EGFR and C-Met fibronectin type III domain binding molecules
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.
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, wherein the first FN3 domain comprises an amino acid sequence of SEQ ID NO: 27, and the second FN3 domain comprises an amino acid sequence of SEQ ID NO: 41.
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 IC 50 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 IC 50 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 IC 50 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 IC 50 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 (K D ) of less than about 1×10 −8 M, and the second FN3 domain binds human c-Met with a K D of less than about 5×10 −8 M, wherein the K D is measured using surface plasmon resonance; or
d) the first FN3 domain binds human EGFR with a K D of between about 2×10 −10 to about 1×10 −8 M, and the second FN3 domain binds human c-Met with a K D of between about 3×10 −10 to about 5 ×10 −8 M, wherein the K D is measured using surface plasmon resonance.
3. The bispecific molecule of claim 1 , wherein the bispecific molecule inhibits NCI-H292 cell proliferation with an IC 50 value that is at least 30-fold less when compared to the IC 50 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.
4. The bispecific molecule of claim 1 , wherein the bispecific molecule
a) inhibits EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with an IC 50 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 IC 50 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 IC 50 value of less than about 9.5×10 −6 M, wherein the cell proliferation is induced with 10% FBS containing 7.5 ng HGF;
d) binds EGFR with a K D of less than about 2.0×10 −8 M; or
e) binds c-Met with a K D of less than about 2.0×10 −8 M, wherein the K D is measured using surface plasmon resonance.
5. The bispecific molecule of claim 1 , 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).
6. The bispecific molecule of claim 1 , 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).
7. The bispecific molecule of claim 6 , 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.
8. The bispecific molecule of claim 1 , wherein the first FN3 domain and the second FN3 domain are coupled by a linker.
9. The bispecific molecule of claim 8 , wherein the linker comprises the amino acid sequence shown in one of SEQ ID NOs: 78-84.
10. The bispecific molecule of claim 1 , further comprising a cysteine linked to the C-terminus of the molecule.
11. The bispecific molecule of claim 1 coupled to a half-life extending moiety.
12. The bispecific molecule of claim 11 , 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.
13. A pharmaceutical composition comprising the bispecific molecule of claim 1 and a pharmaceutically acceptable carrier.