Patent Grant
9403896
The current invention is directed to fusion polypeptides comprising a serpin-finger polypeptide and a second peptide, polypeptide or protein as well as the use of such fusion polypeptides.
The instant application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 15, 2015, is named P4720C1_SL.txt, and is 33,737 bytes in size.
Serine protease inhibitors (serpins) regulate a multitude of physiological pathways, e.g. inflammation, coagulation, fibrinolysis, apoptosis and extracellular matrix remodeling. The reactive loop of a serpin is cleaved by the serine protease and the serine protease is inactivated via disruption of the catalytic site.
Alpha1-antitrypsin is a 394 amino acid, 52 kDa, glycoprotein synthesized by hepatocytes, macrophages and intestinal and bronchial epithelial cells. Crystal structures show that alpha1-antitrypsin is consisting of five beta-sheets, nine alpha-helices and an exposed mobile reactive loop comprising 14 residues that presents a peptide sequence as a pseudo-substrate for the target protease. Cleavage of the scissile reactive bond, denoted as P1-P1′ results in an irreversible conformational change wherein the N-terminal residue of the loop being completely incorporated into the middle of the beta-sheets of alpha1-antitrypsin as strand 4a (Schechter, I., and Berger, A., Biochem. Biophys. Res. Commun. 27 (1967) 157-162; Loebermann, H., et al., J. Mol. Biol. 177 (1984) 531-557; Baumann, U., et al., J. Mol. Biol. 218 (1991) 595-606; Baumann, U., et al., J. Mol. Biol. 226 (1992) 1207-1218; Mourey, L., et al., Biochim. 72 (1990) 599-608; Mourey, L., et al., J. Mol. Biol. 232 (1993) 223-241).
After proteolytic cleavage by its target protease, P1 Met and P1′ Ser are separated and the unprimed active site loop is inserted as strand 4a in the antiparallel beta-sheet A. A peptide with the amino acid sequence of strand 4a, residues 345-358 of human alpha1-antitrypsin Thr-Glu-Ala-Ala-Gly-Ala-Met-Phe-Leu-Glu-Ala-Ile-Val-Met, (Seq ID NO: 89),associates with intact alpha1-antitrypsin and forms a stoichiometric complex with properties similar to cleaved alpha1-antitrypsin (Schulze, A. J., et al., Eur. J. Biochem. 194 (1990) 51-56).
In WO 97/024453 receptor specific chimeric viral surface polypeptides for viral and particle incorporation and internalization in target cells are reported. A covalently attached complex of alpha1-antitrypsin-protease inhibitor with a water soluble polymer is reported in EP 0 147 761. In US 2006/0040867 inhibitors of serine protease activity and their use in methods and compositions for treatment of bacterial infections are reported.
Schule, A. J., et al., report structural transition of alpha1-antitrypsin by a peptide sequentially similar to beta-strand S4A (Eur. J. Biochem. 194 (1990) 51-56). Multifunctional anti-HIV agents based on amino acid sequences present in serpin C-terminal peptides are reported by Congote, L. F., in Anti-infective agents in medicinal chemistry, Bentham Science publishers, Hilversum (NL), 7 (2008) 126-133. Qi, Z., et al. (J. Biol. Chem. 283 (2008) 30376-30384) report rationally-designed anti-HIV peptides containing multifunctional domains as molecule probes for studying the mechanism of action of the first and second generation HIV fusion inhibitors. Methods and compositions for inhibition of membrane fusion-associated events, including HIV transmission, are reported in WO 01/51673. In WO 02/063017 integrin-binding chimeras are reported. Heparin fragments and fractions with anti-HIV action are reported in EP 0 355 905. In WO 00/52034 and U.S. Pat. No. 6,849,605 inhibitors of serine protease activity, methods and compositions for treatment of viral infections are reported.
It has been found that a serpin-finger polypeptide has to have a minimal number of amino acid residues in order to allow for a sufficiently fast association with a serpin. Additionally it has been found that it is beneficial that the amino acid residue at amino acid position 2 of the serpin-finger polypeptide (counted from the N-terminus of the serpin-finger polypeptide) is glutamic acid.
Herein is reported a serpin-finger fusion polypeptide comprising
wherein the further polypeptide can be any polypeptide exerting a biological activity, such as inhibition, activation, binding or labeling.
In one embodiment the serpin-finger fusion polypeptide comprises in addition at least one of
In one embodiment the amino acid sequence of the serpin-finger polypeptide is selected from AGAMFLEAIVM (SEQ ID NO: 01), AAGAMFLEAIVM (SEQ ID NO: 02), TEAAGAMFLEAIVM (SEQ ID NO: 03), AGAMFLEAIVM (SEQ ID NO: 04), TEAAGAMFFEAIPM (SEQ ID NO: 05), TAVVIA (SEQ ID NO: 06), SEAAASTAVVIA (SEQ ID NO: 07), TEAAGATAVVIA (SEQ ID NO: 08), TDAAGATAVVIA (SEQ ID NO: 09), SDAAGAMFLEAI (SEQ ID NO: 10), or SEAAASMFLEAI (SEQ ID NO: 11). In one embodiment the amino acid sequence of the serpin-finger polypeptide is selected from SEAAASTAVVIA (SEQ ID NO: 07) and SEAAASMFLEAI (SEQ ID NO: 11).
In one embodiment the amino acid sequence of the peptidic linker polypeptide is GGSGG (SEQ ID NO: 12), or SGGGGSGGGGSGGGGT (SEQ ID NO: 52), or STT (SEQ ID NO: 75).
In one embodiment the amino acid residue at amino acid position 2 of the serpin-finger polypeptide (counted from the N-terminus of the serpin-finger polypeptide) is glutamic acid.
In one embodiment the serpin-finger polypeptide consists of 8 to 14 amino acid residues, in one embodiment of 10 to 14 amino acid residues, and in one embodiment of 11 to 13 amino acid residues.
In one embodiment the further polypeptide is selected from immunoglobulins, immunoglobulin fragments, hormones, cytokines, growth factors, receptor ligands, receptor agonists, receptor antagonists, enzyme ligands, enzyme agonists, enzyme antagonists, cytotoxic agents, antiviral agents, imaging agents, and enzyme activity modulators.
Herein is reported as an aspect a fusion polypeptide comprising in N- to C-terminal direction a serpin-finger polypeptide fused to a biologically active polypeptide, optionally with a peptidic linker polypeptide in between.
In one embodiment the biologically active polypeptide is an antiviral agent.
In one embodiment the antiviral agent is an HIV fusion inhibitor polypeptide. In one embodiment the amino acid sequence of the HIV fusion inhibitor polypeptide is MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 13).
Another aspect as reported herein is a protein complex of a serpin-finger fusion polypeptide as reported herein and a serpin or a fragment thereof, wherein the fusion polypeptide is incorporated into the middle of beta-sheet A of the serpin as strand 4a.
Another aspect as reported herein is a pharmaceutical composition comprising the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein and optionally a pharmaceutically acceptable carrier. In one embodiment the pharmaceutical composition further comprises an additional therapeutic agent.
Also an aspect as reported herein is the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein for use as a medicament.
Another aspect as reported herein the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein is for use in treating a viral infection.
In one aspect as reported herein the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein is for use in inhibiting cell-cell-membrane fusion or in inhibiting the infection of a cell by a virus. In one embodiment the infection of a cell by a virus is an HIV infection and the serpin-finger fusion polypeptide is a serpin-finger HIV fusion inhibitor polypeptide fusion polypeptide.
One aspect as reported herein is the use of the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein in the manufacture of a medicament. In one embodiment the medicament is for treatment of a viral infection. In another embodiment the medicament is for inhibiting cell-cell-membrane fusion or for inhibiting the infection of a cell by a virus. In one embodiment the viral infection is an HIV infection and the serpin-finger fusion polypeptide is a serpin-finger HIV fusion inhibitor polypeptide fusion polypeptide.
Also an aspect as reported herein is a method of treating an individual having a viral infection comprising administering to the individual an effective amount of the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein. In one embodiment the viral infection is an HIV infection and the serpin-finger fusion polypeptide is a serpin-finger HIV fusion inhibitor polypeptide fusion polypeptide.
A further aspect as reported herein is a method of inhibiting cell-cell-membrane fusion, or a method of inhibiting the infection of a cell by a virus in an individual comprising administering to the individual an effective amount of the serpin-finger fusion polypeptide as reported herein or the protein complex as reported herein to inhibit cell-cell-membrane fusion or to inhibit the infection of a cell by a virus.
Another aspect as reported herein is a mixture comprising a serpin-finger fusion polypeptide as reported herein and a serpin or a fragment thereof, a pharmaceutical composition comprising this mixture and the use thereof as a medicament.
One aspect as reported herein is the use of a serpin or a fragment thereof for the manufacture of a protein complex with a serpin-finger fusion polypeptide comprising in N- to C-terminal direction a serpin-finger polypeptide fused to a biologically active polypeptide.
Another aspect as reported herein is a kit comprising the serpin-finger fusion polypeptide and a serpin in separate containers, optionally a further container for mixing these two components and also optionally an instruction sheet. In one embodiment the kit further comprises a means for detecting the fusion polypeptide in a sample.
Herein is reported a serpin-finger fusion polypeptide comprising
wherein the further polypeptide can be any polypeptide exerting a biological activity, such as inhibition, activation, binding or labeling.
In one aspect herein a protein complex comprising a serpin-finger fusion polypeptide and a serpin is reported, wherein the fusion polypeptide is incorporated into the middle of beta-sheet A as strand 4a of the serpin.
The serpin-finger fusion polypeptide targets and anchors the fused further polypeptide with high affinity and functional spatial orientation to the serpin.
In one embodiment a serpin-finger fusion polypeptide comprising a serpin-finger polypeptide fused to an HIV fusion inhibitor polypeptide via a peptidic linker polypeptide is reported.
The term “serpin-finger polypeptide” as used within the current invention denotes a polypeptide that consists of 8 to 16 amino acid residues derived either from the natural reactive center loop of a serpin or from a synthetic analogue thereof. In one embodiment the amino acid sequence of the polypeptide consists of 11 to 13 amino acid residues. In one embodiment the amino acid sequence of the serpin-finger polypeptide is selected from AGAMFLEAIVM (SEQ ID NO: 01), AAGAMFLEAIVM (SEQ ID NO: 02), TEAAGAMFLEAIVM (SEQ ID NO: 03), AGAMFLEAIVM (SEQ ID NO: 04), TEAAGAMFFEAIPM (SEQ ID NO: 05), TAVVIA (SEQ ID NO: 06), SEAAASTAVVIA (SEQ ID NO: 07), TEAAGATAVVIA (SEQ ID NO: 08), TDAAGATAVVIA (SEQ ID NO: 09), SDAAGAMFLEAI (SEQ ID NO: 10), SEAAASMFLEAI (SEQ ID NO: 11), TIDEKGTEAAGAMFLE (SEQ ID NO: 14), DVFEEGTEASAATAVK (SEQ ID NO: 15), DVDEAGTEAAAATTFA (SEQ ID NO: 16), QLNEEGVDTAGSTGVT (SEQ ID NO: 17), HIGEKGTEAAAVPEVE (SEQ ID NO: 18), EVDERGTEAVAGILSE (SEQ ID NO: 19), EVTEEGVEAAAATAVV (SEQ ID NO: 20), EVTEEGAEAAAATAVV (SEQ ID NO: 21), TVNEEGTQATTVTTVG (SEQ ID NO: 22), EVDENGTQAAAATGAV (SEQ ID NO: 23), EVNEEGTEAAAATAVV (SEQ ID NO: 24), DVNEEGTEAAAGTGGV (SEQ ID NO: 25), EVNESGTVASSSTAVI (SEQ ID NO: 26), DVFEEGTEASAATAVK (SEQ ID NO: 27), EVTEEGTEATAATGSN (SEQ ID NO: 28), EITEDGGDSIEVPGAR (SEQ ID NO: 29), ELSEVGVEAAAATSIA (SEQ ID NO: 30), ELTETGVEAAAASAIS (SEQ ID NO: 31), GTEAAGAMFLEAIPMS (SEQ ID NO: 82), and SGTEAAGAMFLEAIPMS (SEQ ID NO: 83). In one embodiment the amino acid sequence of the serpin-finger polypeptide is AGAMFLEAIVM (SEQ ID NO: 01), or AAGAMFLEAIVM (SEQ ID NO: 02), or TEAAGAMFLEAIVM (SEQ ID NO: 03), or SEAAASTAVVIA (SEQ ID NO: 07), or SEAAASMFLEAI (SEQ ID NO: 11). In one embodiment the serpin-finger polypeptide is derived from alpha1-antitrypsin or antithrombin.
The term “serpin” denotes a superfamily of proteins with diverse functions. Exemplary members of the serpin superfamily are listed in the following. In one embodiment the serpin is selected from alpha1-antitrypsin (serpinA1), antitrypsin-related protein (serpinA2), alpha1-antichymotrypsin (serpinA3), kallistatin (serpinA4), protein C inhibitor (serpinA5), cortisol binding globulin (serpinA6), thyroxine-binding globulin (serpinA7), angiotensinogen (serpinA8), centerin (serpinA9), protein Z-related protease inhibitor (serpinA10), serpinA11, vaspin (serpinA12), serpinA13, monocyte neutrophils elastase inhibitor (serpinB1), plasminogen activator inhibitor-2 (serpinB2), squamous cell carcinoma antigen-1 and -2 (serpinB3 and B4), maspin (serpinB5), PI-6 (serpinB6), megsin (serpinB7), PI-8 (serpinB8), PI-9 (serpinB9), bomapin (serpinB10), serpinB11, yukopin (serpinB12), hurpin/headpin (serpinB13), antithrombin (serpinC1), heparin cofactor II (serpinD1), plasminogen activator inhibitor 1 (serpinE1), glia derived nexin/protease nexin I (serpinE2), pigment epithelium derived factor (serpinF1), alpha2-antiplasmin (serpinF2), complement 1-inhibitor (serpinG1), HSP47 (serpinH1), neuroserpin (serpinI1) and pancpin (serpinI2). A fragment of a serpin is a molecule that still exerts the function to incorporate a serpin-finger fusion polypeptide as reported herein into the middle of beta-sheet A as strand 4a of the serpin fragment.
The term “biologically active polypeptide” as used herein refers to an organic molecule, e.g. a biological macromolecule such as a peptide, protein, glycoprotein, nucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide or protein, that causes a biological effect when administered in or to artificial biological systems, such as bioassays e.g. using cell lines and viruses, or in vivo to an animal, including but not limited to birds or mammals, including humans. This biological effect can be but is not limited to enzyme inhibition or activation, binding to a receptor or a ligand, either at the binding site or circumferential, signal triggering or signal modulation. Biologically active molecules are without limitation for example immunoglobulins, or hormones, or cytokines, or growth factors, or receptor ligands, or agonists or antagonists, or cytotoxic agents, or antiviral agents, or imaging agents, or enzyme inhibitors, enzyme activators or enzyme activity modulators such as allosteric substances. In a one embodiment the biologically active polypeptide is an immunoglobulin, immunoglobulin conjugate, or an HIV fusion inhibitor polypeptide.
A “HIV fusion inhibitor polypeptide” is a polypeptide which inhibits events associated with membrane fusion or the membrane fusion event itself, including, among other things, the inhibition of infection of uninfected cells by a HIV virus due to membrane fusion. The HIV fusion inhibitor polypeptide is in one embodiment a linear polypeptide. For example, it is as in one embodiment derived from the HIV gp41 ectodomain, e.g., such as DP107 or DP178. The amino acid sequence of the HIV fusion inhibitor polypeptide consists of 5 to 100 amino acid residues, in one embodiment of 10 to 75 amino acid residues, in a further embodiment of 15 to 50 amino acid residues. In one embodiment the amino acid sequence of the HIV fusion inhibitor polypeptide is selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 32 to 42. Further examples of HIV fusion inhibitor polypeptides can be found in U.S. Pat. Nos. 5,464,933, 5,656,480, 6,013,263, 6,017,536, 6,020,459, 6,093,794, 6,060,065, 6,258,782, 6,348,568, 6,479,055, 6,656,906, WO 1996/19495, WO 1996/40191, WO 1999/59615, WO 2000/69902, and WO 2005/067960. For example, the amino acid sequences of the HIV fusion inhibitor polypeptide can be selected from the group comprising SEQ ID NO: 1 to 10 of U.S. Pat. No. 5,464,933; SEQ ID NO: 1 to 15 of U.S. Pat. No. 5,656,480; SEQ ID NO: 1 to 10 and 16 to 83 of U.S. Pat. No. 6,013,263; SEQ ID NO: 1 to 10, 20 to 83 and 139 to 149 of U.S. Pat. No. 6,017,536; SEQ ID NO: 1 to 10, 17 to 83 and 210 to 214 of U.S. Pat. No. 6,093,794; SEQ ID NO: 1 to 10, 16 to 83 and 210 to 211 of U.S. Pat. No. 6,060,065; SEQ ID NO: 1286 and 1310 of U.S. Pat. No. 6,258,782; SEQ ID NO: 1129, 1278-1309, 1311 and 1433 of U.S. Pat. No. 6,348,568; SEQ ID NO: 1 to 10 and 210 to 238 of U.S. Pat. No. 6,479,055; SEQ ID NO: 1 to 171, 173 to 216, 218 to 219, 222 to 228, 231, 233 to 366, 372 to 398, 400 to 456, 458 to 498, 500 to 570, 572 to 620, 622 to 651, 653 to 736, 739 to 785, 787 to 811, 813 to 823, 825, 827 to 863, 865 to 875, 877 to 883, 885, 887 to 890, 892 to 981, 986 to 999, 1001 to 1003, 1006 to 1018, 1022 to 1024, 1026 to 1028, 1030 to 1032, 1037 to 1076, 1078 to 1079, 1082 to 1117, 1120 to 1176, 1179 to 1213, 1218 to 1223, 1227 to 1237, 1244 to 1245, 1256 to 1268, 1271 to 1275, 1277, 1345 to 1348, 1350 to 1362, 1364, 1366, 1368, 1370, 1372, 1374 to 1376, 1378 to 1379, 1381 to 1385, 1412 to 1417, 1421 to 1426, 1428 to 1430, 1432, 1439 to 1542, 1670 to 1682, 1684 to 1709, 1712 to 1719, 1721 to 1753, 1755 to 1757 of U.S. Pat. No. 6,656,906; or SEQ ID NO: 5 to 95 of WO 2005/067960.
The term “peptidic linker polypeptide” as used within this application denotes a peptidic linker polypeptide of natural and/or synthetic origin. It consists of a linear amino acid residue chain in which the 20 naturally occurring amino acids are the monomeric building blocks. The chain has a length of 1 to 50 amino acid residues, in one embodiment of 1 to 28 amino acid residues, in another embodiment of 3 to 25 amino acid residues. The peptidic linker polypeptide may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides, such as polypeptides with a hinge-function. The peptidic linker polypeptide has the function to ensure that a polypeptide in a conjugate or fusion each of the conjugated/fused polypeptides can perform its biological activity by allowing each of the polypeptides to fold correctly and to be presented properly. In one embodiment the peptidic linker polypeptide is a “synthetic peptidic linker polypeptide” that is designated to be rich in glycine, glutamine, and/or serine residues. The residues are arranged e.g. in small repetitive units of up to five amino acid residues, such as GGGGS (SEQ ID NO: 90), GGGSG (SEQ ID NO: 91), GGSGG (SEQ ID NO: 12), GSGGG (SEQ ID NO: 92), QQQQG (SEQ ID NO: 93), or SSSSG (SEQ ID NO: 94). The repetitive unit may be repeated for two to five times to form a multimeric unit. At the amino- and/or carboxy-terminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acid residues may be added. Other synthetic peptidic linker polypeptides are composed of a single amino acid residue, that is repeated of from 10 to 20 times and which may comprise at the amino- and/or carboxy-terminal end up to six additional arbitrary, naturally occurring amino acid residues, such as e.g. serine in the linker GSSSSSSSSSSSSSSSG (SEQ ID NO: 61). In one embodiment the peptidic linker polypeptide is selected from antibody hinge region, LSLSPGK (SEQ ID NO: 43), LSPNRGEC (SEQ ID NO: 44), [GQ4]3GNN (SEQ ID NO: 47), LSLSGG (SEQ ID NO: 69), LSLSPGG (SEQ ID NO: 70), G3[SG4]2SG (SEQ ID NO: 73), G3[SG4]2SG2 (SEQ ID NO: 74) or STT (SEQ ID NO: 75). All peptidic linker polypeptides can be encoded by a nucleic acid molecule and therefore can be recombinantly expressed. As the peptidic linker polypeptides are themselves polypeptides, the serpin-finger polypeptide is connected to the peptidic linker polypeptide via a peptide bond that is formed between two amino acids.
The term “into the middle of beta-sheet A as strand 4a” denotes the insertion of a serpin-finger fusion polypeptide between strands 4 and 5 of beta-sheet A of a serpin, e.g. beta-sheet A of alpha1-antitrypsin, or antithrombin.
In a fusion polypeptide comprising a serpin-finger polypeptide fused to a polypeptide with biological activity (optionally via a peptidic linker) the fused polypeptide with biological activity has improved properties compared to the isolated polypeptide. The fusion polypeptide can e.g. be inserted into the beta-sheet A of a serpin, such as alpha1-antitrypsin, to improve the in vivo half-life of the fused polypeptide with biological activity. It has been found that a serpin-finger polypeptide derived from antithrombin inserts well into the beta sheets of alpha1-antitrypsin. This combination is one embodiment of the aspects of the invention. This can e.g. be seen from the in vitro association data presented in Table 3.
In addition it has been found that not the serpin-finger polypeptide with the shortest amino acid sequence of six amino acid residues inserts fastest but a longer one of 11 to 13 amino acid residues length does. Furthermore the amino acid glutamic acid as second amino acid of the serpin-finger polypeptide (counted from the N-terminus of the serpin-finger polypeptide) increases the insertion efficiency of the serpin-finger fusion polypeptide into the serpin beta sheet.
In the following the aspects as reported herein are exemplified with a serpin-finger fusion polypeptide comprising a peptidic linker polypeptide and an HIV fusion inhibitor polypeptide as biologically active polypeptide. The following is presented solely to exemplify the herein reported subject matter and has not to be treated as limitation or restriction.
Different fusion polypeptides have been prepared. The encoding genes have been obtained by chemical gene synthesis and the polypeptides have been recombinantly produced in E. coli. The different fusion polypeptides have been expressed as a construct comprising a streptavidin carrier protein as purification tag, a trypsin cleavage site, a serpin-finger polypeptide, a peptidic linker polypeptide, and a HIV fusion inhibitor polypeptide.
In one embodiment the amino acid sequence of the trypsin cleavage site is GR.
The amino acid sequence of the serpin-finger polypeptide is in one embodiment SEAAASTAVVIA (SEQ ID NO: 07) either with or without N-terminal and/or C-terminal linker whereby the linker is independently and individually selected from S, or G, or SG.
The amino acid sequence of the peptidic linker polypeptide is in one embodiment [SG4]3 (SEQ ID NO: 50), or [SG4]3T (SEQ ID NO: 52), or STT (SEQ ID NO: 75).
The amino acid sequence of fusion polypeptide FP-1 is SEQ ID NO: 76, the amino acid sequence of fusion polypeptide FP-2 is SEQ ID NO: 77, and the amino acid sequence of fusion polypeptide FP-3 is SEQ ID NO: 78. In the fusion polypeptides FP-1 to FP-3 the same serpin-finger polypeptide is conjugated to different HIV fusion inhibitor polypeptides. The fusion polypeptide FP-3 is expressed better in E. coli than FP-2 and in turn FP-2 is expressed better than the fusion polypeptide FP-1. The same sequence of the fusion polypeptides is obtained for the binding to target HIV HR-1 polypeptide (FP-3>FP-2>FP-1) in a BIAcore assay. The same sequence has also been found for the anti-viral activity in a cell-cell-fusion-assay (CCF-assay).
By gel-electrophoresis (urea-PAGE) it has been shown that a protein complex of the fusion polypeptide according to the invention and alpha1-antitrypsin is formed. The stable complex formation between the individual FP-X fusion polypeptides and alpha1-antitrypsin is shown in
As the protein complex and free alpha1-antitrypsin cannot be separated by SDS-PAGE gel electrophoresis a determination of the formation of the protein complex by Western blot with incubation with a HR1-polypeptide-biotin-conjugate has to be performed. An exemplary blot is shown in
Different serpin-finger fusion polypeptides have been assayed for their association rate with the serpin alpha1-antitrypsin. The fastest binders have an in vitro association T1/2 of from 1 to 4 hours.
In Table 4 the binding characteristics of the FP-1 to FP-3 fusion polypeptides compared to the isolated HIV fusion inhibitor polypeptide is shown.
The binding affinity determined by surface plasmon resonance displays similar binding constants for the free fusion inhibitor and the three fusion peptides. From the BIAcore binding diagrams shown in
From the Table 5 it can be seen that the fusion polypeptides FP-1 to FP-3 have comparable antiviral activity as the isolated HIV fusion inhibitor polypeptides.
The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
In some embodiments, the invention relates to:
Materials & Methods
Recombinant DNA techniques
Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The molecular biological reagents were used according to the manufacturer's instructions.
Gene Synthesis
Desired gene segments were prepared by chemical synthesis. Desired gene segments were prepared by gene synthesis. The synthesized gene fragments were cloned into a specified expression vector. The DNA sequence of the subcloned gene fragments were confirmed by DNA sequencing.
Protein Determination
The protein concentration of the conjugate was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.
The fusion polypeptides FP-1, FP-2 and FP-3 were prepared by recombinant means. They were expressed as a larger fusion protein in E. coli using core-streptavidin as a carrier protein for high level expression in E. coli. The desired polypeptides were released by enzymatic cleavage in vitro using either trypsin (FP-1 and FP-2) or the endoproteinase LysC (FP-3).
Design of the Core-streptavidin Carrier Fusion Proteins
The polypeptides FP-1 (SEQ ID NO: 76=GR+SEQ ID NO: 05+SEQ ID NO: 52+SEQ ID NO: 37), FP-2 (SEQ ID NO: 77=GR+SEQ ID NO: 82+SEQ ID NO: 75+SEQ ID NO: 13) and FP-3 (SEQ ID NO: 78=GR+SEQ ID NO: 83+SEQ ID NO: 50+SEQ ID NO: 40) were fused to the core-streptavidin sequence (SEQ ID NO: 81) via a GR or GK protease linker containing a unique trypsin or LysC endoproteinase cleavage site, respectively.
The core-streptavidin fusion genes comprising the core-streptavidin encoding nucleic acid, the short endoproteinase linker encoding nucleic acid (GR), the serpin-finger polypeptide encoding nucleic acid, the linker encoding nucleic acid and the HIV fusion inhibitor peptide encoding nucleic acid were assembled with known recombinant methods and techniques by connection of the according nucleic acid segments. The nucleic acid sequences encoding the polypeptides F1, F2 and F3 were made by chemical synthesis and then ligated into an E. coli plasmid for amplification. The subcloned nucleic acid sequences were verified by DNA sequencing.
Making and Description of the Basic/Starting E. coli Expression Plasmid 4980
Plasmid 4980 (4980-pBRori-URA3-LACI-SAC) is an expression plasmid for the expression of core-streptavidin in E. coli. It was generated by ligation of the 3142 bp long EcoRI/CelII-fragment derived from plasmid 1966 (1966-pBRori-URA3-LACI-T-repeat; reported in EP-B 1 422 237) with the 435 bp long core-streptavidin encoding EcoRI/CelII-fragment.
The core-streptavidin E. coli expression plasmid comprises the following elements:
Plasmid 4981 (4981-SAC-Serpin1-T1249) is the plasmid for the expression of core-streptavidin-FP-1 protein in E. coli. It was prepared by insertion of the following 232 bp long NheI/CelII-F1 gene segment (encoding the F1 polypeptide SEQ ID NO: 84)
gctagcggtc gtaccgaagc cgcgggcgct atgttcctgg
into the 3547 bp long NheI/CelII-4980 plasmid fragment.
b) Plasmid 4982
Plasmid 4982 (4982-SAC-Serpin2-T651) is the plasmid for the expression of core-streptavidin-FP-2 protein in E. coli. It was prepared by insertion of the following 181 bp long NheI/CelII-F2 gene segment (encoding the F2 polypeptide SEQ ID NO: 85)
into the 3547 bp long NheI/CelII-4980 plasmid fragment.
c) Plasmid 4983
Plasmid 4983 (4983-SAC-Serpin3-T2635) is the plasmid for the expression of core-streptavidin-FP-3 protein in E. coli. It was prepared by insertion of the following 232 bp long NheI/CelII-F3 gene segment (encoding the F3 polypeptide SEQ ID NO: 86)
into the 3547 bp long NheI/CelII-4980 plasmid fragment.
For the expression of the core-streptavidin fusion proteins 4981, 4982, and 4983 an E. coli host/vector system was employed which enables an antibiotic-free plasmid selection by complementation of an E. coli auxotrophy (PyrF) (see e.g. EP-B 0 972 838 and U.S. Pat. No. 6,291,245).
The fusion proteins were expressed in the E. coli strain CSPZ-2 (leuB, proC, trpE, thi-1, ΔpyrF).
Transformation and Cell Culturing by Complementation of a pyrF Auxotrophy in Selective Medium
The E. coli K12 strain CSPZ-2 (leuB, proC, trpE, thi-1, ΔpyrF) was transformed with the expression plasmids (4981, 4982, and 4983, respectively) obtained in previous step. The transformed CSPZ-2 cells were first grown at 37° C. on agar plates and subsequently in a shaking culture in M9 minimal medium containing 0.5% casamino acids (Difco) up to an optical density at 550 nm (OD550) of 0.6-0.9 and subsequently induced with IPTG (1-5 mmol/l final concentration).
After an induction phase of 4 to 16 hours at 37° C. the cells were harvested by centrifugation, washed with 50 mmol/l potassium phosphate buffer, pH 6.5, and stored at −20° C. until further processing.
Expression Analysis
For expression analysis cell pellets from 3 OD550 nm units (1 OD550 nm=1 ml cell suspension with an OD at 550 nm of 1) of centrifuged culture medium were re-suspended in 0.25 ml 10 mmol/l potassium phosphate buffer, pH 6.5, and the cells were lysed by ultrasonic treatment (two pulses of 30 sec. at 50% intensity). The insoluble cell components were sedimented (centrifugation 14,000 rpm, 5 min.) and the supernatant was admixed with 1/5 of its volume 5×SDS sample buffer (1×SDS sample buffer: 50 mmol/l Tris-HCl, pH 6.8, 1% SDS, 50 mmol/1DTT, 10% glycerol, 0.001% bromophenol blue). The insoluble cell debris fraction (pellet) was re-suspended in 0.3 ml 1×SDS sample buffer, the samples were incubated for 5 min. at 95° C. and centrifuged again. Subsequently, the proteins were separated by SDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, U. K., Nature 227 (1970) 680-685) and stained with Coomassie Brilliant Blue R dye.
The synthesized core-streptavidin fusion protein was homogeneous and was found exclusively in the insoluble cell debris fraction in the form of insoluble protein aggregates, the so-called inclusion bodies (IBs). The expression yield was comparable within the scope of the measurement accuracy in all clones and was between 30%-60% relative to the total E. coli protein.
Pre-culture:
In order to prepare the pre-culture, 300 ml M9-plus medium (M9 medium supplemented with 0.5% casamino acids and 0.9 g/l Trp, Pro and Leu each) was inoculated with 1 ml of a glycerol stock of E. coli CSPZ-2 transformed with plasmid 4981, 4982, and 4983, respectively, in a 1000 ml Erlenmeyer flask. The culture was incubated for about 6 hours at 37° C. on an excenter shaker with 150 rpm until an OD578 nm of 3.0 was reached.
101 Fed-batch Main Fermentation:
At the beginning of fermentation, the pre-culture was transferred into the 10 liter fermenter. The main culture was grown in defined M9 salt medium containing 1.4% glycerol instead of glucose, 2% casamino acids and 0.1% of the amino acids Trp, Leu and Pro each, up to an OD578 nm of 20. Subsequently, feeding of the culture with a glycerol yeast dosage (stock solution: 30% yeast extract and 33% glycerol) was started, the flow rate of which was varied between 0.8 and 3.5 ml/min depending on the development of the pH value of the culture, thereby avoiding any further addition of correction fluids (H3PO4, KOH). The pH was maintained at pH 7.0, the pO2 value was held at 50% by controlling the stirrer speed. At an OD578 nm of 70 1.5 mmol/l IPTG was added. The fermentation was terminated at an OD578 nm of 160-180.
Harvesting the Biomass:
The content of the fermenter was centrifuged with a flow-through centrifuge (13,000 rpm, 131/h) and the harvested biomass was stored at −20° C. until further processing.
200 g E. coli cells (wet weight) were suspended in one liter 0.1 mol/l Tris-HCl, pH 7.0, at 0° C., 300 mg lysozyme were added and incubated for 20 minutes at 0° C. Subsequently, the cells were completely lysed mechanically by means of high pressure dispersion and the DNA was digested for 30 minutes at 25° C. by adding 2 ml 1 mol/1MgCl2 and 10 mg DNAse. Thereafter, 500 ml 60 mmol/l EDTA, 6% Triton X-100 and 1.5 mol/l NaCl, pH 7.0 were admixed with the lysis solution and incubated for another 30 minutes at 0° C. Subsequently, the insoluble components (cell debris and IBs) were sedimented by centrifugation. The pellet was suspended in one liter 0.1 mol/l Tris-HCl, 20 mmol/l EDTA, pH 6.5, incubated for 30 minutes at 25° C. and the IB preparation was isolated by centrifugation.
The inclusion bodies obtained in the previous example were washed two times each with 100 mM potassium phosphate buffer pH 6.5, 500 mM sodium chloride with 20 mM EDTA, and double distilled water. One gram (wet weight) of pelleted inclusion bodies was dissolved by the addition of 10 ml 30 mM potassium hydroxide solution. After 30 min. of stirring the pH value was changed to pH 8.9 by the addition of 1 M boric acid. After solubilization and pH adjustment the F1 and F2 containing core-streptavidin fusion proteins were enzymatically digested with trypsin (1:25000 w/w) while the F3 containing core-streptavidin fusion protein was enzymatically digested with LysC (1:20000 w/w; 10 μl of a 10 μM LysC solution). The solution was incubated at 15° C. over night. The trypsin digestion was stopped by the addition of a 10-fold molar excess of aprotinin. Residual protease was purified away by rec SerETI affinity chromatography. The LysC digestion of F3 was stopped by rec SerETI affinity chromatography only.
The released F1, F2 and F3 fusion polypeptide was purified by reversed phase chromatography using a Eurospher C8 chromatography column.
25 μM alpha1-antitrypsin in PBS (phosphate buffered saline, 1 mM KH2PO4, 10 mM Na2HPO4, 105 mM NaCl, 2.7 mM KCl) were incubated at 37° C. with four to five times excess of the fusion polypeptide (e.g. 125 μM) for 24 hours. Using a longer incubation time did not result in the further formation of the protein complex. After the incubation the protein complex was separated from non-complexed serpin-finger polypeptide by size exclusion filtration. Fractions comprising molecules of approximately the same molecular weight were combined. The combined fractions were concentrated and a sample was applied to an SDS-PAGE gel. The individual bands on the SDS-PAGE gel were analyzed using the 1D-Image-Master (Amersham Bioscience), the fraction of the protein complex was quantified and molecular weight difference were determined. The activity of the combined and concentrated fractions was determined via BIAcore (see
All surface plasmon resonance measurements were performed on a BIAcore 3000 instrument (GE Healthcare Biosciences AB, Sweden) at 25° C. Chemically prepared HIV HR1 peptide (heptad repeat 1; Biotin-QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKD Q-NH2 (SEQ ID NO: 87)) was immobilized on a CM5 biosensor chip according to the manufacturer instructions (GE Healthcare Biosciences AB, Sweden). The fusion polypeptide was diluted to a concentration of 25 nM and injected over 5 minutes at a flow rate of 50 μl/min. Thereafter the protein complex obtained from different combined fractions was diluted into the same buffer to concentrations of 250 nM and 80 nM and injected over 5 minutes at a flow rate of 50 μl/min. Afterwards the sensor chip was regenerated for 1 minute with PBS, pH 8.0, 0.005% (v/v) Tween 20. Data analysis was performed with the BIAevaluation software (BIAcore, Sweden) (see
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10176617 | Sep 2010 | EP | regional |
This application is a continuation of International Application No. PCT/EP2011/065884, having an international filing date of 13 Sep. 2011, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. 119 to European Patent Application No. 10176617.8 filed 14 Sep. 2010.
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| Number | Date | Country | |
|---|---|---|---|
| 20140045742 A1 | Feb 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2011/065884 | Sep 2011 | US |
| Child | 13830255 | US |
