Pharmaceutically acceptable FN3 polypeptides for human treatments
Disclosed herein are proteins that include an immunoglobulin fold and that can be used as scaffolds. Also disclosed herein are nucleic acids encoding such proteins and the use of such proteins in diagnostic methods and in methods for evolving novel compound-binding species and their ligands.
1 . A pharmaceutically acceptable polypeptide comprising a tenth fibronectin type III ( 10 Fn3) domain, wherein:
(a) the 10 Fn3 domain has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of the human 10 Fn3 domain;
(b) the 10 Fn3 domain binds to a target compound that is not bound by the human 10 Fn3 domain; and
(c) the integrin binding motif, RGD, of the 10 Fn3 domain is replaced by an amino acid sequence as follows: basic amino acid-neutral amino acid-acidic amino acid.
2 . The polypeptide of claim 1 , wherein the pharmaceutically acceptable Fn3 domain binds with a K D of 500 nM or less to the target compound.
3 . The polypeptide of claim 2 , wherein the K D is 1 nM or less.
4 . The polypeptide of claim 2 , wherein binding of the 10 Fn3 domain to the target compound is mediated by one loop of the Fn3 domain.
5 . The polypeptide of claim 2 , wherein the loop is selected from the group consisting of the BC loop, the DE loop and the FG loop.
7 . The polypeptide of claim 2 , wherein binding of the 10 Fn3 domain to the binds to the target compound is mediated by two loops of the Fn3 domain.
8 . The polypeptide of claim 2 , wherein binding of the 10 Fn3 domain to the binds to the target compound is mediated by three loops of the 10 Fn3 domain.
9 . The polypeptide of claim 7 , wherein the BC, DE and FG loops bind to the target compound.
10 . The polypeptide of claim 1 , wherein the FG loop binds to the target compound.
11 . The polypeptide of claim 1 , wherein the FG loop is longer than the FG loop of human 10 Fn3 domain.
12 . The polypeptide of claim 1 , wherein the 10 Fn3 domain has an amino acid sequence that is at least 70% identical to the sequence of a human 10 Fn3 domain.
13 . The polypeptide of claim 1 , wherein the 10 Fn3 domain contains no free sulfhydryl moieties and no disulfide bonds.
14 . The polypeptide of claim 1 , wherein the therapeutic protein is produced by expressing the polypeptide in a prokaryote.
15 . The polypeptide of claim 1 , wherein the 10 Fn3 domain was identified by a screening method comprising:
(i) contacting the target compound with a candidate polypeptide including an 10 Fn3 domain having at least one modified loop under conditions that allow target compound-polypeptide complex formation; and (ii) obtaining the polypeptide which binds to the compound.
16 . The polypeptide of claim 15 , wherein the screening method further comprises: (iii) repeating (i) and (ii) using a further randomized 10 Fn3 domain.
17 . The polypeptide of claim 1 , further comprising a Fc region of an antibody.
18 . The polypeptide of claim 1 , further comprising a complement protein.
19 . The polypeptide of claim 1 , further comprising an albumin protein.
20 . The polypeptide of claim 1 , further comprising a toxin protein.
21 . The polypeptide of claim 1 , further comprising a human Fn3 domain.
22 . The polypeptide of claim 21 , wherein the human Fn3 domain:
(a) has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of a naturally occurring Fn3 domain; and
(b) binds to a second target compound that is not bound by the corresponding naturally-occurring Fn3 domain with a K D of 500 nM or less.
23 . A pharmaceutical composition comprising the polypeptide of claim 1 .
24 . The pharmaceutical composition of claim 23 , wherein the pharmaceutical composition is substantially free of endotoxin.
25 . The pharmaceutical composition of claim 23 , further comprising a physiologically acceptable carrier.