Fast-off rate serum albumin binding fibronectin type III domains
Provided herein are polypeptides which include tenth fibronectin type III domains ( 10 Fn3) that binds to serum albumin. Also provided are fusion molecules comprising a serum albumin binding 10 Fn3 joined to a heterologous protein for use in diagnostic and therapeutic applications.
1. A polypeptide comprising a fibronectin type III tenth ( 10 Fn3) domain, wherein the 10 Fn3 domain binds to human serum albumin (HSA) with a K D 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 10 Fn3 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.
2. The polypeptide of claim 1 , wherein the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with serine (S).
3. The polypeptide of claim 1 , wherein the leucine (L) at position 10 of the CD loop (SEQ ID NO: 28) is substituted with threonine (T).
4. The polypeptide of claim 1 , wherein the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with glutamine (Q).
5. The polypeptide of claim 1 , wherein the FG loop has 1 amino acid substitution relative to the FG loop of the 10 Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23, wherein the lysine (K) at position 12 of the FG loop (SEQ ID NO: 29) is substituted with an amino acid selected from A, T, or S.
6. The polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence at least 90% identical to the non-CD loop regions of SEQ ID NOs: 30-34, 36-40, 42-46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 62, 63, 65-68, 70, 71, 73-76, 78, 79, 81-84, 86, 87, 89-98, 100-109, and 111-118.
7. The polypeptide of claim 1 , wherein the 10 Fn3 domain binds to domain I-II of HSA.
8. The polypeptide of claim 1 , wherein the polypeptide comprises a heterologous protein.
9. A composition comprising the polypeptide of claim 1 , and a carrier.
10. A method of producing a polypeptide comprising a fibronectin type III tenth ( 10 Fn3) domain that binds to human serum albumin, the method comprising culturing a cell comprising a nucleic acid which encodes the polypeptide of claim 1 under conditions suitable for expressing the polypeptide, and purifying the polypeptide.
11. A method of enhancing the half-life of a moiety, comprising linking to the moiety a human serum albumin binding polypeptide of claim 1 .