Stabilized fibronectin based scaffold molecules
Provided herein are proteins comprising a fibronectin based scaffold (FBS) domain, e.g., 10 Fn3 molecules, that bind specifically to a target, and wherein the FBS domain is linked at its C-terminus to a region consisting of PmXn, wherein P is proline, X is any amino acid and wherein n is 0 or an integer that is at least 1 and m is an integer that is at least 1, and wherein the PmXn moiety provides an enhanced property to the FBS domain, e.g., enhanced stability, relative to the protein that is not linked to the PmXn moiety.
1. An isolated fibronectin based scaffold (FBS) protein comprising a 10 Fn3 domain, wherein the 10 Fn3 domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 1, wherein
(i) the C-terminal amino acid at the position corresponding to position 94 of SEQ ID NO:1 is covalently linked through a peptide bond to a moiety consisting of the amino acid sequence PmXn, wherein P is proline, X is any amino acid, m is an integer that is at least 1 and n is 0 or an integer that is at least 1; and
(ii) the PmXn moiety provides an enhanced property to the FBS protein relative to the FBS protein that is not linked to the PmXn moiety.
2. The isolated FBS protein of claim 1 , wherein the moiety consists of P (m is 1 and n is 0).
3. The isolated FBS protein of claim 1 , wherein the moiety consists of PP (m is 2 and n is 0).
4. The isolated FBS protein of claim 1 , wherein the moiety consists of PmXn, wherein n is 1-150.
5. The isolated FBS protein of claim 1 , wherein the moiety consists of PmXn, wherein n is 1-10.
6. The isolated FBS protein of claim 1 , wherein the moiety consists of PmXn, wherein n is 1-5.
7. The isolated FBS protein of claim 1 , wherein the PmXn moiety consists of PI, PC, PID, PIE, PIDK (SEQ ID NO: 61), PIEK (SEQ ID NO: 63), PIDKP (SEQ ID NO: 65), PIEKP (SEQ ID NO: 67), PIDKPS (SEQ ID NO: 69), PIEKPS (SEQ ID NO: 71), PIDKPC (SEQ ID NO: 73), PIEKPC (SEQ ID NO: 75), PIDKPSQ (SEQ ID NO: 77), PIEKPSQ (SEQ ID NO: 79), PIDKPCQ (SEQ ID NO: 81), PIEKPCQ (SEQ ID NO: 83), PHHHHHH (SEQ ID NO: 87) or PCHHHHHH (SEQ ID NO: 86).
8. The isolated FBS protein of any one of claim 1 - 7 , comprising the amino acid sequence LEVVAA(X)uLLISW(X)vYRITY(X)wFTV(X)xATISGL(X)yYTITVY A(X)ZISINYRT (SEQ ID NO: 16), wherein (X)u, (X)v, (X)w, (X)x, (X)y and (X)z consist of the wild-type amino acid sequence (SEQ ID NO: 1) or comprise at least one amino acid difference with the corresponding wild-type sequence.
9. The isolated FBS protein of claim 1 , wherein PmXn is P or PC.
10. The isolated FBS protein of claim 1 , wherein at least one X of PmXn is a cysteine.
11. The isolated FBS protein of claim 10 , wherein the cysteine is conjugated to a heterologous moiety.
12. The isolated FBS protein of claim 11 , wherein the heterologous molecule is a detectable moiety.
13. The isolated FBS protein of claim 11 or 12 , wherein the heterologous molecule is a drug moiety and the drug moiety and the FBS form an FBS-drug conjugate.
14. The isolated FBS protein of claim 1 , wherein the enhanced property conferred by the PmXn moiety is enhanced stability.
15. The isolated FBS protein of claim 14 , wherein enhanced stability is an increase in Tm of at least 1° C., 2° C., 3° C., 4° C., 5° C. or more.
16. The isolated FBS protein of claim 10 , wherein the PmXn moiety is PmCXn or PmXn 1 CXn 2 , wherein C is cysteine and n 1 and n 2 are independently 0 or an integer that is at least 1.
17. The isolated FBS protein of claim 16 , wherein the cysteine is conjugated to a heterologous moiety.
18. The isolated FBS protein of claim 17 , wherein the heterologous molecule is a detectable moiety.
19. The isolated FBS protein of claim 9 , wherein PmXn is P.
20. The isolated FBS protein of claim 9 , wherein PmXn is PC.