FACTOR IX VARIANTS AND USES THEREOF IN THERAPY

Abstract

This invention provides Factor IX variants, molecules comprising the variants, nucleic acids encoding the variants, compositions comprising the variants or the nucleic acids encoding the variants, and their use in methods for the modulation of hemostasis, for example in the prophylaxis or treatment of hemophilia B. The Factor IX variants have improved biological properties relative to other Factor IX variants and/or relative to wild-type Factor IX.

Claims

1. A molecule comprising a Factor IX variant polypeptide comprising histidine at a position corresponding to position 410 of wild-type Factor IX (SEQ ID NO: 1), and comprising an amino acid other than arginine at a position corresponding to position 338 of wild-type Factor IX.

2. The molecule of claim 1, comprising an amino acid selected from the group consisting of valine, threonine, and tryptophan at the position corresponding to position 338 of wild-type Factor IX.

3. (canceled)

4. (canceled)

5. The molecule of claim 1, wherein the Factor IX variant polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.

6. The molecule of claim 1, wherein the molecule further comprises a half-life enhancing portion, selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof, immunoglobulins without antigen binding domain, polyethylene glycol, a C-terminal peptide of human chorionic gonadotropin, and an unstructured recombinant polypeptide.

7. The molecule of claim 6, wherein the molecule further comprises a cleavable peptide linker between the Factor IX variant polypeptide and the half-life enhancing portion.

8. The molecule of claim 1, comprising the Factor IX variant polypeptide of SEQ ID NOs: 11, 12, or 13.

9. The molecule of claim 1, comprising the Factor IX variant polypeptide of SEQ ID NOs: 11, 12, or 13, the linker of SEQ ID NO: 8, and the half-life enhancing portion of SEQ ID NO: 9.

10. The molecule of claim 1, wherein the Factor IX variant polypeptide is an activated version of the Factor IX variant polypeptide.

11. A nucleic acid encoding the molecule of claim 1.

12. A vector comprising the nucleic acid of according to claim 11.

13. A cell comprising the nucleic acid of claim 11.

14. A pharmaceutical composition comprising the molecule of claim 1, and a pharmaceutically acceptable carrier.

15. (canceled)

16. A pharmaceutical composition comprising the nucleic acid of claim 11 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising the cell of claim 13 and a pharmaceutically acceptable carrier.

18. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the molecule of claim 1.

19. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the nucleic acid of claim 11.

20. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the cell of claim 13.

21. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 14.

22. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 16.

23. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 17.

Description

EXAMPLES

[0182] A series of exemplary recombinant FIX variant polypeptides were produced by mutating one or two amino acid positions in human wild-type FIX polypeptide (SEQ ID NO: 1) fused with a recombinant mature human albumin via a cleavable linker (IDELVION®/albutrepenonacog alfa, SEQ ID NO: 10). The recombinant FIX variants were expressed in HEK cells and the cell culture supernatant or purified proteins tested for activity and antigen. The activity to antigen ratios were compared to the corresponding polypeptide comprising wild-type FIX. FIX variants having certain mutations at positions 410 and 338 of wild-type FIX showed a surprisingly high activity, as demonstrated below.

Example 1

[0183] Generation of plasmid DNA, cell transfection and protein expression Plasmid DNA encoding Factor IX or Factor IX-albumin fusion polypeptides (FIX-FP) comprising either wild-type or variant FIX were generated according to standard techniques in the art. The mature wild-type Factor IX polypeptide sequence is shown in SEQ ID NO: 1. Exemplary Factor IX variant polypeptide sequences are shown in SEQ ID NO: 11-14. In particular, E (glutamic acid) at position 410 of wild-type Factor IX (SEQ ID NO: 1) was substituted with H (histidine) or K (lysine), and/or R (arginine) at position 338 of wild-type Factor IX (SEQ ID NO: 1) was substituted with V (valine), W (tryptophan), T (threonine), or L (lysine). Single and double mutants with mutations at positions 338 and/or 410 were generated. The linker and albumin of the Factor IX-FP were as defined in SEQ ID NOs 8 and 9, respectively.

[0184] However, other linkers and half-life enhancing portions can be used (as described in, e.g., reference 1), or they can be omitted.

[0185] Plasmid DNA was cloned in pcDNA3.1 vector and amplified in E. coli XL10-Gold Ultracompetent Cells (Agiland Technologies Cat No.:200315). Plasmid DNA was purified using standard protocols (QIAGEN Plasmid Plus Purification Cat No.: 12945, Hilden, Germany). Transient production of polypeptides was commenced in 250 ml scale, with the Expi293F expression kit (Cat. No. A14635, ThermoFisher). Viable Expi293TM cells in exponential growth phase were collected and re-suspended accordingly to obtain a starting cell density of 2.5×10.sup.6 cells/ml in 2 L shaker flasks (Corning, Lowell, Mass.). Separately, plasmid DNA (125 μg) and Expifectamin™ 293 reagent (675 μl) were diluted in 12.5 ml Opti-MEM® I Reduced serum medium. Diluted Expifectamin™ 293 reagent and plasmid DNA were mixed in equal parts. The complex was added to 225 ml of 62.5×10.sup.7 total viable cells in Expi293TM Expression medium. Expression medium was supplemented with 50 μg/ml Menadione K3 (Sigma Aldrich, Steinheim, Germany). Culture was incubated in an orbital shaker incubator at 37° C. (8% CO.sub.2, 150 rpm). After 17-20 hours, Enhancer I (1.25 ml) and Enhancer II (12.5 ml), which are part of the Expi293 Expression kit, were added to the culture. After a total culture time of 96 hours, the culture supernatant was harvested using appropriate sterile filter. Factor IX protein was then purified as explained in Example 2.

[0186] For experiments that used cell culture supernatants to measure Factor IX activity etc., the FIX-FP wild-type and FIX-FP variant polypeptides were expressed as described above, except that the culture volume was 50 ml and the culture supernatant from the transfected cells was collected at 48 hours. Factor IX activity was assessed in a one stage Factor IX specific clotting assay and antigen levels were determined with a Factor IX specific ELISA, as described below (Examples 3 and 4).

Example 2

[0187] Protein Purification

[0188] Cell culture supernatants containing Factor IX albumin fusion polypeptide, respective Factor IX polypeptide, as described in Example 1 above were applied on a Poros 50HQ column previously equilibrated with 20 mM Hepes, 50 mM NaCl and 12 mmol EDTA buffer pH 6.2. Subsequently, the column was washed with buffer containing 20 mM Hepes, 100 mM NaCl pH 6.2. Elution of the bound FIX fusion polypeptide was achieved by adding 10 mmol CaCl.sub.2 to the washing buffer.

Example 3

[0189] Determination of Factor IX Activity and Antigen

[0190] Factor IX activity was determined as clotting or coagulation activity (FIX:C) using commercially available aPTT reagents (Pathromtin® SL and FIX depleted plasma, Siemens Healthcare). An internal substandard calibrated against the WHO International FIX concentrate Standard was used as a reference.

[0191] Factor IX antigen (FIX:Ag) was determined by an ELISA according to standard protocols known to those skilled in the art. Briefly, microtiter plates were incubated with 1004 per well of the capture antibody (Paired antibodies for Factor IX ELISA (CL20041K), Cedarlane, but other sources of appropriate antibodies may also be applied) overnight at ambient temperature. After washing plates three times with washing buffer B (Sigma T9039) each well was incubated with 200 μL blocking buffer C (Sigma P3688) for one hour at ambient temperature. After another three wash steps with buffer B, serial dilutions of the test sample in buffer B as well as serial dilutions of a substandard (SHP) in buffer B (volumes per well: 100 μL) were incubated for 90 min. at ambient temperature. After three wash steps with buffer B, 100 mL of a 1:200 dilution of the detection antibody (Paired antibodies for Factor IX ELISA, peroxidase labelled, Cedarlane) in buffer B, were added to each well and incubated for another 90 min at ambient temperature. After three wash steps with buffer B, 100 μL of substrate solution (TMB, Siemens Healthcare, OUVF) were added per well and incubated for 30 minutes at ambient temperature in the dark. Addition of 100 μL undiluted stop solution (Siemens Healthcare, OSFA) prepared the samples for reading in a suitable microplate reader at 450 nm wavelength. Concentrations of test samples were then calculated using the standard curve with standard human plasma as reference.

Example 4

[0192] Comparison of Factor IX-Activity/Factor IX-Antigen Ratio of Factor IX Variants Relative to Wildtype

[0193] Factor IX activity and antigen were performed as described in Example 3 above. Factor IX variant specific activity was normalized to Factor IX antigen levels, measured via anti-Factor IX ELISA (ratio of FIX:C to FIX:Ag) to control for experimental variation, thereby representing a measure that is directly proportional to the molar specific activity of the different constructs. The resulting activity of wild-type Factor IX (in this example, IDELVION®, SEQ ID NO: 10) was assigned the value ‘1’. The activity of the Factor IX variants based on IDELVION® are indicated relative to the activity of wild-type Factor IX (IDELVION®).

[0194] Tables 1 and 2 below show the specific activity of various FIX-FP variants relative to wild-type FIX-FP, measured using cell culture supernatants containing the recombinantly expressed proteins (Table 1) or using purified protein (Table 2, 3 and 4).

TABLE-US-00002 TABLE 1 Specific activities of Factor IX variants relative to wild-type Factor IX (Idelvion ®, measured using supernatants. Specific activity relative Construct to wild-type FIX-FP FIX-FP Control (wild-type) 1.00 FIX-FP R338V + E410H 7.77 FIX-FP R338T + E410H 6.75 FIX-FP R338W + E410H 5.32 FIX-FP R338L + E410K 5.58 FIX-FP E410H 2.03 FIX-FP R338V 4.26 FIX-FP R338L 4.76 FIX-FP R338T 2.55

TABLE-US-00003 TABLE 2 Specific activities of Factor IX variants relative to wild-type Factor IX, measured using purified supernatants (anionic exchange). Specific activity relative Construct to wild-type FIX-FP FIX-FP Control (wild-type) 1.00 FIX-FP R338V + E410H 7.47 FIX-FP R338T + E410H 6.68 FIX-FP R338W + E410H 4.02 FIX-FP R338L + E410K 5.48 FIX-FP E410H 3.38 FIX-FP R338V 3.15 FIX-FP R338L 4.59

[0195] Tables 1 and 2 show that FIX-FP variants with certain mutations at positions 338 and/or 410 of wild-type Factor IX yielded greater specific activity than wild-type FIX-FP. Furthermore, double mutants with certain mutations at positions 338 and 410 of wild-type Factor IX (e.g. R338V+E410H, R338V+E410T) yielded greater specific activity than both the wild-type FIX-FP and each of the respective single mutants (e.g. R338V, R338T, E410H). Indeed, the activity of the double mutants can be more than additive (synergistic) relative to the respective single mutants. Additionally, the specific activity of the R338V+E410H, R338T+E410H and R338W+E410H double mutants was higher than the activity of the variant which corresponds to the Factor IX “Padua” mutant, R338L, see reference 35. The specific activity of the R338V+E410H and R338T+E410H double mutants was furthermore higher than the activity of the variant which corresponds to the “Padua” R338L+E410K double mutant.

[0196] In a further experiment, the specific activity of the R338L+E410K and R338L+E410H double mutants produced as described above was determined and compared to the activity of the corresponding R338L single mutant. Table 3 below shows that the R338L+E410H double mutant has a higher specific activity than the R338L single mutant, which itself has a higher specific activity compared to wild-type (as shown in Tables 1 and 2 above). The R338L+E410H double mutant is therefore another useful Factor IX variant.

TABLE-US-00004 TABLE 3 Specific activities of FIX-FP variants relative to R338L, measured using supernatants. Construct Specific activity relative to R338L FIX-FP Control (R338L) 1.00 FIX-FP R338L + E410H 2.10 FIX-FP R338L + E410K 1.34

[0197] Furthermore, Table 3 shows that the E410H mutation, when incorporated into a double mutant, results in an overall higher activity than the E410K mutation in the same double mutants. It will be understood by the skilled person that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

TABLE-US-00005 TABLE 4 Comparison of specific activities of Factor IX variants, relative to wildtype Factor IX measured using purified supernatants (anionic exchange). Construct Specific activity relative to wt-FIX Control FIX-wildtype 1.0 FIX R338V + E410H 6.48 FIX R338T + E410H 8.01

[0198] Table 4 shows, that the specific activity of the double mutants R338V+E410H and R338T+E410H expressed as Factor IX which is not fused to albumin is also higher than the Factor IX wildtype control. Thus, the improved specific activity of the Factor IX mutations of the invention is independent from the albumin fusion.

REFERENCES

[0199] [1] WO 2007/144173 [0200] [2] Beattie & Dugaiczyk (1982) Gene 20:415-422 [0201] [3] WO 2005/024044 [0202] [4] Lichenstein et al. (1994) J. Biol. Chem. 269:18149-18154 [0203] [5] Cooke & David (1985) J. Clin. Invest. 76:2420-2424 [0204] [6] WO 2005/001025 [0205] [7] Powell et al. (2013) N. Engl. J. Med., 369:2313-2323 [0206] [8] Peters et al. (2010) Blood 115:2057-2064 [0207] [9] Shapiro et al. (2012) Blood 119:666-672 [0208] [10] Fares et al. (1992) Proc Natl Acad Sci USA 15; 89(10):4304-4308 [0209] [11] Calo et al. (2015) Precision Medicine, 2, e989 [0210] [12] WO 2011/004361 [0211] [13] Schellenberger et al. (2009) Nature Biotechnology 27, 1186-1190 [0212] [14] WO 2017/024060 [0213] [15] WO 2012/006624 [0214] [16] WO 2015/106052 [0215] [17] Collins et al. (2014) Blood 124:3880-3886 [0216] [18] WO 2006/127896 [0217] [19] WO 2005/055950 [0218] [20] DeFrees et al. (2006) Glycobiology 16(9):833-843 [0219] [21] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [0220] [22] Qiang Wang et al. (2017) Blood December 2017, 130 (Suppl 1), 5562 [0221] [23] Anguela et al. (2013) Blood 122, 3283-3287 [0222] [24] Sharma et al. (2015) Blood 126, 1777-1784 [0223] [25] Barzel et al. (2015) Nature 517, 360-364 [0224] [26] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [0225] [27] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications) [0226] [28] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press) [0227] [29] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed., CRC Press, 1997) [0228] [30] Ausubel et al. (eds) (2002) Short protocols in molecular biology, 5th edition (Current Protocols). [0229] [31] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press) [0230] [32] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag) [0231] [33] Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30 [0232] [34] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489 [0233] [35] Simioni et al. (2009) N Engl J Med. October 22; 361(17):1671-1675

TABLE-US-00006 SEQUENCE LISTING >Human wild-type FIX polypeptide SEQ ID NO: 1 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT >Coding sequence for human wild-type FIX polypeptide SEQ ID NO: 2 ATGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATG GAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGC GGCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGA AAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGT AAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGA AAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCAC AAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTA CTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCA CTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTG AATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACT GCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATT GAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAA CTACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACC CTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACAT CTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGA GATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTC GATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGTA GAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGACCAG TTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAAT ATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTTAA >Human wild-type FIX polypeptide including signal peptide  and propeptide SEQ ID NO: 3 MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERE CMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFG FEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVS VSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVV LNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYN AAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQ YLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISW GEECAMKGKYGIYTKVSRYVNWIKEKTKLT >Coding sequence for human wild-type FIX polypeptide  including signal peptide and propeptide SEQ ID NO: 4 ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATCACCATCTGCCTTTT AGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCATGAAAACGCCAACAAAAT TCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAACC TTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAA ACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCC AATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGT CCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGC AGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGA GGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTG GAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATG TGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCC AATCATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTC CCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAAT GAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTC GCAGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTCG AATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTT CTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGA CAAGGAATACACGAACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAA GAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTG ACCGAGCCACATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTG GCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACT GAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAAT GAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGGAAA AAACAAAGCTCACTTAA >Human wild-type FIXa light chain polypeptide SEQ ID NO: 5 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTR >Human wild-type FIXa heavy chain polypeptide SEQ ID NO: 6 VVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEET EHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGY VSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSG GPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT >Human wild-type FIX polypeptide T148A polymorphic variant SEQ ID NO: 7 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT >Linker SEQ ID NO: 8 PVSQTSKLTRAETVFPDV >Mature human albumin SEQ ID NO: 9 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVD VMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLA ADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPH ECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFS ALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA AFVEKCCKADDKETCFAEEGKKLVAASQAALGL >FIX (wild-type) albumin fusion SEQ ID NO: 10 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTPVSQTSKLTRAETVF PDVDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESA ENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPE VDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDF AAFVEKCCKADDKETCFAEEGKKLVAASQAALGL >FIX variant R338V/E410H SEQ ID NO: 11 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLVSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKHKTKLT >FIX variant R338L/E410H SEQ ID NO: 12 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLTSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKHKTKLT >FIX variant R338W/E410H SEQ ID NO: 13 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLWSTKFTIYNNMFCAGFHEGGRDSCQGDSG GPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKHKTKLT >FIX variant R338L/E410H SEQ ID NO: 14 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLLSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKHKTKLT >FIX variant R338V/E410H albumin fusion SEQ ID NO: 15 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAE NQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV VGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV SGWGRVFHKGRSALVLQYLRVPLVDRATCLVSTKFTIYNNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKHKTKLTPVSQTSKLTRAETVF PDVDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESA ENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPE VDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPK LDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDF AAFVEKCCKADDKETCFAEEGKKLVAASQAALGL >Human IgG1 Fc SEQ ID NO: 16 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Human IgG1 Fc SEQ ID NO: 17 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CTP sequence SEQ ID NO: 18 SSSSKAPPPS >CTP sequence SEQ ID NO: 19 DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL >CTP sequence SEQ ID NO: 20 SSSSKAPPPSLPSPSRLPGPSDTPILPQ >XTEN artificial sequence SEQ ID NO: 21 GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGASS >XTEN artificial sequence SEQ ID NO: 22 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS