Fast-off rate serum albumin binding fibronectin type III domains

A polypeptide comprising a fibronectin type III tenth (10Fn3) domain, wherein the 10Fn3 domain binds to human serum albumin (HSA) with a KD of 1 μM or less and comprises AB, BC, CD, DE, EF, and FG loops, wherein the CD loop has 1 amino acid substitution relative to the CD loop of a 10Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO:

• 23, and wherein the substitution is selected from the group consisting of:
• (i) the arginine (R) at position 2 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from D, E, A, Q, or N;
• (ii) the glutamine (Q) at position 5 of the CD loop (SEQ ID NO:
• 28) is substituted with D;
• (iii) the tyrosine (Y) at position 7 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from S or A;
• (iv) the leucine (L) at position 10 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from S, T, or A;
• (v) the leucine (L) at position 13 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from D, S, or A;
• (vi) the tyrosine (Y) at position 14 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from A or T;
• (vii) the isoleucine (I) at position 15 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from D, S, or A;
• and
• (viii) the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO:
• 28) is substituted with an amino acid selected from Q, E, S, or A.