domain comprises the sequence shown in one of SEQ ID NOs: 32-49, or 111-114.

### Claim 11 (depends on 10)

11) The bispecific molecule of claim 10, wherein the first FN3 domain and the second FN3 domain comprises the amino acid sequence of SEQ ID NOs:
a) 27 and 32, respectively;
b) 27 and 41, respectively;
c) 27 and 40, respectively;
d) 27 and 33, respectively;
e) 107 and 41, respectively;
f) 107 and 40, respectively;
g) 107 and 33, respectively;
h) 107 and 32, respectively;
i) 107 and 114, respectively;
j) 108 and 111, respectively;
k) 108 and 112, respectively;
l) 108 and 113, respectively;
m) 108 and 114, respectively;
n) 109 and 111, respectively;
o) 109 and 112, respectively;
p) 109 and 113, respectively;
q) 109 and 114, respectively;
r) 110 and 111, respectively;
s) 110 and 112, respectively;
t) 110 and 113, respectively; or
u) 110 and 114, respectively.

### Claim 12 (depends on 11)

12) The bispecific molecule of claim 11, wherein the first FN3 domain and/or the second FN3 domain comprises one, two or three substitutions corresponding to substitutions L17A, N46V and E86I in Tencon (SEQ ID NO: 1).

### Claim 13 (depends on 12)

13) The bispecific molecule of claim 12, wherein the first FN3 domain and/or the second FN3 domain is isolated from a library designed based on a Tencon sequence of SEQ ID NO: 1.

### Claim 14 (depends on 1)

14) The bispecific molecule of claim 1, wherein the first FN3 domain and the second FN3 domain are coupled by a linker.

### Claim 15 (depends on 14)

15) The bispecific molecule of claim 14, wherein the linker comprises the amino acid sequence shown in one of SEQ ID NOs: 78-84.

### Claim 16 (depends on 15)

16) The bispecific molecule of claim 15, comprising the amino acid sequence shown in one of SEQ ID NOs: 50-72, 106, 118-121 or 138-165, and optionally comprising a methionine (Met) linked to the N-terminus of the molecule.

### Claim 17 (depends on 16)

17) The bispecific molecule of claim 16, further comprising a cysteine linked to the C-terminus of the molecule.

### Claim 18 (depends on 1)

18) The bispecific molecule of claim 1 coupled to a half-life extending moiety.

### Claim 19 (depends on 18)

19) The bispecific molecule of claim 18, wherein the half-life extending moiety is an albumin binding molecule, a polyethylene glygol (PEG), albumin, or at least a portion of an Fc region of an immunoglobulin.

### Claim 20 (depends on 5)

20) An isolated polynucleotide encoding the bispecific molecule of claim 5.

### Claim 21 (depends on 20)

21) A vector comprising the polynucleotide of claim 20.

### Claim 22 (depends on 21)

22) An isolated host cell comprising the vector of claim 21.

### Claim 23 (depends on 22)

23) A method of producing a bispecific molecule, comprising culturing the isolated host cell of claim 22 under conditions such that the bispecific molecule is expressed, and purifying the bispecific molecule.

### Claim 24 (depends on 1)

24) A pharmaceutical composition comprising the bispecific molecule of claim 1 and a pharmaceutically acceptable carrier.

### Claim 25 (depends on 1)

25) A method of treating a subject having cancer, comprising administering a therapeutically effective amount of the bispecific molecule of claim 1 to a patient in need thereof for a time sufficient to treat cancer.

### Claim 26 (depends on 25)

26) The method of claim 25, wherein cancer is associated with expression or overexpression of EGFR, EGFR variants or c-Met.

### Claim 27 (depends on 25)

27) The method of claim 25, wherein cancer is an epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, gastric cancer, cancer of the thymus, colon cancer, thyroid cancer, liver cancer or sporadic or hereditary papillary renal cell carcinoma (PRCC).

### Claim 28 (depends on 25)

28) The method of claim 25, further comprising administering a second therapeutic agent.

### Claim 29 (depends on 28)

29) The method of claim 28, wherein the second therapeutic agent is a chemotherapeutic agent, an anti-angiogenic agent or a cytotoxic drug, antagonist of Her2, Her3, Her4, VEGF, protein tyrosine kinase inhibitor, gefitinib or erlotinib.

### Claim 30

30) An isolated fibronectin type III (FN3) domain that specifically binds epidermal growth factor receptor (EGFR) and blocks binding of epidermal growth factor (EGF) to EGFR, wherein the FN3 domain is isolated from a library designed based on the Tencon amino acid sequence of SEQ ID NO: 1.

### Claim 31 (depends on 30)

31) The FN3 domain of claim 30, wherein the FN3 domain
a) inhibits EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with an IC50 value of less than about 2.5×10−6 M when measured in A431 cells using 50 ng/mL human EGF;
b) inhibits EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with an IC50 value between about 1.8×10−8 M to about 2.5×10−6 M when measured in A431 cells using 50 ng/mL human EGF;
c) binds human EGFR with a dissociation constant (KD) of less than about 1×10−8 M, wherein the KD is measured using punder the conditions described in Example 3; or
d) binds human EGFR with a KD of between about 2×10−10 to about 1×10−8 M, wherein the KD is measured using surface plasmon resonance as described in Example 3.

### Claim 32 (depends on 30)

32) The FN3 domain of claim 30 comprising an amino acid sequence at least 87% identical to the amino acid sequence of SEQ ID NO: 27.

### Claim 33 (depends on 30)

33) The FN3 domain of claim 30, wherein the FN3 domain comprises
a) an FG loop comprising the amino acid sequence of SEQ ID NO: 179 or the amino acid sequence of SEQ ID NO: 180; and
b) a BC loop comprising the amino acid sequence of SEQ ID NO: 181.

### Claim 34 (depends on 33)

34) The FN3 domain of claim 33, comprising the amino acid sequence of SEQ ID NO: 182 or the amino acid sequence of SEQ ID NO: 183.

### Claim 35 (depends on 34)

35) The FN3 domain of claim 34, wherein the FN3 domain comprises the sequence shown in one of SEQ ID NOs: 18-29, 107-110 or 122-137.

### Claim 36 (depends on 35)

36) The FN3 domain of claim 35, wherein the FN3 domain comprises substitutions at one or more residue positions corresponding to positions 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQ ID NO: 1).

### Claim 37 (depends on 36)

37) The FN3 domain of claim 36, wherein the FN3 domain comprises one or more substitutions corresponding to substitutions L17A, N46V, and E86I in the Tencon amino acid sequence of SEQ ID NO: 1.

### Claim 38 (depends on 32)

38) An isolated polynucleotide encoding the FN3 domain of claim 32.

### Claim 39 (depends on 38)

39) A vector comprising the polynucleotide of claim 38.

### Claim 40 (depends on 39)

40) An isolated host cell comprising the vector of claim 39.

### Claim 41 (depends on 40)

41) A method of producing an FN3 domain that specifically binds EGFR, comprising culturing the isolated host cell of claim 40 under conditions such that the FN3 domain that specifically binds EGFR is expressed, and purifying the FN3 domain.

### Claim 42

42) An isolated fibronectin type III (FN3) domain that specifically binds hepatocyte growth factor receptor (c-Met) and blocks binding of hepatocyte growth factor (HGF) to c-Met.

### Claim 43 (depends on 42)

43) The FN3 domain of claim 42, wherein the FN3 domain
a) inhibits HGF-induced c-Met phosphorylation at c-Met residue Tyrosine 1349 with an IC50 value of less than about 1.5×10−6 M when measured in NCI-H441 cells using 100 ng/mL recombinant human HGF;
b) inhibits HGF-induced c-Met phosphorylation at c-Met residue Tyrosine 1349 with an IC50 value between about 4×10−9 M and about 1.5×10−6 M when measured in NCI-H441 cells using 100 ng/mL recombinant human HGF;
c) binds human c-Met with a KD of less than about 5×10−8 M, wherein the KD is measured under the conditions described in Example 3; or
d) binds human c-Met with a (KD of between about 3×10−10 to about 5×10−8 M, wherein the KD is measured using surface plasmon resonance as described in Example 3.

### Claim 44 (depends on 42)

44) The FN3 domain of claim 42, comprising an amino acid sequence at least 83% identical to the amino acid sequence of SEQ ID NO: 41.

### Claim 45 (depends on 42)

45) The FN3 domain of claim 42, wherein the FN3 domain comprises
a) a C strand and a CD loop comprising the amino acid sequence of SEQ ID NO: 184; and
b) an F strand and an FG loop comprising the amino acid sequence of SEQ ID NO: 185.

### Claim 46 (depends on 45)

46) The FN3 domain of claim 45, comprising the amino acid sequence of SEQ ID NO: 186.

### Claim 47 (depends on 46)

47) The FN3 domain of claim 46, wherein the FN3 domain comprises the sequence shown in one of SEQ ID NOs: 32-49 or 111-114.

### Claim 48 (depends on 47)

48) The FN3 domain of claim 47, wherein the FN3 domain comprises substitutions at one or more residue positions corresponding to positions 11, 14, 17, 37, 46, 73, and 86 of the Tencon amino acid sequence of SEQ ID NO: 1.

### Claim 49 (depends on 48)

49) The FN3 domain of claim 48, wherein the FN3 domain comprises one or more of substitutions corresponding to substitutions L17A, N46V, and E86I of the Tencon amino acid sequence of SEQ ID NO: 1.

### Claim 50 (depends on 42)

50) The FN3 domain of claim 42, wherein the FN3 domain is isolated from a library designed based on the Tencon amino acid sequence of SEQ ID NO: 1.

### Claim 51 (depends on 44)

51) An isolated polynucleotide encoding the FN3 domain of claim 44.

### Claim 52 (depends on 51)

52) A vector comprising the polynucleotide of claim 51.

### Claim 53 (depends on 52)

53) An isolated host cell comprising the vector of claim 52.

### Claim 54 (depends on 53)

54) A method of producing an FN3 domain specifically binding c-Met, comprising culturing the isolated host cell of claim 53 under conditions such that the FN3 domain specifically binding c-Met is expressed, and purifying the FN3 domain.

### Claim 55 (depends on 30, 42)

55) A pharmaceutical composition comprising the FN3 domain of claim 30 or 42 and a pharmaceutically acceptable carrier.

### Claim 56 (depends on 55)

56) A method of treating a subject having cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 55 to a patient in need thereof for a time sufficient to treat cancer.

### Claim 57 (depends on 56)

57) The method of claim 56, wherein cancer is an epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, gastric cancer, cancer of the thymus, colon cancer, thyroid cancer, liver cancer or sporadic or hereditary papillary renal cell carcinoma (PRCC).