Pharmaceutical preparations of 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 pharmaceutical composition for human treatments comprised of: a therapeutic protein comprising a pharmaceutically acceptable fibronectin type III (Fn3) domain and a physiologically acceptable carrier, wherein the Fn3 domain:
(a) has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of a human Fn3 domain; and
(b) binds to a target compound that is not bound by the corresponding human Fn3 domain.
2 . The pharmaceutical composition 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 pharmaceutical composition of claim 2 , wherein the K D is 1 nM or less.
4 . The pharmaceutical composition of claim 1 , wherein the Fn3 domain comprises at least five mutations, wherein said five mutations are positioned in one or more loops of the Fn3 domain.
5 . The pharmaceutical composition of claim 2 , wherein binding of the Fn3 domain to the target compound is mediated by one loop of the Fn3 domain.
6 . The pharmaceutical composition 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 pharmaceutical composition of claim 2 , wherein binding of the Fn3 domain to the binds to the target compound is mediated by two loops of the Fn3 domain.
8 . The pharmaceutical composition of claim 2 , wherein binding of the Fn3 domain to the binds to the target compound is mediated by three loops of the Fn3 domain.
9 . The pharmaceutical composition of claim 8 , wherein the BC, DE and FG loops bind to the target compound.
10 . The pharmaceutical composition of claim 1 , wherein the Fn3 domain is a 10 Fn3 domain and 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.
11 . The pharmaceutical composition of claim 10 , wherein the Fn3 domain has an amino acid sequence that is at least 70% identical to the sequence of a human 10 Fn3 domain.
12 . The pharmaceutical composition of claim 10 , wherein the integrin binding motif, RGD, of the Fn3 domain is replaced by an amino acid sequence as follows: basic amino acid-neutral amino acid-acidic amino acid.
13 . The pharmaceutical composition of claim 1 , wherein the Fn3 domain contains no free sulfhydryl moieties and no disulfide bonds.
14 . The pharmaceutical composition of claim 1 , wherein the therapeutic protein is produced by expressing the polypeptide in a prokaryote.
15 . The pharmaceutical composition of claim 1 , wherein the Fn3 domain was identified by a screening method comprising:
(i) contacting the target compound with a candidate polypeptide including an 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 pharmaceutical composition of claim 15 , wherein the screening method further comprises: (iii) repeating (i) and (ii) using a further randomized Fn3 domain.
17 . The pharmaceutical composition of claim 1 , wherein the therapeutic protein further comprises a Fc region of an antibody.
18 . The pharmaceutical composition of claim 1 , wherein the therapeutic protein further comprises a complement protein.
19 . The pharmaceutical composition of claim 1 , wherein the therapeutic protein further comprises an albumin protein.
20 . The pharmaceutical composition of claim 1 , wherein the therapeutic protein further comprises a toxin protein.
21 . The pharmaceutical composition of claim 1 , wherein the therapeutic protein further comprises a second Fn3 domain.
22 . The pharmaceutical composition of claim 21 , wherein the second 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 . The pharmaceutical composition of claim 22 , wherein the first and second Fn3 domains bind to the same target compound.
24 . The pharmaceutical composition of claim 10 , wherein the pharmaceutical composition is substantially free of endotoxin.
25 . A pharmaceutical composition for human treatment comprised of: a therapeutic protein comprising a pharmaceutically acceptable fibronectin type III (Fn3) domain and a physiologically acceptable buffer, wherein the Fn3 domain:
(a) has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of a human Fn3 domain; and
(b) binds to a target compound that is not bound by the corresponding human Fn3 domain.
26 . The pharmaceutical composition of claim 25 , wherein the pharmaceutically acceptable Fn3 domain binds with a K D of 500 nM or less to the target compound.
27 . The pharmaceutical composition of claim 25 , wherein the Fn3 domain is a 10 Fn3 domain and 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.
28 . The pharmaceutical composition of claim 27 , wherein the Fn3 domain has an amino acid sequence that is at least 70% identical to the sequence of a human 10 Fn3 domain.
29 . The pharmaceutical composition of claim 25 , wherein the Fn3 domain contains no free sulfhydryl moieties and no disulfide bonds.
30 . The pharmaceutical composition of claim 25 , wherein the therapeutic protein is produced by expressing the polypeptide in a prokaryote.
31 . The pharmaceutical composition of claim 30 , wherein the pharmaceutical composition is substantially free of endotoxin.
32 . A pharmaceutical composition comprised of: a therapeutic protein comprising a pharmaceutically acceptable fibronectin type III (Fn3) domain, wherein the pharmaceutical composition is substantially free of endotoxin and wherein the Fn3 domain:
(a) has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of a human Fn3 domain; and
(b) binds to a target compound that is not bound by the corresponding human Fn3 domain.
33 . The pharmaceutical composition of claim 32 , wherein the pharmaceutically acceptable Fn3 domain binds with a K D of 500 nM or less to the target compound.
34 . The pharmaceutical composition of claim 32 , wherein the Fn3 domain is a 10 Fn3 domain and 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.
35 . The pharmaceutical composition of claim 32 , wherein the Fn3 domain has an amino acid sequence that is at least 70% identical to the sequence of a human 10 Fn3 domain.
36 . The pharmaceutical composition of claim 32 , wherein the therapeutic protein is produced by expressing the polypeptide in a prokaryote.
37 . The pharmaceutical composition of claim 32 , wherein the FG loop binds to the target compound.