Patent ID: 8921304

Claim:
A non-naturally occurring protein that binds to the extradomain B (ED-B) of fibronectin, said protein comprising two monomeric ubiquitin units linked together in a head-to-tail arrangement, wherein: (i) each monomeric ubiquitin unit of said protein has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1; and amino acid substitutions selected from the group consisting of positions corresponding to residues 2, 4, 6, 8, 62, 63, 64, 65, and 66 of SEQ ID NO: 1, and further wherein: (1) the amino acid substitutions in the first monomeric ubiquitin unit are the amino acid substitutions K6W, L8W, K63R, E64K, S65F, and T66P and the amino acid substitutions in the second monomeric ubiquitin unit are the amino acid substitutions K6T, L8Q, Q62W, K63S, E64N, and T66E, optionally Q2R; or (2) the amino acid substitutions in the first monomeric ubiquitin unit are the amino acid substitutions Q2T, F4W, K6H, Q62N, K63F, E64K, S65L, and T66S and the amino acid substitutions in the second monomeric ubiquitin unit are the amino acid substitutions K6X, L8X, Q62X, K63X, E64X, S65X, and T66X, optionally Q2X, wherein X can be any amino acid; (ii) said protein has a specific binding affinity to said ED-B domain of fibronectin of K d =10 −7 -10 −12 M; and (iii) said protein exhibits a monovalent binding activity with respect to said ED-B domain of fibronectin.