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Building IP: BMY Patent Application re "SERUM ALBUMIN-BINDING FIBRONECTIN TYPE III DOMAINS"
SERUM ALBUMIN-BINDING FIBRONECTIN TYPE III DOMAINS The present invention relates to polypeptides which include tenth fibronectin type III domains (.sup.10Fn3) that binds to serum albumin, with south pole loop substitutions. The invention further relates to fusion molecules comprising a serum albumin-binding .sup.10Fn3 joined to a heterologous protein for use in diagnostic and therapeutic applications.
1. A polypeptide comprising a fibronectin type III tenth domain (.sup.10Fn3) wherein the .sup.10Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops, b) a CD loop with an altered amino acid sequence relative to the sequence of the corresponding CD loop of the human .sup.10Fn3 domain, c) wherein the polypeptide binds to human serum albumin with a K.sub.D of less than 500 nM, and d) wherein the .sup.10Fn3 domain further binds to one or more of rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin with a K.sub.D of less than 500 nM. 2. The polypeptide of claim 1, wherein the .sup.10Fn3 domain binds to: a) rhesus serum albumin and cynomolgus serum albumin, b) mouse serum albumin and rat serum albumin, or c) rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin. 3. The polypeptide of claim 1, wherein the .sup.10Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4. 4. The polypeptide of claim 1, wherein the .sup.10Fn3 domain binds to domain I-II of HSA. 5. The polypeptide of claim 1, wherein the CD loop comprises an amino acid sequence according to the formula G-X.sub.1-X.sub.2-V-X.sub.3-X.sub.4-X.sub.5-S-X.sub.6-X.sub.7-G-X.sub.8-X- .sub.9-Y-X.sub.10-X.sub.11-X.sub.12-E (SEQ ID NO: 170), wherein, (a) X.sub.1 is selected from the group consisting of R or W; (b) X.sub.2 is selected from the group consisting of H, E, D, Y, or Q; (c) X.sub.3 is selected from the group consisting of Q or H; (d) X.sub.4 is selected from the group consisting of I, K, M, Q, L, or V; (e) X.sub.5 is selected from the group consisting of Y, F, or N; (f) X.sub.6 is selected from the group consisting of D, V, or E; (g) X.sub.7 is selected from the group consisting of L, W, or F; (h) X.sub.8 is selected from the group consisting of P or T; (i) X.sub.9 is selected from the group consisting of L or M; (j) X.sub.10 is selected from the group consisting of I or V; (k) X.sub.11 is selected from the group consisting of Y or F; and (l) X.sub.12 is selected from the group consisting of T, S, Q, N, or A. 6. (canceled) 7. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence at least 95% identical to the non-CD loop regions of SEQ ID NOs: 81, 88, 23-80, 82-87, 89-100, 184-209 and 235-260. 8. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 81, 88, 23-80, 82-87, 89-100, 184-209 and 235-260. 9-14. (canceled) 15. A composition comprising a polypeptide of claim 1 and a carrier. 16. An isolated nucleic acid molecule encoding the polypeptide of claim 1. 17. (canceled) 18. An expression vector comprising the nucleotide sequence encoding the polypeptide of claim 1. 19. A cell comprising a nucleic acid molecule of claim 16 or expression vector of claim 18. 20. A method of producing the polypeptide of claim 1 comprising culturing the cell of claim 19 under conditions suitable for expressing the polypeptide, and purifying the polypeptide. 21. A conjugate comprising the polypeptide of claim 1 and at least one additional moiety. 22. The conjugate of claim 21, wherein the additional moiety is a small organic molecule, a nucleic acid, a peptide or a protein. 23. The conjugate of claim 21, wherein the additional moiety is linked to the polypeptide of claim 1 via a polypeptide linker having a protease site. 24. The conjugate of claim 21, wherein the additional moiety is a detectable moiety. 25. The conjugate of claim 24, wherein the detectable moiety is a radioisotope or fluorescent compound. REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 16/549,462 (Allowed), filed Aug. 23, 2019, which is a divisional of U.S. patent application Ser. No. 15/127,166, filed Sep. 19, 2016, now U.S. Pat. No. 10,442,851, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2015/021535, filed Mar. 19, 2015, which claims priority to U.S. Provisional Application No. 61/968,181 entitled "Novel Serum Albumin-Binding Fibronectin Type III Domains" filed Mar. 20, 2014. The contents of the aforementioned applications are hereby incorporated by reference. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 15, 2021, is named SequenceListingMXI536USDVCN.txt and is 200,688 bytes in size. BACKGROUND [0003] Inadequate half-lives of therapeutics often necessitate their administration at high frequencies and/or higher doses, or the use of sustained release formulations, in order to maintain serum levels necessary for therapeutic effects. Yet, this is often associated with negative side effects. For example, frequent systemic injections present considerable discomfort to the subject, and pose a high risk of administration-related infections, and may require hospitalization or frequent visits to the hospital, in particular when the therapeutic is to be administered intravenously. Moreover, in long term treatments, daily intravenous injections can also lead to considerable side effects of tissue scarring and vascular pathologies caused by the repeated puncturing of vessels. Similar problems are known for all frequent systemic administrations of therapeutics, such as, for example, the administration of insulin to diabetics, or interferon drugs to patients suffering from multiple sclerosis. All these factors lead to a decrease in patient compliance and increased costs for the health system. [0004] This application provides compounds that increase the serum half-life of various therapeutics, compounds having increased serum half-life, and methods for increasing the serum half-life of therapeutics. Such compounds and methods for increasing the serum half-life of therapeutics can be manufactured in a cost effective manner, possess desirable biophysical properties (e.g., Tm, substantially monomeric, or well-folded), and are of a size small enough to permit tissue penetration. SUMMARY [0005] The invention is based, at least in part, on the discovery of novel south pole-based serum albumin binding fibronectin type III tenth domain (.sup.10Fn3) containing Adnectins (PKE2 Adnectins), which provide enhanced properties over prior north pole-based serum albumin binding .sup.10Fn3 domain containing Adnectins. [0006] In one aspect, the invention provides a polypeptide comprising a .sup.10Fn3 domain, wherein the .sup.10Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops, b) a CD loop with an altered amino acid sequence relative to the sequence of the corresponding CD loop of the human .sup.10Fn3 domain, and c) wherein the polypeptide binds to human serum albumin with a K.sub.D of less than 500 nM. [0007] In certain embodiments, the .sup.10Fn3 domain further binds to one or more of rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin. For example, the .sup.10Fn3 domain may bind to HSA, rhesus serum albumin, and cynomolgus serum albumin, or the .sup.10Fn3 domain may bind to HSA, rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin. In some embodiments, the .sup.10Fn3 domain binds to the corresponding serum albumin with a K.sub.D of less than 500 nM, for example, a K.sub.D of less than 100 nM, or even a K.sub.D less than 10 nM. In some embodiments, the .sup.10Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4. [0008] In certain embodiments, the .sup.10Fn3 domain binds to domain I-II of USA. [0009] In certain embodiments, the serum half-life of the polypeptide comprising the .sup.10Fn3 domain in the presence of human serum albumin is at least 10 hours, such as at least 20 hours, or at least 30 hours. [0010] In certain embodiments, the CD loop comprises an amino acid sequence according to the formula G-X.sub.1-X.sub.2-V-X.sub.3-X.sub.4-X.sub.5-S-X.sub.6-X.sub.7-G-X.sub.8-X- .sub.9-Y-X.sub.10-X.sub.11-X.sub.12-E (SEQ ID NO: 170), wherein, [0011] (a) X.sub.1 is selected from the group consisting of R or W; [0012] (b) X.sub.2 is selected from the group consisting of H, E, D, Y, or Q; [0013] (c) X.sub.3 is selected from the group consisting of Q or H; [0014] (d) X.sub.4 is selected from the group consisting of I, K, M, Q, L, or V; [0015] (e) X.sub.5 is selected from the group consisting of Y, F, or N; [0016] (f) X.sub.6 is selected from the group consisting of D, V, or E; [0017] (g) X.sub.7 is selected from the group consisting of L, W, or F; [0018] (h) X.sub.8 is selected from the group consisting of P or T; [0019] (i) X.sub.9 is selected from the group consisting of L or M; [0020] (j) X.sub.10 is selected from the group consisting of I or V; [0021] (k) X.sub.11 is selected from the group consisting of Y or F; and [0022] (l) X.sub.12 is selected from the group consisting of T, S, Q, N, or A. [0023] In a preferred embodiment, (a) X.sub.1 is R; (b) X.sub.2 is E; (c) X.sub.3 is Q; (d) X.sub.4 is K; (e) X.sub.5 is Y; (f) X.sub.6 is D; (g) X.sub.7 is L or W; (h) X.sub.8 is P; (i) X.sub.9 is L; (j) X.sub.10 is I; (k) X.sub.11 is Y; and (l) X.sub.12 is Q or N. [0024] In yet a further preferred embodiment, (a) X.sub.1 is R; (b) X.sub.2 is E; (c) X.sub.3 is Q; (d) X.sub.4 is K; (e) X.sub.5 is Y; (f) X.sub.6 is D; (g) X.sub.7 is L; (h) X.sub.8 is P; (i) X.sub.9 is L; (j) X.sub.10 is I; (k) X.sub.11 is Y; and (l) X.sub.12 is Q. [0025] In yet a further preferred embodiment, (a) X.sub.1 is R; (b) X.sub.2 is E; (c) X.sub.3 is Q; (d) X.sub.4 is K; (e) X.sub.5 is Y; (f) X.sub.6 is D; (g) X.sub.7 is W; (h) X.sub.8 is P; (i) X.sub.9 is L; (j) X.sub.10 is I; (k) X.sub.11 is Y; and (l) X.sub.12 is N. [0026] In certain embodiments, the CD loop comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-125. In a preferred embodiment, the CD loop comprises the amino acid sequence set forth in SEQ ID NO: 106 or 113. [0027] In certain embodiments, the invention provides a polypeptide comprising a .sup.10Fn3 domain comprising (i) a CD loop comprising an amino acid sequence having the consensus sequence of SEQ ID NO: 170 or the amino acid sequence of any one of SEQ ID NOs: 101-125 and (ii) an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-CD loop regions of SEQ ID NOs: 23-100, 184-209 and 235-260 or that differs from one of SEQ ID NOs: 23-100, 184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260 or that differs from one of SEQ ID NOs: 23-100, 184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids. Amino acid differences may be substitutions, additions or deletions. [0028] In certain aspects, the invention provides a fusion polypeptide comprising a fibronectin type III tenth (.sup.10Fn3) domain and a heterologous protein, wherein the .sup.10Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops, b) a CD loop with an altered amino acid sequence relative to the sequence of the corresponding loop of the human .sup.10Fn3 domain, and c) wherein the polypeptide binds to human serum albumin with a K.sub.D of less than 500 nM. [0029] In certain embodiments, the fusion polypeptide comprises an albumin binding Adnectin comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260 or that differs from one of SEQ ID NOs: 23-100, 184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids. In a preferred embodiment, the fusion polypeptide comprises an albumin binding Adnectin comprising the amino acid sequence of SEQ ID NO: 55, 81, 190 or 241. In yet another preferred embodiment, the fusion polypeptide comprises an albumin binding Adnectin comprising the amino acid sequence of SEQ ID NO: 62, 88, 197 or 248. [0030] In certain embodiments, the fusion polypeptide comprises an albumin binding Adnectin and a heterologous moiety, wherein the heterologous moiety is a therapeutic moiety. [0031] In certain embodiments, the heterologous protein is a polypeptide comprising a .sup.10Fn3 domain. In some embodiments, the .sup.10Fn3 domain binds to a target protein other than serum albumin. In one embodiment, the .sup.10Fn3 domain binds to PCSK9 (i.e., a PCSK9 Adnectin), and comprises an amino acid sequence at least 70%. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 167 or that differs from SEQ ID NO: 167 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids. [0032] In certain embodiments, the fusion polypeptide is a PCSK9-PKE2 tandem Adnectin comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168, 169 or 261 or that differs from one of SEQ ID NOs: 168, 169 or 261 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids (and may or may not comprise an N-terminal methionine). [0033] In certain embodiments, the serum half-life of the fusion polypeptide in the presence of mouse serum albumin is at least 10 hours. In some embodiments, the serum half-life of the fusion polypeptide in the presence of cynomolgus serum albumin is at least 50 hours. In certain embodiments, the serum half-life of the fusion polypeptide in the presence of mouse or cynomolgus serum albumin is 10-100 hours, such as 10-90 hours, 10-80 hours, 10-70 hours, 10-60 hours, 10-50 hours, 10-40 hours, 10-30 hours, 10-20 hours, 50-100 hours, 60-100 hours, 70-100 hours, 80-100 hours, 90-100 hours, 20-90 hours, 30-80 hours, 40-70 hours, or 50-60 hours. [0034] In certain aspects, the invention provides a PKE2 Adnectin or PCSK9-PKE2 tandem Adnectin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-100, 168, 169, 184-209, 235-260, and 261. [0035] In certain aspects, the invention provides a composition comprising any one of the albumin binding Adnectins or fusion proteins comprising such, as described herein, and a carrier. [0036] In certain aspects, the invention provides an isolated nucleic acid molecule encoding any one of the albumin binding Adnectins or fusion proteins comprising such, as described herein, for example, those set forth in SEQ ID NOs: 126-151 and 172, expression vectors encoding the nucleic acid molecules, and cells comprising the nucleic acid molecules. Also provided are nucleic acids comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to any of these nucleotide sequences described herein, or which differ therefrom in at most 1-5, 1-10, 1-50 or 1-100 nucleotides. [0037] In certain aspects, the invention provides a method of producing the albumin binding Adnectins or fusion proteins comprising such described herein, comprising culturing the cell comprising the nucleic acid molecules encoding the same under conditions suitable for expressing the Adnectins or fusion proteins, and purifying the same. |
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