Fast-off rate serum albumin binding fibronectin type iii domains

Abstract

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  or 1µM or less and comprises AB, BC, CD, DE, EF, and FG loops, wherein the  10 Fn3 domain comprises the amino acid sequence of SEQ ID NO: 23, but wherein the CD loop has 1, 2, or 3 amino acid substitutions, and the FG loop optionally has 1, 2, or 3 amino acid substitutions , and wherein the serum half-life of a moiety that is linked to the  10 Fn3 domain is extended relative to that of the moiety that is not linked to the  10 Fn3 domain. 
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide binds to HSA with an off-rate (kd) that is greater than the kd of the  10 Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23. 
     
     
         3 . The polypeptide of  claim 1 , wherein the polypeptide binds to HSA with an on-rate (ka) that is within 10-fold of the ka of the  10 Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23. 
     
     
         4 - 6 . (canceled) 
     
     
         7 . The polypeptide of  claim 1 , wherein the CD loop has 1 amino acid substitution and the FG loop has 1 substitution relative to the CD loop and FG loop of the  10 Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23. 
     
     
         8 . (canceled) 
     
     
         9 . The polypeptide of  claim 1 , wherein the CD loop has 1 amino acid substitution, 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.   
     
     
         10 . The polypeptide of  claim 9 , wherein:
 (a) the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with serine (S);   (b) the leucine (L) at position 10 of the CD loop (SEQ ID NO: 28) is substituted with threonine (T); and/or   (c) the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with glutamine (Q).   
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The polypeptide of claim  61 , 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. 
     
     
         14 . The polypeptide of  claim 7 , wherein
 a) the 1 amino acid substitution in the CD loop 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; and 
   b) the lysine at position 12 of the FG loop (SEQ ID NO: 29) is substituted with an amino acid selected from A, T, or S.   
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The polypeptide of  claim 1 , wherein the  10 Fn3 domain binds to domain I-II of HSA. 
     
     
         18 - 20 . (canceled) 
     
     
         21 . The polypeptide of  claim 1 , wherein the polypeptide comprises a heterologous protein. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . A composition comprising the polypeptide of  claim 1 , and a carrier. 
     
     
         25 . An isolated nucleic acid molecule encoding the polypeptide of  claim 1 . 
     
     
         26 . An expression vector comprising the nucleotide sequence encoding the polypeptide of  claim 1 . 
     
     
         27 . A cell comprising the nucleic acid molecule of  claim 25  . 
     
     
         28 . 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 the cell of  claim 27  under conditions suitable for expressing the polypeptide, and purifying the polypeptide. 
     
     
         29 . A method of enhancing the half-life of a moiety, comprising linking to the moiety a human serum albumin binding polypeptide of  claim 1 . 
     
     
         30 . A method of detecting the presence of a protein in a sample comprising:
 (a) contacting the sample with a fusion molecule comprising a heterologous molecule which binds to the protein and a fibronectin type III domain tenth ( 10 Fn3) domain, wherein the  10 Fn3 domain binds to human serum albumin (HSA) with a K D  of 1µM or less and comprises AB, BC, CD, DE, EF, and FG loops, wherein the  10 Fn3 domain comprises the amino acid sequence of SEQ ID NO: 23, but wherein the CD loop has 1, 2, or 3 amino acid substitutions and the FG loop optionally has 1, 2, or 3 amino acid substitutions, and wherein the serum half-life of a moiety that is linked to the  10 Fn3 domain is extended relative to that of the moiety that is not linked to the  10 Fn3 domain, under conditions which promote the formation of a complex between the fusion molecule and protein, and   (b) detecting the complex.   
     
     
         31 . A method of treating a disease comprising administering to a subject in need thereof the polypeptide of  claim 1 . 
     
     
         32 . The polypeptide of  claim 31 , wherein the polypeptide comprises a second  10 Fn3 domain which binds to a target of interest.