EA041874B1 - FACTOR VIII COMPOSITIONS AND METHODS FOR PRODUCING AND USE OF SIMILAR - Google Patents

Description

Cross-reference to related applications

The present patent application claims priority to US Provisional Application Serial No. 61/599400, filed February 15, 2012, which is incorporated herein by reference.

Sequence listing

This appendix contains a listing of sequences that have been presented in ASCII format using EFS-Web and are incorporated herein by reference in their entirety. The specified ASCII copy, created on July 11, 2012, is named 32887346.txt and has a size of 13344768 bytes.

State of the art

Factor VIII is an important component of the intrinsic pathway of the blood coagulation cascade. In the circulation, factor VIII mainly forms a complex with von Willebrand factor. After activation by thrombin (factor IIa), it dissociates from the complex to interact with factor IXa in the internal coagulation cascade, which in turn activates factor X. After removal of von Willebrand factor from the complex, activated factor VIII is proteolytically inactivated by activated protein C (APC), factor Xa and factor IXa, and are rapidly cleared from the circulation. When complexed with the normal von Willebrand factor protein, the half-life of factor VIII is approximately 12 hours, while in the absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham EG, et al., Br J Haematol. ( 1982) 52(2):259-267).

In hemophilia, blood clotting is impaired due to the absence of certain clotting factors in the blood plasma. Hemophilia is a factor VIII deficiency, and is a sex-linked recessive X-chromosome disorder that represents 80% of cases of hemophilia. The standard of care for the management of hemophilia A is replacement therapy with recombinant factor VIII concentrates. Subjects with severe hemophilia A have circulating levels of procoagulant factor VIII below 1-2% of normal, and are typically on prophylactic therapy to maintain factor VIII above 1% between doses, which is usually accomplished by providing factor VIII between two and three once a week. Individuals with moderately severe hemophilia (factor VIII levels are 2-5% of normal) account for 25-30% of hemophilia cases and exhibit bleeding after minor trauma. Individuals with mild hemophilia A (factor VIII levels are 5-40% of normal) account for 15-20% of all cases of hemophilia, and bleeding does not begin until after major trauma or surgery.

In vivo activity of exogenously provided factor VIII is limited both by the short protein half-life and by inhibitors that bind to factor VIII and reduce or suppress hemostatic function.

Up to 30% of patients with hemophilia receiving exogenous factor VIII increase an IgG immune response directed to factor VIII (Towfighi, F., et al. Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity. Acta Haematol (2005) 114:84-90), which can lead to complete inhibition of its procoagulant activity and/or provide more rapid excretion of factor VIII (Briet E et al. High titer inhibitors in severe haemophilia A. A meta -analysis based on eight-term follow-up studies concerning inhibitors associated with crude or intermediate purity factor VIII products Throm Haemost (1994) 72: 162-164. IgG antibodies, which are called FVIII inhibitors, primarily target the A2, A3, and C2 domains (Scandella D et al. Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood (1989) 74:1618-1626), but may also oppose the A1, B, and C1 domains. Thus, treatment options for patients with FVIII inhibitors are limited.

Large proteins such as factor VIII are typically administered intravenously so that the drug is directly available in the bloodstream. It has previously been shown that unmodified Factor VIII given intramuscularly results in a maximum circulating level of only 1.4% of normal plasma levels (Pool et al., Ineffectiveness of Intramuscularly Injected Factor VIII Concentrate in Two Hemophilic Patients. New England J. Medicine (1966) 275(10):547-548). Compositions that can be administered by other than intravenous route could greatly simplify their use, improve safety and lead to significant cost savings.

Chemical modification of a therapeutic protein can alter its in vivo elimination rate and subsequent serum half-life. One example of a general modification is the addition of a polyethylene glycol (PEG) moiety, typically in combination with a protein, via an aldehyde or N-hydroxysuccinimide (NHS) group by reacting the PEG with an amino group (eg, with a lysine side chain or N-terminus). Despite this, the conjugation step can lead to the formation of mixtures of heterogeneous products that require extraction, purification and/or other additional methods, all of which necessarily affect product yield and quality control. In addition, the pharmacological function of clotting factors may be hindered if the amino acid side chains in the immediate area of its binding site are modified by

- 1 041874 method of pegylation. Other approaches include the genetic fusion of the Fc domain to the therapeutic protein, which increases the size of the therapeutic protein, hence reducing the rate of excretion through the kidneys. In some cases, the Fc domain confers the ability to bind to and be processed from lysosomes by the FcRn receptor, resulting in an increased pharmacokinetic half-life. Unfortunately, during recombinant expression, the Fc domain does not fold efficiently and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodies must be solubilized and the functional protein must be renatured from the misfolded aggregate, which is laborious, inefficient and costly.

The essence of the invention

The present invention relates to novel coagulation factor VIII fusion protein compositions and uses thereof. In particular, the compositions provided herein are primarily used to treat or improve conditions associated with hemophilia A, factor VIII deficiencies, clotting disorders, and coagulopathy. In a separate aspect, the present invention provides isolated fusion protein compositions that contain factor VIII (FVIII) and one or more of the extended recombinant polypeptides (XTEN), while the fusion protein exhibits procoagulant activity. The XTEN subject used to construct such fusion proteins is typically a polypeptide with a non-repetitive sequence and an unstructured conformation. In a particular embodiment of the present invention, one or more XTENs are linked to a clotting factor FVIII (CF) selected from natural human factor VIII, factor VIII B-domain deleted sequences (FVIII BDD) and sequence variants thereof (all of the above together FVIII or CF), which results in the recombinant factor VIII-XTEN fusion protein (CFXTEN). The factor VIII CFXTEN polypeptide component comprises an A1 domain, an A2 domain, a C1 domain, a C2 domain, and optionally a B domain or a portion thereof. In certain embodiments of the present invention, FVIII is further characterized by transdifferentiating the aforementioned domains so that they contain the acidic spacer a1, a2 and a3. In an additional embodiment, the present invention relates to pharmaceutical compositions that contain hybrid proteins and their use in methods and regimens for the treatment of conditions associated with factor VIII. CFXTEN formulations have enhanced pharmacokinetic and pharmacological properties compared to non-XTEN-related FVIII, which may allow for more convenient dosing and improved efficacy.

In a first aspect, the present invention relates to recombinant factor VIII fusion proteins that comprise a factor VIII polypeptide and one or more factor VIII-linked extended recombinant polypeptides (XTEN). In certain embodiments, the present invention provides recombinant factor VIII fusion proteins that comprise a factor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain that contains an a1 acidic spacer, an A2 domain that contains an a2 acidic spacer, an A3 domain that contains an a3 acidic spacer, a C1 domain, a C2 domain, and optionally all or part of the B domain, and characterized in that said at least one XTEN is linked to said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) in the B domain of said factor VIII polypeptide if all or part of the B domain is present; (iii) in the A1 domain of said factor VIII polypeptide; (iv) in the A2 domain of said factor VIII polypeptide; (v) in the A3 domain of said factor VIII polypeptide; (vi) in the C1 domain of said factor VIII polypeptide; (vii) in the C2 domain of said factor VIII polypeptide; (viii) at the N-terminus of said factor VIII polypeptide, or (ix) between two domains of said factor VIII polypeptide, characterized in that the fusion protein retains at least about 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100, 200, 300, 400, or 500% procoagulant activity, as measured by an in vitro coagulation activity assay, compared to the corresponding non-XTEN factor VIII. In a separate embodiment of the present invention, in the previous recombinant factor VIII fusion protein, at least one XTEN is linked to said factor VIII polypeptide within a site, or within 1-6 amino acids of a site selected from Table 1. 5, tab. 6, tab. 7, tab. 8 and tab. 9. In additional embodiments, the present invention provides recombinant factor VIII fusion proteins that comprise a factor VIII polypeptide and at least a first extended recombinant polypeptide (XTEN) wherein said factor VIII polypeptide comprises an A1 domain that contains an acidic spacer a1, an A2 domain that contains an a2 acidic spacer, an A3 domain that contains an a3 acidic spacer, a C1 domain, a C2 domain, and optionally all or part of the B domain, and characterized in that said first XTEN is associated with said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) in the B domain of said factor VIII polypeptide if all or part of the B domain is present; (iii) in the A1 domain of said factor VIII polypeptide; (iv) in the A2 domain of said factor VIII polypeptide; (v) in the A3 domain of said factor VIII polypeptide; (vi) in the C1 domain of said factor VIII polypeptide; or (vii) in the C2 domain of said polypeptide

- 2 041874 factor VIII; and if, compared with the corresponding non-XTEN factor VIII protein, fusion protein (a) retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 200%, 300%, 400% or 500% procoagulant activity in the in vitro coagulant activity assay described herein or other similar assays known in the art, and/or (b) exhibit reduced binding to an anti-Factor VIII antibody in the in vitro binding assay described in this document, or other similar assays known in the art. In a further embodiment of the present invention, in the previous recombinant factor VIII fusion protein, at least one XTEN is linked to said factor VIII polypeptide within a site or within 1-6 amino acids of a site selected from Table 1. 5, 6, 7, 8, and 9. In additional embodiments, the present invention provides recombinant factor VIII fusion proteins that comprise a factor VIII polypeptide and at least a first extended recombinant polypeptide (XTEN), characterized in that said factor VIII polypeptide contains a domain A1, which contains the a1 acidic spacer, an A2 domain, which contains the a2 acidic spacer, an A3 domain, which contains the a3 acidic spacer, a C1 domain, a C2 domain, and optionally all or part of the B domain, and, characterized in that the specified first XTEN associated with the specified factor VIII polypeptide within the insertion site, selected from the table. 6 and tab. 7 and, characterized in that the fusion protein retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% procoagulant activity, as measured by in vitro coagulation activity assay described herein, or other similar assays known in the art, compared to the corresponding non-XTEN factor VIII protein. Non-limiting examples of factor VIII protein not associated with XTEN include native FVIII, BDD FVIII, pBC100, and sequences from Table. 1. In a further embodiment of the recombinant factor VIII fusion protein, the factor VIII polypeptide has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity with a sequence selected from the group consisting of sequences from table. 1, the sequence is illustrated in FIG. 3 and the sequence is illustrated in FIG. 4, with optimal alignment. In yet another further embodiment of the present invention, the fusion protein comprises at least another similar XTEN linked to said Factor VIII polypeptide at the C-terminus of said Factor VIII polypeptide, or within, or optionally replaces, the B domain of said Factor VIII polypeptide. In a specific embodiment of the present invention, the fusion protein contains at least one XTEN sequence that resides within, or optionally replaces, the B domain of said Factor VIII polypeptide. In a further specific embodiment of the present invention, the fusion protein at the C-terminus of said factor VIII polypeptide contains at least one XTEN sequence linked to said factor VIII polypeptide. In a specific embodiment of the present invention, the recombinant factor VIII fusion protein comprises a variant of human factor VIII without the B domain, with the deletion of the B domain beginning at the first position near amino acid residue 741 to about 750 and ending at the second position at amino acid residue c number 1635 to about 1648 relative to the full length human factor VIII sequence as illustrated in FIG. 3. In a further embodiment of the present invention, the recombinant factor VIII fusion protein comprises a first XTEN sequence linked to said factor VIII polypeptide at the C-terminus of said factor VIII polypeptide and at least a second XTEN within or replacing the B domain of said factor VIII polypeptide, characterized in that the second XTEN is linked to the C-terminal end near amino acid residue number 741 to about 750 and to the N-terminal end of amino acid residue number 1635 to about 1648 relative to the full-length human factor VIII sequence, as illustrated in FIG. . 3, characterized in that the total length of XTEN is at least about 100 amino acid residues. In a separate embodiment of the present invention, in the previous fusion protein, the second XTEN binds factor VIII amino acids between N745 and P1640, or between S743 and Q1638, or between P747 and V1642, or between N745 and Q1656, or between N745 and S1657, or between N745 and T1667, or between N745 and Q1686, or between R747 and V1642, or between T751 and T1667. In a specific embodiment of the present invention, the recombinant factor VIII fusion protein comprises a sequence that has at least about 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least least about 99%, up to about 100% sequence identity, compared with a sequence of comparable length, selected from the table. 21, with optimal alignment. In a further embodiment of the present invention, the recombinant factor VIII fusion protein comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally

- 3 041874 specifically a fifth XTEN and optionally a sixth XTEN, with each of the second, third, fourth, fifth, or sixth XTEN linked to said factor VIII polypeptide at the second, third, fourth, fifth, or sixth site selected from the group that consists of the insertion site from the table. 5, 6, 7, 8 and 9; location within 6 amino acids of amino acid residues 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 of mature factor VIII; location between any two adjacent domains of said Factor VIII polypeptide, wherein said two adjacent domains are selected from the group consisting of A1 and A2 domains, A2 and B domains, B and A3 domains, A3 and C1 domains, and C1 and C2; a location in the B domain of said factor VIII polypeptide, characterized in that the second XTEN is linked to the C-terminus near amino acid residue 741 to about 750 and to the N-terminus of amino acid residue 1635 to about 1648 of the natural factor VIII sequence ; and the C-terminus of said factor VIII polypeptide. In a specific embodiment of the present invention, the first XTEN is separated from the second XTEN by at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids. In a separate embodiment of a recombinant factor VIII fusion protein that contains at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN, and optionally a sixth XTEN, each XTEN has at least about 80% sequence identity , or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about

100% sequence identity, compared to XTEN of comparable length, selected from the group that consists of sequences from table. 4, tab. 13, tab. 14, tab. 15, tab. 16 and tab. 17, with optimal alignment. In yet another further embodiment of a recombinant factor VIII fusion protein that comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN, and optionally a sixth XTEN, In preferred embodiments of the present invention, the recombinant fusion protein Factor VIII exhibits a terminal half-life of at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to a subject, with said subject selected from a human and a mouse with two factor VIII/von Willebrand factor knockout genes. Additionally, in embodiments of the present invention under this item, the fusion protein exhibits reduced binding to an anti-factor VIII antibody, or retains a higher procoagulant activity, or both, compared to the corresponding non-XTEN-bound factor VIII. In a particular embodiment of the present invention, the procoagulant activity of the recombinant factor VIII fusion protein is at least 30%, or 40%, 50%, 80%, 100%, 200%, 300%, 400%, or 500% more procoagulant activity in the presence of an anti-FVIII antibody, compared to the corresponding Factor VIII not associated with XTEN, if each is analyzed by an in vitro coagulation activity assay. In a separate embodiment of the present invention, reduced fusion protein binding to an anti-factor VIII antibody is determined using the Bethesda assay, using an anti-factor VIII antibody selected from the group consisting of the antibodies in Table 1. 10 and a polyclonal antibody from a hemophilia A patient with factor VIII inhibitors, characterized in that reduced binding and retained procoagulant activity of the fusion protein is indicative of a lower Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15 , 20, 30, 40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion protein, compared with that for non-XTEN factor VIII.

In a particular embodiment of the present invention, the recombinant factor VIII fusion protein may, for example, comprise one or more XTENs, wherein the XTEN has at least about 80% sequence identity, or about 90%, or about 91%, or about 92% , or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity, compared to one or more XTEN comparable length, selected from the table. 4, 13, 14, 15, 16 and 17, with optimal alignment. In additional aspects, the present invention relates to recombinant factor VIII fusion proteins that contain FVIII and one or more XTEN in specific N- to C-terminal conformations. In a separate embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula I: (XTEN) _x -CF-(XTEN) _y I where independently for each occurrence, CF is factor VIII as defined herein, which contains sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% , or at least about 99% or 100% sequence identity with sequences from the table. 1; x is either 0 or

- 4 041874

1 and y is either 0 or 1, where x+y>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. Accordingly, the CFXTEN fusion composition may have the XTEN-CF, XTEN-CF-XTEN, or CF-XTEN conformations. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula II:

(XTEN)x-(S) _x -(CF)-(XTEN) _y II where CF is Factor VIII as defined herein, which contains sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequences, given in table. 1; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain cleavage sequences or amino acids compatible with restriction sites; x is either 0 or 1 and y is either 0 or 1, where x+y>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein, wherein the fusion protein has the formula III:

(XTEN)x-(S) _x -(CF)-(S) _y -(XTEN) _y III where CF is Factor VIII as defined herein, which contains sequences that are at least about 80% or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequence presented in table. 1; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain cleavage sequences or amino acids compatible with restriction sites; x is either 0 or 1 and y is either 0 or 1, where x+y>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula IV:

(Al)-(XTEN)u-(A2)-(XTEN)v-(B)-(XTEN) _w -(A3)-(XTEN)x-(Cl)-(XTEN) _y -(C2)-( XTEN)z IV where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; A3 is the A3 domain of FVIII; B is the B domain of FVIII, which may be a fragment or a splice variant of the B domain; C1 is the C1 domain of FVIII; C2 is the C2 domain of FVIII; v is either 0 or 1; w is either 0 or 1; x is either 0 or 1; y is either 0 or 1; y is either 0 or 1, provided that u+v+χ+y+z>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of Formula V:

(XTEN)t-(S) _a -(Al)-(S)b-(XTEN) _u -(S) _b -(A2)-(S) _c -(XTEN)v-(S)c-(B )-(S)d-(XTEN) _w -(S)d-(A3)(S) _e -(XTEN)x-(S) _e -(Cl)-(S)f-(XTEN) _y -( S)f-(C2)-(S) _g -(XTEN)z V where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; A3 is the A3 domain of FVIII; B is the B domain of FVIII, which may be a fragment or a splice variant of the B domain; C1 is the C1 domain of FVIII; C2 is the C2 domain of FVIII; S is a spacer region that has 1 to about 50 amino acid residues

- 5 041874 kov, which may optionally contain cleavage sequences or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that t + u + v + w + x + y + z >1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In an additional embodiment of formula V, the spacer region is a glycine or a sequence selected from table. 11 and 12.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula VI:

(XTEN)u-(S)a-(Al)-(S)b-(XTEN) _v -(S)b-(A2)-(S)c-(XTEN)w-(S)c-(A3 )-(S)d-(XTEN)x(S)d-(Cl)-(S)e-(XTEN) _y -(S)e-(C2)-(S)f-(XTEN) _z VI where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; A3 is the A3 domain of FVIII; C1 is the C1 domain of FVIII; C2 is the C2 domain of FVIII; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain cleavage sequences or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that u + v + w + x + y + z >1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In an additional embodiment of formula V, the spacer region is a glycine or a sequence selected from table. 11 and 12.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of Formula VII:

(SP)-(XTEN)x-(CS)x-(S)x-(FVIII_l-745)-(S) _y -(XTEN) _y -(S) _y -(FVIII_1640-2332)-(S) _z -(CS)z-(XTEN)z VII where SP is the signal sequence, preferably with the sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is the cleavage sequence shown in Table. 12, S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain amino acids compatible with restriction sites, FVIII_1-745 is residues 1-745 of FVIII and FVIII_1640-2332 is residues 1640 -2332 FVIII, x is either 0 or 1, y is either 0 or 1, and z is either 0 or 1, where x+y+z>2; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity of the sequences shown in table. 4. In a separate embodiment of Formula VII, the spacer region is GPEGPS (SEQ ID NO: 1612). In an additional embodiment of Formula V, the spacer region is a glycine or a sequence selected from Table 1. 11 and 12.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula VIII:

(Al)-(S)a-(XTEN) _v -(S)a-(A2)-(Bl)-(S)b-(XTEN) _w -(S)b-(B2)-(A3)- (S)c-(XTEN)x-(S)c(Cl)-(S)d-(XTEN) _y -(S)d-(C2)-(S)e-(XTEN) _;

VIII where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; B1 is a fragment of the B domain, which may be from residue 741 to 743-750 FVIII, or alternatively from about residue 741 to about residue 745 FVIII; B2 is a fragment of the B domain, which may be from residues 1635-1686 to 1689 FVIII, or alternatively from about residue 1640 to about residue 1689 FVIII; A3 is the A3 domain of FVIII; C1 is the C1 domain of FVIII; C2

- 6 041874 is the C2 domain of FVIII; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain cleavage sequence(s) or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that u + v + w + x + y + z >1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In a separate embodiment of formula VIII, the spacer region is GPEGPS (SEQ ID NO: 1612). In an additional embodiment of Formula V, the spacer region is a glycine or a sequence selected from Table 1. 11 and 12. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula IX:

(Al _N )-(S) _a -(XTEN)t-(S)b-(Alc)-(A2N)-(S)c-(XTEN)u-(S)d-(A2c)-(BN) -(S)e-(XTEN)v-(S)f-(Bc)(A3 _N )-(S)g-(XTEN) _w -(S)h-(A3c)-(ClN)-(S) i-(XTEN) _x -(S) _j -(Clc)-(C2 _N )-(S) _k -(XTEN) _y -(S)i(C2c)-(S) _m -(XTEN)z IX where A1N is a fragment of the A1 domain from a residue with a number of at least 1 (numbering relative to native, mature FVIII) to a residue with a number of no more than 371, A1 _c is a fragment of the A1 domain from a residue with a number of at least 2 to a residue with a number no more than 372, with the condition that the sequence of the fragment A1N, duplicated in A1 _c , is not a fragment; A2N is a fragment of the A2 domain from a residue with a number of at least 373 to a residue with a number of no more than 739, A2 _c is a fragment of the A2 domain from a residue with a number of at least 374 to a residue with a number of no more than 740, with the condition, that the sequence of fragment A2N, duplicated in A2 _c , is not a fragment; B _N is a fragment of domain B from a residue with a number of at least 741 to a residue with a number of no more than 1647, B _c is a fragment of domain B from a residue with a number of at least 742 to a residue with a number of no more than 1648, with the condition that the sequence BN duplicates the fragment in Bc is not a fragment; A3N is a fragment of the A3 domain from a residue with a number of at least 1649 to a residue with a number of no more than 2019, A3 _c is a fragment of the A3 domain from a residue with a number of at least 1650 to a residue with a number of no more than 2019, with the condition, that the fragment sequence A3 _N duplicated in A3 _c is not a fragment; C1N is a fragment of the C1 domain from a residue with a number of at least 2020 to a residue with a number of no more than 2171, C1 _c is a fragment of the C1 domain from a residue with a number of at least 2021 to a residue with a number of no more than 2172, with the condition, that the C1N fragment sequence duplicated in C1 _c is not a fragment; C2N is a fragment of the C2 domain from a residue with a number of at least 2173 to a residue with a number of no more than 2331, C2c is a fragment of the C2 domain from a residue with a number of at least 2174 to a residue with a number of no more than 2332, with the proviso that the C2N fragment sequence duplicated in C2c is not a fragment; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain cleavage sequences or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; l is either 0 or 1; m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that t + u + v + w + x + y + z >1; and XTEN is an extended recombinant polypeptide as described herein that contains, but is not limited to, sequences that have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity, compared to one or more comparable XTEN length selected from the table. 4. In a separate embodiment of formula IX, the spacer region is GPEGPS (SEQ ID NO: 1612). In an additional embodiment of Formula V, the spacer region is a glycine or a sequence selected from Table 1. 11 and 12. In a further embodiment of Formula IX, Z is 1. In a further embodiment of the Formula IX fusion protein, V is 1 and XTEN

- 7 041874 is linked to the C-terminal end of amino acid residue numbers from about 741 to about 750 and to the N-terminal end of amino acid residue numbers from 1635 to about 1648 relative to the full-length human factor VIII sequence, as illustrated in FIG. 3. In a further embodiment of the Formula IX fusion protein, the sum of t, u, v, w, x, y, and z is equivalent to 2, 3, 4, 5, or 6. In a further embodiment of Formula IX, the sum of t, u, v, w , x, y, and z is equivalent to 2, v is 1, and z is 1. In a further embodiment of the Formula IX fusion protein, the sum of t, u, v, w, x, y, and z is equivalent to 3, each of v and z is equivalent to 1, and either t, u, w, x, or y is 1. In a further embodiment of formula IX, the sum of t, u, v, w, x, y, and z is equivalent to 4, each of v, w, and z is equivalent to 1 , and two of t, u, x, or y are 1. In a further embodiment of the Formula IX fusion protein, the total length of XTEN is from about 84 to about 3000 amino acid residues. In a further embodiment of formula IX, at least one XTEN is administered directly downstream of amino acids that correspond to an amino acid in mature natural human factor VIII selected from the group consisting of amino acid residue numbers 32, 220, 224, 336, 339 , 399, 416, 603, 1656, 1711, 1725, 1905, and 1910. In an additional embodiment of the Formula IX fusion protein, each XTEN is associated with the specified fusion protein within the sites selected from Table. 5, tab. 6, tab. 7, tab. 8 and tab. 9. In a further embodiment of the Formula IX fusion protein, each XTEN has at least about 80%, or about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100 % sequence identity, compared with XTEN of comparable length, selected from the group, which consists of sequences from table. 4, 13, 14, 15, 16 and 17, with optimal alignment.

In a further embodiment of the CFXTEN composition, the present invention provides a first recombinant Factor VIII polypeptide of formula X:

(Al) - al - (A2) - a2 - [B] X and a second polypeptide that contains the formula XI:

a3 - (AZ) - (Cl) - (C2) XI where the first polypeptide and the second polypeptide are fused or exist as a heterodimer; where A1 is the A1 domain of factor VIII; A2 is the A2 domain of factor VIII; [B] is the B domain of factor VIII, a fragment thereof, or is deleted; A3 is the A3 domain of factor VIII; C1 is the C1 domain of factor VIII; C2 is the C2 domain of factor VIII; a1, a2 and a3 are acidic spacer sites; where the domain A1 contains the region of the XTEN loop available for embedding-1 (A1-1) and the region of the XTEN loop available for embedding-2 (A1-2); where the A2 domain contains the XTEN loop region available for embedding-1 (A2-1) and the XTEN loop region available for embedding-2 (A2-2); where the A3 domain contains the region of the XTEN loop available for embedding-1 (A3-1) and the region of the XTEN loop available for embedding-2 (A3-2); where the sequence XTEN is introduced into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1 or A3-2; and while the recombinant factor VIII protein exhibits procoagulant activity. In a separate heterodimer embodiment, the first polypeptide and the second polypeptide from the same polypeptide chain comprise the formula (A1) - a1 - (A2) - a2 - [B] - [a3] - (A3) - (C1) - (C2). In a specific embodiment of the above, fused means a peptide bond.

In a further embodiment of the CFXTEN composition, the present invention provides a first recombinant Factor VIII polypeptide of formula X:

(Al) - al - (A2) - a2 - [B] X and a second polypeptide that contains the formula XI:

a3-(AZ)-(C1)-(C2) XI wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; where A1 is the A1 domain of factor VIII; A2 is the A2 domain of factor VIII; [B] is the B domain of factor VIII, a fragment thereof, or is deleted; A3 is the A3 domain of factor VIII; C1 is the C1 domain of factor VIII; C2 is the C2 domain of factor VIII; a1, a2 and a3 are acidic spacer sites; where the sequence XTEN is entered into a3; and wherein the recombinant factor VIII protein exhibits procoagulant activity. In a separate embodiment of the heterodimer, the first polypeptide and the second polypeptide from the same polypeptide chain comprise the formula (A1) - a1 - (A2) - a2 - [B] - [a3] - (A3) - (C1) - (C2). In a particular embodiment of the foregoing, fused means a peptide bond.

In embodiments of the above formulas, X and XI polypeptides, insertable XTEN loops are contained within conformationally labile open surface loop structures, and are characterized in that A1-1 is located between beta strand 1 and beta strand 2, A12 is located between beta strand 11 and beta strand 12, A2-1 is located between beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-1 is located between beta strand

- 8 041874 and beta-strand 39, A3-2 is located between beta-strand 45 and beta-strand 46, in accordance with the secondary structure of mature factor VIII, which is saved with accession number 2R7E of the DSSP database. In additional embodiments of the above polypeptide formulas X and XI, the conformationally labile open surface loop structure that contains A1-1 corresponds to a region in natural mature human factor VIII from amino acid about 15 to amino acid about 45. In additional embodiments of the above polypeptide formulas X and XI A1-1 corresponds to a region in natural mature human factor VIII from about amino acid 18 to about amino acid 41. natural mature human factor VIII from about amino acid 201 to about amino acid 232. In additional embodiments of the above polypeptide formulas X and XIA1-2 corresponds to a region in natural mature human factor VIII from about amino acid 218 to about amino acid 229. In addition In additional embodiments of the above polypeptide formulas X and XI, the conformationally labile open surface loop structure that contains A2-1 corresponds to a region in natural mature human factor VIII from about amino acid 395 to about amino acid 421. In further embodiments of the above polypeptide formulas X and XI A2-1 corresponds to a region in natural mature human factor VIII from about amino acid 397 to about amino acid 418. Human VIII from about amino acid 577 to about amino acid 635. In additional embodiments of the above polypeptide formulas X and XI, A2-2 corresponds to a region in natural mature human factor VIII from about amino acid 595 to about amino acid 607. In addition In further embodiments of the above polypeptide formulas X and XI, the conformationally labile open surface loop structure that contains A3-1 corresponds to a region in natural mature human factor VIII from about amino acid 1705 to about amino acid 1732. In further embodiments of the above polypeptide formulas X and XI A3-1 corresponds to a region in natural mature human factor VIII from about amino acid 1711 to about amino acid 1725. mature human factor VIII from about amino acid 1884 to about amino acid 1917. In additional embodiments of the above polypeptide formulas X and XI, A3-2 corresponds to a region in natural mature human factor VIII from about amino acid 1899 to about amino acid 1911 In additional embodiments of the above polypeptide formulas X and XI, the XTEN sequence is inserted into at least two of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2. In additional embodiments of the above polypeptide formulas X and XI, the XTEN sequence is inserted directly downstream of amino acids that correspond to an amino acid in mature natural human factor VIII selected from the group consisting of amino acid residue numbers 32, 220, 224, 336, 339 , 399, 416, 603, 1656, 1711, 1725, 1905, and 1910. In additional embodiments of the above formulas X and XI polypeptides, an additional XTEN sequence is introduced into the acidic region of spacer a3. In additional embodiments of the above polypeptide formulas X and XI, the amino acid corresponding to amino acid 1656 is inserted into the additional XTEN sequence directly into the acidic spacer a3 in the forward direction. -1 (A1-1), and an XTEN loop region available for insertion-2 (A1 -2), characterized in that domain A2 contains an XTEN loop region available for insertion-1 (A2-1) and an XTEN loop region embed-2 accessible (A2-2), and characterized in that the A3 domain contains an embed-in-1 accessible XTEN loop region (A3-1) and an embed-in-2 accessible XTEN loop region (A3-2) , and characterized in that the additional XTEN sequence is introduced into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1 or A3-2. In additional embodiments of the above polypeptide formulas X and XI, an amino acid is inserted directly into the additional sequence of XTEN in the forward direction, which corresponds to an amino acid in mature natural human factor VIII, selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905, and 1910. In previous embodiments of formula X and XI polypeptides, the fusion protein exhibits at least about 30, 40, 50, 60, 70, or 80, or 90% procoagulant activity of the corresponding factor VIII, not associated with XTEN, characterized in that the procoagulant activity is analyzed using an in vitro coagulation activity assay.

In all embodiments of the present invention, the polypeptide may, for example, exhibit an in vitro procoagulant activity that is greater than 0.5 IU/mL, or 1.0, or 1.5, or 2.0 IU/mL, when expressed in cell culture medium. and analyzed using an in vitro coagulation assay

- 9 041874 activities. Procoagulant activity can be measured using a chromogenic assay, a one-step coagulation activity assay (eg, aPTT), or both.

In certain embodiments of the present invention, characterized in that the recombinant factor VIII fusion protein comprises factor VIII and at least the first and second XTEN, with at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids distinguish at least the first XTEN from at least the second XTEN.

In preferred embodiments of the present invention, a recombinant factor VIII fusion protein that comprises factor VIII, at least a first XTEN and optionally at least a second, or optionally at least a third, or optionally at least a fourth XTEN, the fusion protein exhibits reduced binding to the anti-factor VIII antibody, compared to the corresponding non-XTEN-bound factor VIII. Reduced binding is assessed using either an in vivo or in vitro assay. In a specific embodiment of the present invention, the in vitro assay is an ELISA assay wherein the binding of the anti-FVIII antibody to the fusion protein is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or at least at least about 40% or more compared to FVIII not associated with XTEN. In a further embodiment of the present invention, the in vitro assay is a Bethesda assay, with reduced binding of the fusion protein evidenced by a lower Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion protein compared to non-XTEN factor VIII. In in vitro assays, the anti-factor VIII antibody is selected from the antibody in Table. 10 and a polyclonal antibody from a hemophilia A patient with factor VIII inhibitors. In specific embodiments, a recombinant factor VIII fusion protein that comprises factor VIII and at least a first and second XTEN that exhibits reduced binding to a factor VIII inhibitor antibody, the first XTEN is linked to said factor VIII polypeptide in the C2 domain of said factor VIII polypeptide, and the second XTEN is associated with said factor VIII polypeptide within the A1 or A2 domain of said factor VIII polypeptide, characterized in that said fusion protein exhibits reduced binding to a factor VIII inhibitor antibody compared to the corresponding non-XTEN-bound factor VIII, wherein that the factor VIII inhibitor antibody is capable of binding to an epitope that is located within the A1, A2 or C2 domain, and furthermore that the fusion protein exhibits procoagulant activity. In a separate embodiment of the above fusion protein, the second XTEN is linked to said factor VIII polypeptide in the A2 domain of the factor VIII polypeptide and the factor VIII inhibitor antibody is linked to the A2 domain of the factor VIII polypeptide. In a further embodiment of the above fusion protein, the second XTEN is linked to said factor VIII polypeptide in the C2 domain of the factor VIII polypeptide and the factor VIII inhibitor antibody is linked to the C2 domain of the factor VIII polypeptide. The binding of an anti-factor VIII antibody to the fusion protein is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, compared to the corresponding non-XTEN-bound factor VIII , provided that analyzed using the analysis of ELISA, given that the anti-factor VIII antibody is selected from the group that consists of antibodies in table. 10 and a hemophilia A subject polyclonal antibody with factor VIII inhibitors. The previous fusion proteins may further comprise at least three XTENs, characterized in that at least a third XTEN is bound to factor VIII within a site selected within or replacing domain B, at the C-terminus, and at, or within, 1 , 2, 3, 4, 5 or 6 amino acids of the insertion site selected from the table. 7 or table. 9. In embodiments with reduced binding to anti-factor VIII antibody, the fusion protein has a higher procoagulant activity in the presence of anti-FVIII antibody by at least 10, 20, 30, 40, 50, 80, 100, 200, 300, 400 or 500% or more compared to the corresponding non-XTEN factor VIII when analyzed by an in vitro coagulation activity assay (eg, chromogenic or one-step coagulation activity assay).

In all embodiments of the present invention, the XTEN of the fusion protein may, for example, be characterized in that the XTEN contains at least 36, or at least 42, or at least 72, or at least 96, or at least 144, or at least 288, or at least 400, or at least 500, or at least 576, or at least 600, or at least 700, or at least 800, or at least 864, or at least 900 or at least 1000 or at least 2000 or up to about 3000 amino acid residues or even more residues; the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) residues is at least about 80%, or at least about 90%, or at least at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the total XTEN amino acid residues; XTEN is essentially non-repetitive such that (i) XTEN does not contain three contiguous amino acids that are identical if the amino acids are not serine; (ii) at least about 80% of the XTEN sequence consists of non-overlapping motifs in sequence

- 10 041874 steps, each of the sequence motifs contains from about 9 to about 14 or about 12 amino acid residues, which consist of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine ( T), glutamate (E) and proline (P), characterized in that any two adjacent amino acid residues occur no more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has less than 10 subsequences; XTEN has greater than 90%, or greater than 95%, or greater than 99% random spiral formation as determined by the GOR algorithm; XTEN has less than 2% alpha helices and 2% beta sheets as determined by the Chou-Fasman algorithm; XTEN needs a predictive T-cell epitope if analyzed by the TEPITOPE algorithm, characterized in that the TEPITOPE cut-off value in case of said prediction using said algorithm has a threshold of -9, and said fusion protein exhibits a finite half-life that is longer than at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours , or at least about 144 hours, or at least about 21 days or more. In a particular embodiment of the present invention, the recombinant factor VIII fusion protein comprises at least a second, or at least a third, or at least a fourth XTEN, which may be identical to or different from other XTENs. In another approach, at least each of the first, at least the second, or at least the third, or at least the fourth XTEN of the CFXTEN fusion protein has at least about 80% sequence identity, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity, by compared with one or more XTEN of comparable length, selected from the table. 4, tab. 13, tab. 14, tab. 15, tab. 16 and tab. 17, with optimal alignment. In another different approach, at least each of the first, at least the second, or at least the third, or at least the fourth XTEN of the CFXTEN fusion protein has at least 90%, or about 91%, or about 92%, or about 93% or about 94% or about 95% or about 96% or about 97% or about 98% or about 99% or up to about 100% sequence identity compared to a sequence selected from AE42_1, AE42_2 , AE42_3, AG42_1, AG42_2, AG42_3, AG42_4, AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, AG144_F, AG144_3, AG144_4, AE288_1, AE288_2, AG288_1 and AG288_2.

In a separate embodiment of the present invention, the factor VIII component of the recombinant factor VIII fusion protein CFXTEN contains one, two or three amino acid substitutions selected from residues R1648, Y1680 and R1689, numbering relative to mature human factor VIII, characterized in that the substitutions are selected from alanine, glycine and phenylalanine. Non-limiting examples of these substitutions include R1648A, Y1680F, and R1689A.

In a further embodiment of the present invention, the CFXTEN fusion protein exhibits an average molecular weight ratio of at least about 1.3, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about 10, as measured by gel permeation chromatography or a comparable method.

In certain embodiments of the CFXTEN fusion protein, one or more XTENs are fused to FVIII via one or two cleavage sequences, each of which is cleaved by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, MMP-12, MMP13, MMP-17, and MMP-20, wherein cleavage at the cleaved sequence with a mammalian protease releases sequence factor VIII from the XTEN sequence, and while the released factor VIII sequence exhibits an increase in procoagulant activity compared to the uncleaved fusion protein. In a separate embodiment of the present invention, the cleavage sequence(s) are cleaved with factor XIa.

According to another approach, CFXTEN fusion proteins contain at least three XTENs located at different locations on the factor VIII polypeptide, with said different locations being selected from: the location of the insertion at or within 1 to 6 amino acids from a site selected from tab. 5, tab. 6, tab. 7, tab. 8 and tab. 9; location at, or within, 1 to 6 amino acids of amino acid residue 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905, and 1910 of mature factor VIII; location between any two adjacent domains in Factor VIII sequences, with said two adjacent domains selected from the group consisting of A1 and A2, A2 and B, B and A3, A3 and C1, and C1 and C2; location within an intraclonal deletion of the B domain starts at position one from amino acid residue number 741 to about 750 and ends at position two at amino acid residue number 1635 to about 1648,

- 11 041874 to the full length human factor VIII sequence as illustrated in FIG. 3 and the C-terminus of the factor VIII sequence, with the combined length of several XTENs being at least about 100 to about 3000 amino acid residues and the fusion protein retaining at least about 30%, or about 40%, or about 50 %, or about 60%, or about 70%, or about 80%, or about 90% of procoagulant activity compared to the corresponding non-XTEN factor VIII, with procoagulant activity assayed by an in vitro coagulation activity assay . In a specific embodiment of the above, the fusion protein exhibits a prolonged terminal half-life when administered to a subject, compared to a corresponding factor VIII polypeptide that lacks said XTEN, wherein said fusion protein exhibits a terminal half-life of at least about 3 hours, or 4 h, or 6 h, or 12 h, or 13 h, or 14 h, or 16 h, or 24 h, or 48 h, or 72 h, or 96 h, or 120 h, or 144 h, or 7 days, or 14 days, or 21 days when administered to a subject. In a specific embodiment of the present invention, the subject is selected from the group consisting of a human and a mouse with two factor VIII/von Willebrand factor gene knockouts. In a separate embodiment of the above, the fusion protein does not contain a sequence selected from GTPGSGTASSSP(SEQ

ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34), and GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS

EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59).

In a further embodiment of the above, the fusion protein does not contain XTEN sequences that consist of GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS

EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59),

PGS SPS ASTGTGPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGATGSPGS SPS ASTGTGPG

ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS

PGTSSTGSPGTPGSGTASSS (SEQ ID NO: 71), or

PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATTGSPGTPGSGTASSSPG

SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATTGSPGSSPSASTGTGPGSS

PS ASTGTGPGASPGTS STGSPGTPGSGTAS S SPGS STPSGATGSPGS SPS ASTGTGPGS SPS

ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS

STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS (SEQ ID NO: 80).

In a further aspect, the present invention relates to CFXTEN fusion proteins with improved pharmacokinetic properties, which contain improved parameters compared to non-XTEN FVIII, while the improved properties include, but are not limited to, a longer terminal half-life, greater area under the curve, increased time that blood concentration remains within the therapeutic window, increased time between successive doses results in blood concentrations within the therapeutic window, and a reduced dose in ME over the time it is administered, compared to non-XTEN-related FVIII and still results in blood concentrations above the threshold concentration required for a procoagulant effect. In certain embodiments, the CFXTEN fusion proteins exhibit a prolonged terminal half-life when administered to a subject, compared to the corresponding factor VIII polypeptide that lacks said XTEN. The subject may be a human or a mouse, such as a factor VIII/von Willebrand factor knockout mouse. In a specific embodiment of the above, CFXTEN, when administered to a subject, exhibits a terminal half-life that is at least about two times, or about three times, or about four times, or about five times, or about 10 times, or about 20 times longer than with the corresponding factor VIII, not associated with XTEN. In a particular embodiment of the present invention, the CFXTEN fusion protein exhibits a terminal half-life of at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours. h or 72 h or 96 h or 120 h or 144 h or 7 days or 14 days or 21 days when administered to a subject. In additional embodiments of the present invention, the improved pharmacokinetic property of the fusion proteins of an embodiment of the present invention is the property of maintaining the circulating blood concentration of the procoagulant fusion protein in a subject in need thereof above a threshold concentration of 0.01 IU/mL, or 0.05 IU/ ml or 0.1 IU/ml or 0.2 IU/ml or 0.3 IU/ml or 0.4 IU/ml or 0.5 IU/ml for a period that is at least about twice, or at least about three times, or at least about four times, or

at least about five times, or at least about six times, or at least about eight times, or at least about ten times, or at least about 20 times, or at least about 40 times , or at least about 60 times longer than the corresponding non-XTEN FVIII, and administered to the subject at a comparable dose. The increase in half-life and time spent above the threshold concentration allows for less frequent dosing and reduced amounts of fusion protein (substance equivalent) administered to a subject compared to the corresponding non-XTEN FVIII. In a separate embodiment of the present invention, administration of the inventive fusion protein to a subject using a therapeutically effective dosing regimen results in a gain in time of at least two times, or at least three times, or at least four times, or at least five times, or at least six times, or at least eight times, or at least 10 times, or at least about 20 times, or at least about 40 times, or at least about 60 times or more between at least two consecutive peaks of Cmax and/or lowest Cmin for blood levels of the fusion protein compared to the corresponding non-XTEN FVIII and administered to the subject using a similar dosing regimen.

In preferred embodiments of the present invention, the CFXTEN fusion proteins retain at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% procoagulant activity compared to with the appropriate non-XTEN factor VIII, while procoagulant activity is assayed by an in vitro coagulation activity assay, such as, but not limited to, a chromogenic assay or a one or two step coagulation assay.

According to another approach, the present invention provides recombinant factor VIII fusion proteins that comprise a factor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain, an A2 domain, an A3 domain, a C1, a C2 domain, and optionally all or part of a B domain, wherein said at least one XTEN is linked to said Factor VIII polypeptide at an insertion site selected from residue 18-32 or 40 or 211 -224 or 336-403 or 599 or 745-1640 or 1656-1728 or 1796-1804 or 1900-1912 or 2171-2332; and wherein the fusion protein retains at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of procoagulant activity compared to the corresponding factor VIII , not associated with XTEN. In a specific embodiment of the above, the fusion protein comprises at least a second XTEN, or at least a third or at least a fourth XTEN, wherein the XTEN is bound to Factor VIII within a site, or within 1-6 amino acids of a site selected from tab. 5, tab. 6, tab. 7, tab. 8 and tab. 9. In a further embodiment, the present invention provides a recombinant factor VIII fusion protein that further comprises at least a second XTEN or at least a third or at least a fourth XTEN associated with said FVIII polypeptide within an insertion site selected from Table . 5, tab. 6, tab. 7, tab. 8, tab. 9 at or within 6 amino acids to the N- or C-terminal side of the insert location at one or more of the insert locations of FIG. 8 and within one or more of the insertion ranges of FIG. 9, with at least two XTENs separated by an amino acid sequence of at least 100 to about 400 amino acids.

The present invention provides CFXTEN wherein XTEN has XTEN ratio limits of at least 2.3 or at least 2.5 and is separated by an amino acid sequence of at least about 20 amino acid residues, or at least about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400 amino acid residues. In additional embodiments of the present invention, the CFXTEN is at least four XTENs, while having XTEN ratio limits of at least 2.3, or at least 2.5, or at least 2.8, and while at least three of the four XTENs associated with the fusion protein are separated by an amino acid sequence of at least about 20 amino acid residues, or at least about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400 amino acid residues, and the fourth XTEN is bound within the B domain (or fragment thereof) or within the C domain (or end thereof).

In certain embodiments of the present invention, the claimed compositions are designed to have a reduced binding affinity for an excretion receptor in a subject, compared to the corresponding FVIII not bound to XTEN. In a specific embodiment of the present invention, the CFXTEN fusion protein exhibits a binding affinity for the FVIII clearance receptor in the range of about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or from about 2% to about 10% binding affinity of the corresponding FVIII not bound to XTEN. In a further embodiment of the present invention, the affinity reduced fusion protein in the case of the excretion receptor has reduced active clearance and

- 13 041874 a corresponding increase in half-life of at least about 2 times, or 3 times, or at least 4 times, or at least about 5 times, or at least about 6 times, or at least about 7 times, or at least about 8 times, or at least about 9 times, or at least about 10 times, or at least about 12 times, or at least about 15 times, or at least about 17 times, or at least about 20 times longer than the corresponding FVIII that is not associated with XTEN.

In a separate embodiment of the present invention, a recombinant factor VIII fusion protein is provided that comprises FVIII and one or more XTENs, wherein the fusion protein exhibits a solubility increased by at least three times, or at least about four times, or at least about five times, or at least about six times, or at least about seven times, or at least about eight times, or at least about nine times, or at least about ten times times, or at least about 15 times, or at least 20 times, or at least 40 times, or at least 60 times under physiological conditions, compared to FVIII not associated with XTEN.

In an additional aspect, the present invention provides a pharmaceutical composition that contains a fusion protein according to any of the embodiments of the present invention described herein and a pharmaceutically acceptable carrier.

In a further embodiment, the present invention provides a method for treating coagulopathy in a subject, which comprises administering to said subject a composition that contains a clotting effective amount of a pharmaceutical composition. In a specific embodiment of the method, after said administration, the blood concentration of procoagulant factor VIII is maintained at about 0.05, or 1, or 1.5 IU/ml or more, for at least 48 hours after said administration. In a further embodiment, the present invention provides a method for clotting blood in a subject, which comprises contacting a clotting effective amount of a pharmaceutical composition with blood.

In a further embodiment, the present invention provides a method for treating coagulopathy in a subject with circulating factor VIII inhibitors, which comprises administering to said subject a composition that contains a therapeutically effective amount of a pharmaceutical composition of CFXTEN, wherein the composition exhibits greater procoagulant activities in said subject, compared with a composition that contains the corresponding factor VIII, not associated with XTEN, and administered using a similar amount. In a separate embodiment of the method, the coagulopathy is hemophilia A. In a further embodiment of the present invention, the coagulopathy is the result of trauma, surgery, or infection.

The present invention provides a method of treating a bleeding event in a subject which comprises administering to said subject a composition that contains a clotting effective amount of a CFXTEN pharmaceutical composition, wherein the clotting effective amount of the fusion protein stops the bleeding event for a period that is at least three times, or at least four times, or at least five times longer than the corresponding non-XTEN factor VIII, which is administered using a similar amount to the specified subject. Non-limiting examples of relevant factor VIII, not associated with XTEN, contain natural FVIII, the sequence of the table. 1, BDDFVIII and pCB0114 FVIII.

In a further embodiment, the present invention provides a recombinant factor VIII CFXTEN fusion protein for use in a pharmaceutical regimen for treating a patient with hemophilia A, said regimen comprising a pharmaceutical composition that contains the CFXTEN fusion protein. In a separate embodiment of the pharmaceutical regimen, the regimen further comprises the step of determining the amount of pharmaceutical composition that contains CFXTEN necessary to allow hemostasis to be achieved in a hemophilia A patient. composition in two or more successive doses to a subject in an effective amount, wherein administration results in at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or An 80% or 90% greater improvement in at least one, two, or three parameters associated with hemophilia A disease compared to non-XTEN factor VIII, and administered using a similar dose. Non-limiting examples of improved parameters include FVIII procoagulant blood concentration, reduced partial prothromboplastin activation (aPTT) assay time, reduced one- or two-stage coagulation activity assay time, delayed onset of a bleeding event, reduced chromogenic assay time, reduced bleeding assay time, resolution of a bleeding episode, or reduced titer of Bethesda to natural FVIII.

In additional aspects, the present invention provides an isolated nucleotide sequence that encodes fusion proteins of any of the fusion protein embodiments.

- 14 041874 protein CFXTEN. In a separate embodiment of the present invention, the isolated nucleotide is a complementary sequence that encodes a CFXTEN fusion protein according to embodiments of the present invention. In a separate embodiment of the present invention, the isolated nucleotide further comprises a sequence that encodes a signal sequence, wherein said sequence is ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT(SEQIDNO: 1613) or its complementary sequence. In an additional embodiment, the present invention provides an expression vector that contains a nucleotide encoding a fusion protein, or its complementary sequence. In an additional embodiment, the present invention provides an isolated host cell that contains the above expression vector. In a further embodiment, the present invention provides a method for producing a fusion protein according to any of the embodiments of the present invention, which comprises providing a host cell that contains an expression vector; culturing the host cell to ensure production of the fusion protein; and recovering the fusion protein.

In a separate embodiment, the present invention provides an isolated fusion protein comprising a polypeptide that has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity, compared with a sequence of comparable length, selected from the table. 21, with optimal alignment.

In a further embodiment, the present invention provides an isolated nucleotide that contains a polynucleotide sequence selected from (a) a sequence that has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or up to about 100% sequence identity, compared to a sequence of comparable length selected from Table . 21 at optimal alignment, or (b) the complement of polynucleotide (a). In a further embodiment of the present invention, the isolated nucleotide contains the sequence

ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613) linked to the 5' end of the nucleotide (a) or sequence complement linked to the 3' end (b).

It is specifically contemplated that recombinant factor VIII fusion proteins may exhibit one or more or any combination of the properties disclosed herein.

Inclusion in the description of the invention of information by reference

All publications, patents, and patent applications referred to in this specification are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually identified as being incorporated by reference.

Brief description of graphic materials

The features and advantages of the present invention may be further explained with reference to the following detailed description and the accompanying drawings, which set forth illustrative embodiments of the present invention. Fig. 1 illustrates a schematic representation of the structure of FVIII and the spatial arrangement of the domains during processing and folding, and is intended to represent both the native FVIII variant and the variant without the B domain. The A1 domain varies from 1 to 372 residues (numbering relative to the mature form of the NCBI Protein FVIII sequence RefSeq NP_000123 and spans a1 residues), A2 domain varies from residue 373 to residue 740, B domain varies from residue 741 to residue 1648, A3 domain varies from residue 1649 to 2019 (covers a3 acidic region), C1 domain changes in the range from 2020 to 2172, and the C2 domain varies in the range from 2173 to 2332 residues. The BDD variants contain deletions ranging from 741 to 1648, leaving some or no remaining residues, with the remaining non-limiting BDD sequence being SFSQNPPVLKRHQR (SEQ ID NO: 1614). Fig. 1A illustrates the domain structure of single chain FVIII before processing. Arrows indicate sites on residues R372, R740, R1648 and R1689 that are cleaved upon processing and converting FVIII to FVIIIa. Fig. 1B illustrates a FVIII molecule that is processed into a heterodimer by cleavage of the R1648 residue, with the acidic region a3a of the A3 domain indicated at the N-terminus of A3. Fig. 1C illustrates a FVIII molecule that is processed into the FVIIIa heterotrimer by cleavage at R372, R740 and R1689 residues.

Fig. 2 is a collapse cascade diagram that shows the inner and outer branches that lead to a common path.

Fig. 3 illustrates the amino acid sequence of mature human factor VIII (SEQ ID NO: 1592).

Fig. 4 illustrates the sequence of factor VIII with deletions of part of the B domain (SEQ ID NO: 1593).

- 15 041874

Fig. 5 illustrates several examples of the CFXTEN FVIII conformation associated with XTEN (the latter is shown as thick, wavy lines). In all cases, FVIII can be either natural, or the BDD form of FVIII, or a single chain form in which the entire B domain, which contains natural cleavage sites, is removed. Fig. 5A illustrates, from left to right, three variants of single chain factor VIII with XTEN linked to the N-terminus, C-terminus, and two XTEN linked to the N- and C-terminus. Fig. 5B illustrates six variants of a mature FVIII heterodimer with, from left to right, XTEN linked to the N-terminus of the A1 domain; XTEN associated with the C-terminus of the C2 domain; XTEN associated with the N-terminus of the A1 domain and the C-terminus of the C2 domain; XTEN associated with the N-terminus of the A1 domain and with the N-terminus of the A3 domain; XTEN linked to the C-terminus of the C2 domain and to the N-terminus of the A3 domain via residual amino acids of the B domain; and XTEN linked to the N-terminus of the A1 domain, the C-terminus of the A2 domain via residual amino acids of the B domain, and to the C-terminus of the C2 domain. Fig. 5C illustrates, from left to right, three variants of single chain factor VIII: XTEN bound to the N-terminus of the A1 domain, XTEN bound within the outer loop of the A1 domain, and XTEN bound within the outer loop of the A3 domain; XTEN bound within the outer loop of the A2 domain, XTEN bound within the outer loop of the C2 domain, and XTEN bound to the C-terminus of the C2 domain; XTEN bound to the N-terminus of the A1 domain and within the outer loop of the C1 domain and to the C-terminus of the C domain. FIG. 5D illustrates six variants of a mature FVIII heterodimer with, from left to right, XTEN bound to the N-terminus of the A1 domain, XTEN bound within the outer loop of the A1 domain, and XTEN bound within the outer loop of the A3 domain; XTEN bound within the outer loop of the A2 domain and XTEN bound within the outer loop of the C1 domain and XTEN bound to the C-terminus of the C2 domain; XTEN associated with the N-terminus of the A1 domain, XTEN associated within the outer loop of the A1 domain, XTEN associated within the outer loop of the A3 domain, and XTEN associated with the C-terminus of the C2 domain; XTEN linked to the N-terminus of the A1 domain, XTEN linked to the N-terminus of the A3 domain via residual amino acids of the B domain, and XTEN linked within the outer loop of the C2 domain; XTEN linked within the outer loop of the A2 domain, XTEN linked to the N-terminus of the A3 domain via residual amino acids of the B domain, XTEN linked within the outer loop of the C1 domain, and XTEN linked to the C-terminus of the C2 domain; and XTEN linked in the B domain or between remnants of the B domain of the BDD variant (and the present invention also contemplates a variant in which XTEN replaces the entire scope of the B domain, which contains all natural cleavage sites, linking the A2 and A3 domains, resulting in a single-stranded form factor VIII). This figure also embodies all variants in which one or more XTEN sequences are introduced into the B domain and the resulting fusions are cleaved at one or more sites (eg, at the R1648 site) during intracellular processing.

Fig. 6 is a graphical representation of a CFXTEN construct with XTEN introduced into the B domain and linked to the C-terminus of the C2 domain, which illustrates the unstructured characteristic of XTEN that results in random coiling that can span regions of factor VIII close to XTEN. In the lower part, the graphic material illustrates that if XTEN is in a random helix, it can adopt a conformation that leads to steric hindrance that blocks the binding of factor VIII inhibitor antibodies that might otherwise have affinity for epitopes close to the XTEN site of insertion.

Fig. 7 is a graphical representation of various assays performed on the FVIII sequence without B domains to identify XTEN insertion sites within the FVIII sequence. Each of the A-H lines is represented at an arbitrary y-axis value scale across the entire BDD FVIII sequence, so that the minimum values represent areas of highly predictive tolerance to the XTEN insert, with residue numbers on the x-axis. Line A illustrates domain boundaries; all breaks in this line represent boundaries that XTEN can accept. Line B illustrates exon boundaries; those. each lineage stage represents a new exon. Line C illustrates regions that were not visible on the X-ray structure due to the lack of order in the crystal. The lines marked D represent several predictions that were calculated using the appropriate FoldIndex programs taken from the internet site bip.weizmann.ac.il/fldbin/findex (last visited 23 February 2011) (see Jaime Prilusky, Clifford E Felder, Tzviya Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man, Jacques S. Beckmann, Israel Silman, and Joel L. Sussman, 2005, bioinformatics based on the Kyte & Doolitlle algorithm, as well as RONN found on the strubi website .ox.ac.uk/RONN (accessed 23 February 2011) (see Yang, Z.R., Thomson, R., McMeil, P. and Esnouf, R.M. (2005) RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins Bioinformatics 21: 3369-3376 Lines E and F are calculated based on multiple alignments of FVIII gene sequences from 11 mammals available in GenBank Line E represents single residue retention Line F represents conservatism 3 amino acid segments of FVIII. Lines G and H represent breaks and insertions observed in multiple sequence alignments of 11 mammalian FVIII genes. Line J lists the XTEN insertion points by number of amino acids, which were obtained by combining many of the above measurements.

Fig. 8 illustrates sites in FVIII sequences without B domains (SEQ ID NO: 1594) defined

- 16 041874 divided as the active insertion point in the case of XTEN using the information illustrated in FIG. 8 and as confirmed in the assays in Example 34.

Fig. 9 illustrates the range of sites in FVIII sequences without B domains (SEQ ID NO: 1595) identified for XTEN insert using the information illustrated in FIG. 8 and or in Example 34 plus the range of amino acids near each insertion point that are considered suitable for XTEN insertion.

Fig. 10 is an assembly diagram of a CFXTEN library created by defining insertion points as described in the case of FIG. 7 followed by insertion of individual XTEN (black bars) at various insertion points using molecular biology techniques. The constructs are expressed and reconstituted, then FVIII activity and pharmacokinetic properties are evaluated to identify those CFXTEN conformations that result in improved properties.

Fig. 11 is an assembly diagram of a CFXTEN component library in which FVIII BDD domain segments, either single or associated with different XTEN lengths (black bars), are assembled in combinatorial form into libraries of CFXTEN-encoding genes that can then be assayed for FVIII activity and pharmacokinetic properties to identify CFXTEN conformations that lead to improved properties.

Fig. 12 illustrates several examples of conformations of CFXTEN with XTEN (shown in thick, wavy lines) with some XTENs that are secreted by the insertion of cleavage sequences (indicated by black triangles) that are cleavable with procoagulant proteases. Fig. 12A illustrates scFVIII with two final secreted XTENs. Fig. 12B illustrates a similar conformation to FIG. 12A, but with an additional non-secreted XTEN binding the A3 and C1 domains. Fig. 12C illustrates a mature FVIII heterodimer with two final secreted XTENs. Fig. 12D illustrates the same conformation as 10C but with additional non-secreted XTEN binding the A3 and C1 domains.

Fig. 13 is a schematic block diagram of representative steps for assembling, obtaining, and evaluating XTEN.

Fig. 14 is a schematic block diagram of representative assembly steps for a CFXTEN polynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are annealed into sequence motifs 502, such as a 12 amino acid (12-mer) motif, which is ligated with additional sequence motifs from the library to create a pool that spans the desired length of XTEN 504, and also ligated with a lower concentration of oligo-containing BbsI and KpnI restriction sites 503. The resulting pool of ligation products is gel-purified and the band is cut to the desired XTEN length, resulting in an isolated XTEN gene with stopper sequence 505. The XTEN gene is cloned into a spacer vector. In this case, the vector encodes an additional CBD 506 sequence and the GFP 508 gene. BbsI/HindIII cleavage is then performed to remove 507 and 508 and place a stop codon. The resulting product is then cloned into a BsaI/HindIII cleavable vector that contains the gene encoding FVIII, resulting in the 500 gene encoding the FVIII-XTEN fusion protein.

Fig. 15 is a schematic flow diagram of representative assembly steps of a gene encoding a fusion protein that contains CF and XTEN, its expression and recovery as a fusion protein, and its evaluation as a CFXTEN candidate product.

Fig. 16 illustrates the use of an XTEN donor sequence to generate processed XTEN. Fig. 16A illustrates the sequence of AG864 (SEQ ID NO: 1596), with the underlined sequence used to generate sequence length 576 (SEQ ID NO: 1597). Fig. 16B illustrates the AG864 sequence (SEQ ID NO: 1598), with the underlined sequence used to generate sequence length 288 (SEQ ID NO: 1599). Fig. 16C illustrates the AG864 sequence (SEQ ID NO: 1600), with the underlined sequence used to generate sequence length 144 (SEQ ID NO: 1601). Fig. 16D illustrates the AE864 sequence (SEQ ID NO: 1602), with the underlined sequence used to generate sequence length 576 (SEQ ID NO: 1603). Fig. 16E illustrates the AE864 sequence (SEQ ID NO: 1604), with the underlined sequence used to generate sequence length 288 (SEQ ID NO: 1605). Fig. 16F illustrates the AE864 sequence (SEQ ID NO: 1606) which is used to generate four sequences of length 144 (SEQ ID NOS 1607-1610, respectively, in order of appearance) (a double underline indicates the first amino acid in sequences 144 and a single underline represents the balance of this sequences).

Fig. 17 is a schematic representation of the structure of factor VIII-XTEN expression vectors with various strategies for introducing XTEN elements into the FVIII coding sequence. Fig. 17A illustrates an expression vector that encodes a 3'-fused XTEN sequence that encodes FVIII. Fig. 17B illustrates an expression vector that encodes an XTEN element inserted in the middle of a coding sequence that encodes a single FVIII. Fig. 17C illustrates an expression vector that encodes two elements

- 17 041874 menta XTEN: one inserted inside the FVIII coding sequence and the other fused to the 3' end of the FVIII coding sequence.

Fig. 18 illustrates a method for assembling hybrid genes that encode XTEN. In this case, the genes are assembled from 6 main fragments, and each fragment is available in 4 different codon variants (A, B, C and D). This allows for the theoretical diversity of 4096 in the assembly of 12-amino acid motifs.

Fig. 19 illustrates the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeys after single doses of various GFP compositions associated with unstructured polypeptides of various lengths administered either subcutaneously or intravenously as described in Example 41. The compositions are GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 and XTEN_AD836-GFP. Blood samples are analyzed at various time points after injection and plasma GFP concentration is measured by ELISA using a polyclonal anti-GFP antibody for capture and biotinylated production of the same polyclonal antibody for detection. The results are presented as plasma concentration versus time (h) after dosing and demonstrate in particular a significant increase in half-life for XTEN_AD836-GFP, the formulation with the longest XTEN sequences. The construct with the shortest sequence length, GFP-L288, has the shortest half-life.

Fig. 20 illustrates SDS-PAGE of samples from a stability study of the XTEN_AE864 fusion protein fused to the N-terminus of GFP (see Example 42). GFP-XTEN is incubated in cynomolgus monkey plasma and rat kidney lysate for up to 7 days at 37°C. In addition, GFP-XTEN, which is administered to the cynomolgus macaque, is also evaluated. Samples were taken at 0, 1 and 7 days and analyzed by SDS PAGE followed by detection using Western blot with anti-GFP antibody.

Fig. 21 illustrates the results of GPC analysis of samples of the Glucagon-XTEN construct as measured against known molecular weight protein standards, with a graphical representation of the results as absorbance versus retention volume as described in Example 40. The Glucagon-XTEN constructs are is 1) glucagon-Y288; 2) glucagon Y-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results indicate an increase in average molecular weight with increasing length of the XTEN fragment (for data, see example 40).

Fig. 22 illustrates the results of a Western blot of cell culture expressed proteins of cells that have been transformed with the designated constructs (Example 25). Samples in lanes 1-12 are: MW standards, FVIII (42.5 ng), pBC0100B, pBC0114A, pBC0100, pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0149 and pBC0146, respectively. Lanes 8, 9 and 12 show bands corresponding to FVIII with C-terminal XTEN288, with an estimated molecular weight of 95 kDa. Lanes 7 and 11 show bands corresponding to FVIII with C-terminal XTEN42, with an estimated molecular weight of 175 kDa. Lanes 2-6 show bands corresponding to FVIII and the heavy chain. Lanes 10 and 23 show heavy chain bands. Lane 7 illustrates the band corresponding to the heavy chain and bound XTEN42.

Fig. 23 illustrates the results of the analysis of FVIII in samples obtained from mice with two knockout factors, FVIII and von Willebrand factor, with hydrodynamic injection of plasmid DNA, as described in example 36.

Fig. 24 is a graphical and tabular display of the pharmacokinetic properties of rBDD-FVIII and purified CFXTEN fusion proteins pBC0145 and pBC0146 (with C-terminal XTEN) administered to mice with either HemA or FVIII/VWF dual knockout genes as described in Example 30, which show an improved half-life of CFXTEN in both strains of mice.

Fig. 25 is a graphical and tabular display of the pharmacokinetic properties of rBDD-FVIII and the CFXTEN pSD0050 and pSD0062 fusion proteins (with internally administered XTEN) administered to mice with either HemA (FIG. 25A) or FVIII/VWF gene knockouts (FIG. 25B ), using cell culture PK analysis in HemA mice. The dose, 5-minute recovery and half-life (T1/2) as described in Example 32 are shown, which highlight the improved recovery and half-life of CFXTEN compared to the FVIII positive control in both strains of mice.

Fig. 26 is a graphical representation of the titration of the FVIII inhibitor GMA8021 using pBC0114 BDD-FVIII and the CFXTEN LSD0049.002 construct with three 144 amino acid XTEN inserts at residues 18, 745, and 2332. The data indicate a shift in the amount of antibody required to inhibit CFXTEN by up to 50%. level, compared with the positive control FVIII in μg/ml to the right by about 0.7 orders of magnitude.

Fig. 27 is a flow chart of the SegScore algorithm. The figure uses the following designations: i, j - counters that are used in control systems passing through the entire sequence; HitCount - This variable is a counter that keeps track of how many times a subsequence collides in a block with an identical subsequence; SubSeqX - this variable contains the subsequence that is checked for redundancy; SubSeqY - this variable contains the subsequence that SubSeqX checks again; BlockLen this variable contains the block length defined by the user; SegLen - this variable contains

- 18 041874 segment length. The program is hard-coded to generate results in the case of subsequences with lengths 3, 4, 5, 6, 7, 8, 9, and 10; Block - this variable contains a string with the length BlockLen. The string consists of letters from the input sequence XTEN and is determined by the position of the counter i; SubSeqList is a list that contains all the results of the generated subsequences. Fig. 28 illustrates the application of the SegScore algorithm to a hypothetical 11 amino acid XTEN (SEQ ID NO: 1591) in order to determine repeatability. The XTEN sequence consists of N amino acids divided into N-S+1 subsequences of length S (in this case S=3). Pairwise comparison of all subsequences is carried out and the average number of identical subsequences is calculated in order to result in a number of subsequences of 1.89.

Fig. 29 is a plot of individual construct magnitudes of the ratio of FVIII activity in assayed CFXTEN to that of the pBC114 positive FVIII control after exposure to FVIII antibody GMA8021, grouped according to the amount of XTEN in the fusion protein construct (see Example 28). The results show a significant linear relationship between the ability of CFXTEN to maintain FVIII activity with an increase in the number of inserted XTENs.

Fig. 30 illustrates the primary sequence and domain structure of a mature human FVIII construct without B domains (BDD) (Example 46). The location of the introduced NheI and ClaI restriction sites is illustrated. It should be noted that the amino acid numbering corresponds to amino acid positions in the primary sequence of mature FVIII (FIG. 30). Individual domains are delimited by gray lines/boxes with the domain name in gray text. Acidic regions (a1, a2, a3) are indicated by dotted frames. Solid wedges/triangles indicate thrombin cleavage sites upon FVIII activation to FVIIIa. The open wedges/triangle indicate the site of intracellular proteolytic processing into the double-stranded form of FVIII. Hexagons indicate N-linked glycosylation sites. Circles indicate Tyr sulfation sites. Unique non-natural restriction sites (NheI, GCTAG; ClaI, ATCGAT) introduced into cDNA to facilitate XTEN insertion/recombination are shown in gray with double underlining.

Fig. 31 illustrates a graphical representation of the FVIII construct described in FIG. 30, indicating the organization of the domain and the location of natural and non-natural restriction sites.

Fig. 32 illustrates the graphical output of ASAView for the 2R7E, 3CDZ, and PM0076106 structural datasets. Available surface adsorption (ASA) is shown for amino acids in the A1, A2, A3, C1 and C2 domains. Analyzes are performed in X-ray crystallographic coordinates 3CDZ (Ngo et al., Structure 16: 597-606 (2008)) and 2R7E (Shen et al., Blood 111:1240-1247 (2008)) contributed to the Protein Data Bank provided by Research Collaboratory for Structural Bioinformatics (RCSB; http://www.rcsb.org/pdb), as well as in atomic coordinates PM0076106 in the case of a predicted refined FVIII structure derived from a molecular dynamics simulation study (Venkateswarlu, BMC Struct. Biol. 10:7 (2010)) contributed to the Protein Model Database (http://mi.caspur.it/PMDB/main.php) provided by the Consorzio Interuniversitario per le Applicazioni di Supercalcolo per Universita e Riserca (CASPUR) and the Department of Biochemical Sciences of the University of Rome.

Fig. 33 illustrates a structural representation of the location of XTEN insertion sites. The central graphic corresponds to the crystal structure of FVIII (PDB: 2R7E) surrounded by a detailed view of the A1, A2, A3, C1 and C2 domains. Beta strands and alpha helices are shown as a band representation. The hinges are shown as alpha carbon tubes. Amino acids at XTEN insertion sites are shown as a spherical representation of CPK. The number of each image indicates the location of the XTEN insertion sites as numbered in FIG. thirty.

Fig. 34 illustrates a structural representation of the location of the XTEN insertion sites shown in FIG. 33, with the resulting recombinant FVIII protein showing FVIII activity.

Fig. 35 illustrates a structural representation of the location of the XTEN insertion sites shown in FIG. 34, with the resulting recombinant FVIII protein showing FVIII activity.

Fig. 36 illustrates a structural representation of the location of the XTEN insertion sites shown in FIG. 35, with the resulting recombinant FVIII protein showing FVIII activity.

Fig. 37 illustrates a multiple sequence alignment of ClustalW sequences of FVIII domains A1, A2, A3, C1 and C2 that demonstrates the location of XTEN inserts that result in recombinant FVIII proteins displaying FVIII activity (black box, white text) or not displaying FVIII activity (grey box, thumbnail).

Fig. 38 illustrates a DSSP graphical representation of the secondary structure of two polypeptide chains in the crystal structure of naturally active human FVIII deposited under code 2R7E at the Protein Data Bank (see Example 47). The amino acid sequence numbering is the same as in the protein sequence in FIG. 30. Beta list areas shown filled

- 19 041874 arrows and are marked from β1 to β66. The location of the XTEN loops available for embedding is indicated by hatched frames. The A1 domain of the XTEN loop available for embedding is designated Loop A1-1 and Loop A1-2. The A2 domain of the XTEN loop available for embedding is designated Loop A2-1 and Loop A22. The A3 domain of the XTEN loop available for embedding is designated Loop A3-1 and Loop A3-2.

Fig. 39 illustrates a DSSP graphical representation of the secondary structure of two polypeptides in the crystal structure of naturally active human FVIII deposited under code 2R7E at the Protein Data Bank (see Example 47). The amino acid sequence numbering is the same as in the protein sequence in FIG. 30. The beta sheet regions are shown with filled arrows and labeled β1 to β66. The location of the XTEN loops available for embedding is indicated by hatched frames. The A1 domain of the XTEN loop available for embedding is designated Loop A1-1 and Loop A1-2. The A2 domain of the XTEN loop available for embedding is designated Loop A2-1 and Loop A22. The A3 domain of the XTEN loop available for embedding is designated Loop A3-1 and Loop A3-2.

Fig. 40 illustrates a multiple alignment of ClustalW sequences of FVIII domains A1, A2, A3, C1, and C2 that demonstrates the location of XTEN inserts resulting in recombinant FVIII proteins displaying FVIII activity (black box, white text) or not displaying FVIII activity (grey box, bold). text). Locations of XTEN loops available for embedding are indicated by dotted boxes (see example 47).

Fig. 41. FIG. 41A is a front view of a structural representation of human FVIII (PDB:2R7E) showing the location of the A1, A2, A3, C1 and C2 domains (outlined in dotted lines) and the location of XTEN loops available for insertion, A1-1, A1-2, A2 -1, A2-2, A3-1 and A3-2 are highlighted as spherical representations of the CPK. Fig. 41B is a side view of a structural representation of human FVIII (PDB:2R7E) showing the location of the A1, A2, A3, C1 and C2 domains (outlined in dotted lines) and the location of XTEN loops available for insertion, A11, A1-2, A2-1 , A2-2, A3-1 and A3-2 are highlighted as spherical representations of the CPK.

Fig. 42 illustrates a plan view of the structural representation of the A domains of isolated human FVIII (PDB:2R7E), which demonstrates the location of the XTEN loops available for insertion highlighted in CPK spherical views. Fig. 42B, 42D and 42F show a side view of the structural representations of the A domains of isolated human FVIII (PDB:2R7E), which demonstrate the location of the XTEN loops available for insertion highlighted as CPK spherical representations.

Fig. 43 illustrates the sequences of various factor VIII B domain deletions and individual mutations. Lines 4-10 show various deletions of the B domain with the indicated XTEN linking the residual flanking B domain or residues of the A3 domain. The R1648A mutation is indicated by an arrow on lines 5 and 8, while the Y1680F mutation is indicated by an arrow on lines 8-10.

Fig. 44 is a histogram of chromogenic assay and aPTT activity assay of various CFXTENs with single XTEN inserts (Example 49).

Fig. 45 is a histogram of chromogenic assay and aPTT activity assay of various CFXTENs with 2 XTEN inserts (Example 49).

Fig. 46 is a histogram of chromogenic assay and aPTT activity assay of various CFXTENs with 3 XTEN inserts (Example 49).

Fig. 47 is a plot of plasma levels in DKO mice of various CFXTEN administrations with single XTEN inserts compared to the BDD-FVIII control, showing the 10- to 20-fold longer half-life achieved with XTEN insert at various sites (Example 50).

Fig. 48 is a graph of plasma levels in DKO mice of various CFXTEN-treated mice with one, two, and three XTEN inserts compared to the BDD-FVIII control, showing the increase in half-life achieved with the addition of additional XTEN inserts compared to single or two inserts (example 51).

Fig. 49 is plotted inhibition curves for the remaining factor VIII procoagulant activity in samples analyzed in the Bethesda assay with three sera from hemophiliacs (Fig. 49A-C) or anti-FVII sheep (Fig. 49D) described in Example 52, which clearly shows a leftward shift of the inhibition curve for two CFXTEN molecules compared to FVIII not bound to XTEN.

- 20 041874

Detailed description of the invention

Before embodiments of the present invention are described, it should be understood that such embodiments of the present invention are provided by way of example only, and that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. Numerous variations, changes and substitutions will be apparent to those skilled in the art without departing from the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. While methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, the description of the patent, including definitions, will be controlled. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Numerous variations, changes and substitutions will be apparent to those skilled in the art without departing from the invention.

Definitions

In the context of the present invention, the following terms have the meanings assigned to them, unless otherwise indicated.

As used in the specification and claims, the singular forms include references to the plural unless the context clearly dictates otherwise. For example, the term cell contains many cells, including their mixtures.

The terms polypeptide, peptide, and protein are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also contain an amino acid polymer that has been modified, for example, by disulfide bonding, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a label component.

The term amino acid, as used herein, refers to any naturally occurring and/or non-natural or synthetic amino acid, including, but not limited to, both D and L optical isomers and analogs of amino acids and peptidomimetics. Amino acids are designated using standard one- or three-letter codes.

The term domain, when used in relation to a Factor VIII polypeptide, refers to either the entire domain or a functional fragment thereof, e.g., the full length or functional fragments of the A1 domain, A2 domain, A3 domain, B domain, C1 domain, and/or the C2 domain of Factor VIII .

The term natural L-amino acid means the L forms of the optical isomer of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D ), serine (S), and threonine (T).

The term non-naturally occurring, when applied to sequences and as used herein, means polypeptide or polynucleotide sequences that are unparalleled, not complementary, or do not have a high degree of homology with a wild-type or naturally occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may exhibit no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity compared to the natural sequence when properly aligned.

The terms hydrophilic and hydrophobic refer to the degree of affinity that a substance has with water. A hydrophilic substance has a high affinity for water, tending to dissolve in, mix with, or be wetted by water, whereas a hydrophobic substance has essentially no affinity for water, tends to reflect rather than absorb water, and tends not to dissolve or mix. with, or not wetted by water. Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is the scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is given in Hopp, TP, et al., Proc Natl Acad Sci U S (1981) 78:3824. Examples of hydrophilic amino acids are arginine, lysine, threonine, alanine, asparagine and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, serine, and glycine. Examples of hydrophobic amino acids are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine and valine.

A fragment, when applied to a protein, is a truncated form of a naturally occurring biologically active protein that retains at least a portion of its therapeutic and/or biological activity. A variant, when applied to a protein, is a protein with sequence homology to a naturally occurring biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein.

- 21 041874 on. For example, an alternative protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity as compared to the corresponding biologically active protein. As used herein, the term biologically active protein fragment includes proteins altered intentionally, such as by site-directed mutagenesis, coding gene synthesis, insertions, or accidentally, through mutations.

The term sequence variant means polypeptides that have been altered from their natural or parental sequence by one or more amino acid insertions, deletions, or substitutions. Inserts may be located at one or both ends of the protein, and/or may be located in internal regions of the amino acid sequence. A non-limiting example is the insertion of an XTEN sequence into a protein sequence with a biologically active function. For deletion variants, one or more amino acid residues of the polypeptide are removed as described herein. Deletion variants thus contain all fragments of a functional polypeptide sequence. In substitution variants, one or more amino acid residues of the polypeptide are removed and replaced with alternative residues. In a particular aspect, the substitutions are conservative in nature, and conservative substitutions of this type are well known in the art. As used herein, the internal XTEN corresponds to the XTEN sequences that are introduced into the clotting factor sequence. An internal XTEN can be constructed by inserting an XTEN sequence into the sequence of a clotting factor such as FVIII, or by inserting between two adjacent amino acids in a domain (intradomain) or between two domains (interdomain) of a clotting factor, or where XTEN partially replaces the internal sequence clotting factor.

As used herein, a terminal XTEN corresponds to an XTEN sequence that has been fused to or at the N- or C-terminus of a clotting factor or to a proteolytic cleavage sequence or linker at the N- or C-terminus of a clotting factor. The terminal XTEN may be fused to the natural end of the clotting factor. In addition, the terminal XTEN may replace part of the terminal clotting factor sequence.

The term XTEN secretion site corresponds to a CFXTEN fusion protein cleavage sequence that can be recognized and cleaved by mammalian proteases, affecting the secretion of XTEN or a portion of XTEN from the CFXTEN fusion protein. As used herein, a mammalian protease means a protease that typically exists in body fluids, cells, or tissues of mammals. XTEN secretion sites can be designed to be cleaved by various mammalian proteases (also known as XTEN secretion proteases) such as FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), elastase-2, MMP-12, MMP13, MMP -17, MMP-20, or any protease that is present in the clotting process. Other equivalent proteases (endogenous or exogenous) that are capable of recognizing a particular cleavage site can be used. The cleavage sites can be adjusted and adapted to the proteases used.

The term within, if corresponding to, a first polypeptide that is linked to a second polypeptide, encompasses a junction that connects the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, as well as the insertion of the first polypeptide into the sequence of the second polypeptide. For example, if XTEN is bound within a factor VIII polypeptide domain, XTEN may be linked to the N-terminus, C-terminus, or may be introduced into said domain.

As used herein, the term site, when used in relation to an XTEN insertion site within, or with, a biological polypeptide, such as factor VIII, represents the amino acid position at which the XTEN is bound. When numbered sites are described, such as the first, second, third, fourth, fifth, or sixth XTEN insertion site within, or at, factor VIII, each site is understood to represent a distinct factor VIII site; for example, the second site is a different location in factor VIII from the first site, the third site is different from the second and first, and so on.

The activity or procoagulant activity, when applied to the form(s) of the CFXTEN polypeptide provided herein, corresponds to the ability to bind to the target substrate of the coagulating protein or cofactor and provide a coagulation event, whether measured by an in vitro, ex vivo, or in vivo assay. Such assays include, but are not limited to, one-step clotting activity assays, two-step clotting activity assays, chromogenic assays, and ELISA assays. Biological activity corresponds to an in vitro or in vivo biological function or effect, which includes, but is not limited to, either receptor or ligand binding, or effect on clotting, generally known in the art in the case of clotting factor FVIII, or cellular, physiological or a clinical response that includes stopping the bleeding event.

As used herein, the term ELISA corresponds to an enzyme-linked immunosorbent assay as described herein or as otherwise known in the art.

- 22 041874

The host cell contains a single cell or cell culture that may or has been a recipient for dependent vectors. Host cells contain the progeny of a single host cell. The offspring may not necessarily be completely identical (in morphology or genetically from a common complement of DNA) to the original parent cell through natural, accidental or intentional mutation. The host cell contains cells naturally transfected with the vector of this invention.

Isolated (isolated), when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and isolated and/or isolated from a component of its natural environment. Contaminant components of its natural environment are substances that would normally interfere with diagnostic or therapeutic applications of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As will be appreciated by those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require isolation to distinguish it from its natural counterpart. In addition, a concentrated, separated, or diluted polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, differs from its natural counterpart in that the concentration or number of molecules per unit volume is generally greater than that of its naturally occurring counterpart. . In general, a polypeptide created by a recombinant method and expressed in a host cell is considered to be isolated.

An isolated polynucleotide, or a polypeptide-encoding nucleotide, or other polypeptide-encoding nucleotide, is a nucleotide molecule that is identified and separated from at least one contaminating nucleotide molecule to which it is normally associated in the natural source of the polypeptide-encoding nucleotide. An isolated polypeptide-encoding nucleotide molecule differs in form or condition from that which exists in naturally occurring cells. An isolated polypeptide-encoding nucleotide molecule is thus distinct from a particular polypeptide-encoding nucleic acid molecule as it exists in natural cells. Despite this, an isolated polypeptide-encoding nucleotide molecule contains polypeptide-encoding nucleotide molecules contained in cells that normally express the polypeptide if, for example, the nucleotide molecule is in a chromosomal or extrachromosomal location different from that in natural cells.

The fusion protein contains at least one fusion polypeptide that contains at least one region in the sequence in a position other than that which occurs in nature. Regions can typically exist on separate proteins and are combined into a fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. The fusion protein can be created, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired ratio.

Conjugated, linked, fused, and hybrid are used interchangeably herein. These terms refer to the joining together of two or more chemical elements, sequences or components by any methods that involve chemical conjugation or recombinant methods. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. In general, operably linked means that the DNA sequences, when linked, are contiguous, and in the reading phase or within the reading frame. In-frame fusion refers to the joining of two or more open reading frames (ORFs) to form a continuous long ORF, such that the correct reading frame of the original ORFs is maintained. Thus, the resulting recombinant fusion protein is a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORF (segments that are usually connected differently than in nature).

In the context of polypeptides, a linear sequence or sequence is an N- to C-terminal order of amino acids in a polypeptide in which residues that are adjacent to each other in the sequences are contiguous in the primary structure of the polypeptide. A partial sequence is a linear sequence of a portion of a polypeptide known to contain additional residues in one or both directions.

Heterological means derived from an entity that is genotypically different from the entity to which it is being compared. For example, a glycine-rich sequence removed from its natural coding sequence and operably linked to a coding sequence other than the natural sequence is a heterologous glycine-rich sequence. The term heterologous, as applied to a polynucleotide, polypeptide, means that the polynucleotide or polypeptide is derived from an entity that is genotypically different from the other entity to which it is being compared.

The terms polynucleotides, nucleotides, nucleic acids and oligonucleotides are used interchangeably. They refer to the polymeric form of nucleic acids of any length, either

- 23 041874 deoxyribonucleic acids, or ribonucleic acids, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) are determined from linkage analysis, exons, introns, template RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe and primers. The polynucleotide may comprise altered nucleic acids such as methylated nucleic acids and nucleotide analogs. If present, modification of the nucleotide structure may be carried out before or after the assembly of the polymer. The sequence of nucleic acids may be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeled moiety.

The term polynucleotide complement means a polynucleotide molecule having a complementary base sequence and reverse orientation compared to a reference sequence such that it can hybridize to the reference sequence with full fidelity.

Recombinant when applied to a polynucleotide means that which is the product of various combinations of in vitro cloning, restriction and/or ligation steps and other methods that result in a construct that can potentially be expressed as a recombinant protein in a host cell.

The terms gene and gene fragment are used interchangeably herein. They refer to a polynucleotide that contains at least one open reading frame that is capable of encoding a particular protein after it has been transcribed and translated. The gene or gene fragment may be genomic or cDNA, provided that the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A fusion gene is a gene that consists of at least two heterologous polynucleotides that are linked to each other.

Homology, homologous, or sequence identity refers to sequence similarity or interchangeability between two or more polynucleotide sequences, or between two or more polypeptide sequences. When used to determine sequence identity, similarity, or homology between two different amino acid sequences, a program such as BestFit, default settings can be used, or an appropriate substitution matrix, such as blosum45 or blosum80, can be selected to optimize results of identity, similarity, or homology. Preferably, polynucleotides that are homologous are those that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more more preferably 95%, most preferably 97%, still most preferably 98%, and still most preferably 99% sequence identity, compared to those sequences. Polypeptides that are homologous preferably have sequence identities that are at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95-99%, and most preferably 100% identical.

Ligation corresponds to the formation of phosphodiester bonds between two nucleotide fragments or genes, linking them together. To ligate DNA fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends will be matched directly after endonuclease cleavage. Despite this, it may be necessary to first convert the sticky ends usually obtained after endonuclease digestion to blunt ends to make them compatible for ligation.

The terms stringent conditions or stringent hybridization conditions refer to conditions under which a polynucleotide will hybridize to its target sequence to a detectably higher degree than other sequences (eg, at least 2 times background). As a rule, the accuracy of hybridization is expressed, in particular, in relation to the temperature and salt concentration at which the washing step is carried out. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically the concentration of Na ion (or other salts) is from about 0.01 to 1.0 M at a pH of 7.0 to 8.3 and the temperature is at least about 30°C in the case of short polynucleotides (eg, 10 to 50 nucleic acids) and at least about 60°C in the case of long polynucleotides (eg, more than 50 nucleic acids) - for example, stringent conditions may include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and three washes, 15 min each, in 0.1 x SSC/l% SDS at 60°C to 65°C . In addition, temperatures around 65°C, 60°C, 55°C or 42°C may be used. The SSC concentration can vary from about 0.1 to 2xSSC, with SDS being about 0.1%. Similar temperatures

- 24 041874 washes, as a rule, choose from about 5 to 20°C below the melting point of a particular sequence at a certain ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The equation for calculating Tm and nucleotide hybridization conditions is well known and can be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ^3rd edition, Cold Spring Harbor Laboratory Press, 2001. Typically, a blocking agent is used to block non-specific hybridization. Such blocking agents contain, for example, degraded and denatured DNA from salmon milt at about 100-200 µg/ml. An organic solvent such as formamide at a concentration of about 35-50% v/v can also be used under certain circumstances, for example in the case of RNA:DNA hybridizations. Useful variations of these washing conditions will be apparent to those of ordinary skill in the art.

The terms percent identity, percent sequence identity, and % identity, as applied to a polynucleotide sequence, refer to the percentage of residue matches between at least two polynucleotide sequences that align using a standardized algorithm. Such an algorithm can insert, in a standardized and reproducible manner, gaps into the compared sequences in order to optimize the alignment of the two sequences, and hence achieve a more meaningful comparison of the two sequences. Percent identity is measured over the length of a defined entity polynucleotide sequence, or is measured over a shorter length, such as the length of a fragment that is derived from the largest defined polynucleotide sequence, such as a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 contiguous residues. Such lengths are illustrative only and it should be understood that any fragment length based on the sequences presented herein, in tables, figures, or sequence listing, can be used to describe the length at which percent identity can be measured. Percent sequence identity is calculated by comparing two optimally aligned sequences in the comparison window, determining the number of matching positions (where identical residues occur in both polypeptide sequences), dividing the number of matching positions by the total number of positions in the comparison window (i.e. window size) and multiplying the result by 100 to obtain percent sequence identity. If it is necessary to compare sequences with different lengths, the shortest sequence determines the length of the comparison window. When calculating sequence identity, conservative substitutions are ignored.

Percent (%) sequence identity, with respect to the polypeptide sequences defined herein, is defined as the percentage of amino acid residues in the target sequence that is identical to the amino acid residues of a second, control, polypeptide sequence, or portion thereof, after sequence alignment and the introduction of gaps, if necessary, to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways that are known to those skilled in the art, for example using open source software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. Percent identity can be measured over the entire length of a defined polypeptide sequence, or can be measured over a shorter length, such as the length of a fragment taken from a larger, defined polypeptide sequence, such as a fragment thereof, of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. Such lengths are illustrative only, and it should be understood that any fragment length represented by sequences shown herein, in tables, figures, or sequence listing, can be used to describe the length for which percent identity can be measured.

The term uniqueness, as used herein in the context of a polypeptide, refers to the absence or limitation of the degree of internal homology in a peptide or polypeptide sequence. The term substantially non-repetitive may mean, for example, that there are few, or none, instances of four contiguous amino acids in a sequence that are identical types of amino acids, or a polypeptide that has a subsequence count (defined below) of 10 or less, or that is not there is a pattern in order, from the N- to the C-terminus, of the sequence motifs that make up the polypeptide sequence. The term repeatability, which is used herein in the context of a polypeptide, corresponds to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a repeating sequence may contain several identical

- 25,041,874 copies of short amino acid sequences. For example, a polypeptide sequence of interest can be divided into n-mer sequences and the number of identical sequences can be counted. Frequently repeating sequences contain a high proportion of identical sequences, while non-repeating sequences contain few identical sequences. The sequence may contain multiple copies of shorter sequences, of definite or variable length, or motifs in which the motifs themselves have non-repeating sequences, making the full-length polypeptide essentially non-repetitive. The length of the polypeptide within which non-repeatability is measured can vary from 3 amino acids to about 200 amino acids, from about 6 to about 50 amino acids, or from about 9 to about 14 amino acids. Repeatability, as used in the context of a polynucleotide sequence, corresponds to the degree of internal homology in a sequence, such as, for example, the frequency of identical nucleotide sequences of a given length. Repeatability can, for example, be measured by analyzing the frequency of identical sequences.

The vector is a nucleotide molecule, preferably self-replicating in an appropriate host, which transports the administered nucleotide molecule into and/or between host cells. The term includes vectors that function primarily for the insertion of DNA or RNA into a cell, replication vectors that function primarily for DNA or RNA replication, and expression vectors that function for transcription and/or translation of DNA or RNA. In addition, contain vectors that provide more than one of the above features. An expression vector is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into polypeptide(s). An expression system generally refers to a suitable host cell that contains an expression vector that can be operated to produce the desired expression product.

Serum resistance to degradation as applied to a polypeptide corresponds to the ability of polypeptides to withstand degradation in blood or its components, which typically contain proteases in serum or plasma. Serum resistance to degradation can be measured by pooling the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically on varying numbers of days (e.g., 0.25, 0.5, 1, 2, 4, 8, 16 days), typically around 37°C. Samples for these time points can be Western blotted and protein is detected with antibody. The antibody can be labeled on a protein. If the protein in the immunoblot shows a distinct band, provided that the size of the protein is identical to the size of the injected protein, then no degradation occurs. In this exemplary method, the point in time at which 50% of the protein is cleaved, as judged by Western blotting or equivalent methods, is the degradation half-life of the serum or the serum half-life of the protein.

The term t _1/2 that is used in this document means the final half-life, calculated as ln(2)/K _el . K _el is the terminal elimination rate constant calculated by linear regression of the linear portion of the log concentration versus time. The half-life generally corresponds to the time it takes for half of the amount of an administered substance introduced into a living organism to be metabolized or eliminated by conventional biological methods. The terms t _1/2 , terminal half-life, half-life, and circulating half-life are used interchangeably herein.

Active clearance means mechanisms by which a protein is removed from the bloodstream, other than filtration or clotting, and which includes removal from the bloodstream by cells, receptors, metabolism, or protein degradation.

Average molecular weight ratio and average molecular weight are related terms referring to the degree of relative increase or decrease in apparent molecular weight exhibited by a particular amino acid sequence. Average molecular weight is determined by gel permeation chromatography (SEC) or similar methods, by comparison with globular protein standards, and is measured in units of apparent kDa. The average molecular weight ratio is the ratio between apparent molecular weight and actual molecular weight; the latter is predicted by addition based on amino acid composition, calculated by the molecular weight of each type of amino acid in the composition, or by estimation against molecular weight standards in SDS gel electrophoresis.

The terms hydrodynamic radius or Stokes radius are the effective radius (R _h in nm) of a molecule in solution, measured assuming that this body moves through the solution and is resisted by the viscosity of the solution. In embodiments of the present invention, measurements of the hydrodynamic radius of XTEN fusion proteins correlate with an apparent molecular weight ratio, which is a more intuitive measure. Hydrodynamic radius pro

- 26 041874 theine affects its diffusion rate in an aqueous solution, as well as its ability to penetrate macromolecular gels. The hydrodynamic radius of a protein is determined by its molecular weight as well as its structure, including shape and compactness. Methods for determining hydrodynamic radius are well known in the art, such as using gel permeation chromatography (SEC) as described in US Pat. Nos. 6,406,632 and 7,294,513. with the smallest hydrodynamic radius. Some proteins adopt a random and open, unstructured or linear conformation, and as a result have a much larger hydrodynamic radius than typical globular proteins of similar molecular weight.

Physiological conditions refer to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions in a living subject. Many physiologically appropriate conditions have been created for use in in vitro assays. Typically, the physiological buffer contains a physiological salt concentration and is adjusted to a neutral pH in the range of about 6.5 to about 7.8, and preferably about 7.0 to about 7.5. Many physiological buffers are given in Sambrook et al. (2001). The physiologically appropriate temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.

A functional group is a chemical structure that can be connected to a second functional group. Examples of functional groups are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups. Some functional groups may be activated to facilitate association with a second functional group. Non-limiting examples of activation are the reaction of a carboxyl group with a carbodiimide, the transformation of a carboxyl group into an activated ester, or the transformation of a carboxyl group into an azide function.

A controlled release agent, a delayed release agent, a depocomposition, and a sustained release agent are used interchangeably to refer to an agent capable of increasing the duration of release of a polypeptide of the present invention, as compared to the duration of secretion when the polypeptide is administered in the absence of the agent. Various embodiments of the present invention may have different secretion rates resulting in different therapeutic amounts.

The terms antigen, target antigen, and immunogen are used interchangeably herein to refer to a binding structure or determinant whose antibody fragment or antibody fragment therapeutic is bound to, or has specificity for, it.

The term payload as used herein corresponds to a protein or peptide sequence that has biological or therapeutic activity; analogue of the pharmacophore of small molecules. Examples of payloads include, but are not limited to, clotting factors, cytokines, enzymes, hormones, and blood and growth factors.

The term antagonist as used herein includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a native polypeptide disclosed herein. Methods for identifying polypeptide antagonists may comprise contacting a natural polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide. In the context of the present invention, antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecule that reduces the effect of a biologically active protein.

The term agonist, which is used in its broadest sense, includes any molecule that mimics the biological activity of the native polypeptide disclosed herein. Suitable agonist molecules particularly comprise an antibody agonist or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, and the like. Methods for detecting natural polypeptide agonists may comprise interacting the natural polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities typically associated with the natural polypeptide.

As used herein, treat or cure or mitigate or ameliorate is used interchangeably and means administering a drug or biological agent to achieve a therapeutic effect, to cure or reduce the severity of an existing disease, or to achieve a prophylactic effect, prevent or reduce the likelihood of occurrence or severity occurrence of the disease. By therapeutic effect is meant the elimination or reduction of the intensity of the underlying disease to be treated, or one or more physiological symptoms associated with the cause, so that improvement is observed.

- 27 041874 is given to the subject, despite the fact that the subject may still be affected by the underlying disease.

A therapeutic effect or therapeutic effect, as used herein, corresponds to a physiological effect, which includes, but is not limited to, alleviating, ameliorating, or preventing disease in humans or other animals, or otherwise enhancing the physical or mental well-being of humans or animals, as a result of the introduction of the fused protein of the present invention, in addition to the ability to induce the production of antibodies against antigenic epitope, which has a biologically active protein. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom of a disease (e.g., bleeding in a subject diagnosed with hemophilia A), or to a subject reporting one or more physiological symptoms of the disease, although the diagnosis of the disease may be and was not placed.

The terms therapeutically effective amount and therapeutically effective dose, as used herein, refer to the amount of drug or biologically active protein, either alone or as part of a fusion protein composition, that is capable of having a measurable, beneficial effect on any symptom, aspect, measurable parameter. or a description of the disease state or disease when administered to a subject in single or multiple doses. Such an effect does not have to be absolute to be beneficial. Determination of a therapeutically effective amount is within the ability of a person skilled in the art, especially in light of the presence of a detailed description in this document.

The term therapeutically effective dosing regimen as used herein corresponds to a schedule of sequentially administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as part of a fusion protein composition, with the doses are given in therapeutically effective amounts to result in a sustained beneficial effect on any symptom, aspect, measurable parameter, or characteristic of the disease state or disease.

I) General methods.

The practice of the present invention uses, unless otherwise indicated, conventional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genetics, and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ^3rd edition, Cold Spring Harbor Laboratory Press, 2001; Current protocols in molecular biology, FM Ausubel, et al. eds., 1987; Series Methods in Enzymology, Academic Press, San Diego, CA.; PCR 2: a practical approach, MJ MacPherson, BD Hames and GR Taylor eds., Oxford University Press, 1995; Antibodies, a laboratory manual Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; Goodman &Gilman's The Pharmacological Basis of Therapeutics, ^11th Edition, McGraw-Hill, 2005; and Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, ^4th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated herein in their entirety by reference.

II) Coagulation factor VIII.

The present invention relates in particular to compositions that contain a factor VIII clotting factor (CF) associated with one or more extended recombinant proteins (XTEN), resulting in a CFXTEN fusion protein composition. As used herein, CF corresponds to factor VIII (FVIII) or mimetics, sequence variants, and truncated FVIII variants, as described below.

Factor VIII or FVIII or FVIII protein means a coagulation factor protein, species (including human, porcine, canine, rat, or mouse FVIII proteins) and sequence variants thereof, which contain, but are not limited to, a single-stranded 2351 amino acid precursor protein ( with a 19 amino acid hydrophobic signal sequence), a 2332 amino acid mature factor VIII cofactor protein of approximately 270-330 kDa with an A1-A2B-A3-C1-C2 domain structure, and a non-enzymatic active or cofactor form of FVIII (FVIIIa), which represents is a circulating heterodimer of two chains that form, as a result of proteolytic cleavage after R1648, the heavy chain form, consisting of A1-A2-B (within 90-220 kD) 1-1648 amino acids (numbering relative to the mature form of FVIII) and the light chain A3- C1-C2 in 80 kDa 1649-2232 amino acids, each of which is schematically illustrated in FIG. 1. In addition, and as used here, each domain of A1, A2, and A3 spans acidic spacer sites; acidic areas a1, a2, and a3, respectively. Thus, it is to be understood that the CFXTEN constructs described as having A1, A2, A3, B, C1 and C2 domains contain acidic regions a1, a2 and a3. As used herein, a factor VIII or FVIII or FVIII polypeptide also contains variant forms that contain substitutions, additions and/or deletions for proteins as long as the variant retains the desired biological activity, such as procoagulant activity. Huge numbers of functional FVIII variants have been constructed and can be used as recombinant FVIII proteins as described herein. See PCT Application Nos. WO 2011/069164 A2, WO 2012/006623 A2, WO 2012/006635 A2 or WO 2012/006633 A2, all of which

- 28 041874 incorporated herein by reference in their entirety. Very many functional variants of FVIII are known. In addition, hundreds of non-functional FVIII mutations have been identified in hemophiliacs. See, for example, Cutler et al., Hum. Mutat. 19:274-8 (2002), incorporated herein by reference in its entirety. In addition, comparisons between FVIII from humans and other species have identified conserved residues that may be required for function. See, for example, Cameron et al., Thromb. haemost. 79:317-22 (1998) and US 6251632, incorporated herein by reference in their entirety.

In a separate embodiment of the present invention, human factor VIII domains are defined using the following amino acid residues: A1, Ala1-Arg372 residues; A2, Ser373Arg740 residues; B, Ser741-Arg1648 residues; A3, Ser1649-Asn2019 residues; C1, Lys2020-Asn2172 residues; C2, Ser2173-Tyr2332 residues. The A3-C1-C2 sequence contains Ser1649-Tyr2332 residues. In a further embodiment of the present invention, Arg336-Arg372 residues typically correspond to the a1 region and Arg372 is cleaved with thrombin. In certain embodiments of the present invention, the a2 region is part of the A1 domain. In a further embodiment of the present invention, Glu1649-Arg1689 residues correspond to the acidic region of a3. In certain embodiments, the a3 acidic region is part of the A3 domain. In a further embodiment of the present invention, the natural FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, a2 and a3 are structurally related domains A, B is domain B, C1 and C2 are structurally related C domains, and a1, a2 and a3 are acidic spacer regions. In the above formula and with reference to the primary position of the amino acid sequence in FIG. 30, human FVIII domain A1 extends from Ala1 to around Arg336, spacer a1 extends from around Met337 to around Arg372, domain A2 extends from around Ser373 to around Tyr719, spacer a2 extends from around Glu720 to around Arg740, domain B extends from around Ser741 to about Arg1648, the a3 spacer region extends from about Glu1649 to about Arg1689, the A3 domain extends from about Ser1690 to about Asn2019, the C1 domain extends from about Lys2020 to about Asn2172, and the C2 domain extends from about Ser2173 to Tyr2332 (Saenko et al ., 2005, J Thromb Hemostasis, 1, 922-930). In addition to specific proteolytic cleavage sites, the designation of the location of the boundaries between domains and regions of factor VIII in different literature sources may vary. The boundaries marked here are therefore indicated as approximate using the term about.

Such factor VIII contains truncated sequences, such as BDD sequences without B domains, in which some or most of the B domain sequence is removed (such as the BDD sequences disclosed or described in US Pat. Nos. 6,818,439 and 7,632,921). An example of a FVIII BDD is REFACTO® or XYNTHA® (recombinant FVIII BDD) which contains the first polypeptide corresponding to amino acids 1 to 743 in FIG. 30 fused to a second polypeptide corresponding to amino acids 1638 to 2332 in FIG. 30. Exemplary FVIII BDD constructs that can be used to generate the recombinant proteins of the present invention comprise, but are not limited to, FVIII with an amino acid deletion corresponding to amino acids 747-1638 of mature human FVIII (Fig. 30) (Hoeben R.C., et al. J. Biol Chem 265 (13): 7318-7323 (1990, incorporated herein by reference in their entirety), and FVIII with an amino acid deletion corresponding to amino acids 771-1666 or amino acids 868-1562 of mature human FVIII (Fig. 30) (Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in their entirety).

In addition, sequences that contain heterologous amino acid insertions or substitutions (eg, aspartic acid is replaced by valine at position 75), or single chain FVIII (scFVIII) in which the heavy and light chains are covalently linked via a linker. As used herein, FVIII should be any functional form of a factor VIII molecule with typical characteristics of coagulation factor VIII capable of modifying human factor VIII deficiencies when administered to such a subject, e.g., a subject with hemophilia A. FVIII or sequence variants are isolated, determined, and clone as described in US patent or applications No. 4757006; 4965199; 5004804; 5198349, 5250421; 5919766; 6228620; 6818439; 7138505; 7632921; and 20100081615. Human factor VIII is encoded by a single copy gene that is located at the tip of the long arm of the X chromosome (q28). It contains about 186,000 base pairs (bp) and makes up approximately 0.1% of the X chromosome (White, G.C. and Shoemaker, C.B., Blood (1989) 73:1-12). The human FVIII amino acid sequence is decoded from cDNA as shown in US Pat. No. 4,965,199, which is incorporated herein by reference in its entirety. Natural mature human FVIII derived from the cDNA sequence (ie without the secretory signal peptide but before other post-translational processing) is illustrated in FIG. 3.

The mature factor VIII mRNA encoding DNA is located in 26 individual exons ranging in size from 69 to 3106 bp. The 25 intermediate intron regions share a range of exons in size from 207 to 32400 bp. A complete gene consists of approximately 9 kb. exon and 177 kb. intron. Three repeat

- 29,041,874 shared A domains share approximately 30% sequence homology. The B domain contains 19 of the approximately 25 predicted glycosylated sites, and the A3 domain is believed to contain the von Willebrand factor binding site. The paired C domains follow the A3 domain and share approximately 37% homology with each other (White, G.C. and Shoemaker, C.B., Blood (1989) 73:1-12).

The B domain shares the A2 and A3 domain of natural factor FVIII in the newly synthesized single-chain precursor molecule. According to various sources, the precise boundaries of the B domain extend from amino acids 712 to 1648 of the precursor sequence (Wood et al., Nature (1984) 312:330-337) or amino acids 741-1648 (Pipe, SW, Haemophilia (2009) 15:1187-1196 and US Pat. No. 7,560,107) or amino acids 740-1689 (Toole, JJ. Proc. Natl. Acad. Sci. USA (1986) 83:5939-5942). As used herein, the B domain refers to amino acids 741-1648 of mature factor VIII. As used herein, a FVIII or FVIII BDD B domain deletion means a FVIII sequence with any amino acid, fragment, or all amino acids 741 to 1648 deleted. , as used in this document) and C-terminus (B2, as used in this document). In a separate FVIII BDD variant, the remaining B domain amino acids are SFSQNPPVLKRHQR (SEQ ID NO: 1614). In a separate embodiment of BDD FVIII, residue B1 is SFS and residue B2 is QNPPVLKRHQR (SEQ ID NO: 1615). In an additional embodiment of FVIII BDD, residue B1 is SFSQN (SEQ ID NO: 1616) and residue B2 is PPVLKRHQR (SEQ ID NO: 1617). Factor VIII without B-domain, FVIII, or BDD FVIII can have complete or partial deeds, disclosed in US patents No. 6316226, 6346513, 7041635, 5789203, 60604447, 5595886, 62228620, 5972885, 6048720, 51111111, 511111, 5111111, 511111111111111A 4,868,112 and 6,458,563, each of which is incorporated herein by reference in its entirety. In certain embodiments of the present invention, the Factor VIII sequence without the B domain of the present invention contains any of the deletions disclosed in Col. 4, line 4 to count. 5, line 28 and examples 1-5 of US Pat. No. 6,316,226 (and also in US 6,346,513). In a further embodiment of the present invention, Factor VIII without B domains is S743/Q1638 Factor VIII without B domains (SQ variant of Factor VIII) (for example, Factor VIII that has a deletion from amino acid 744 to amino acid 1637, for example, Factor VIII which has amino acids 1-743 and amino acids 1638-2332 of full-length factor VIII). In certain embodiments of the present invention, Factor VIII without the B domain of the present invention has a deletion disclosed in Col. 2, lines 26-51 and examples 5-8 of US Pat. No. 5,789,203 (also US Pat. No. 6,060,447, US Pat. In certain embodiments of the present invention, Factor VIII without the B domain has the deletion described in Col. 1, line 25 to qty. 2, line 40 of US Pat. No. 5,972,885; count 6, lines 1-22 and Example 1 of US Pat. No. 6,048,720; count 2, lines 17-46 of US Pat. No. 5,543,502; count 4, line 22 to col. 5, line 36 of US Pat. No. 5,171,844; count 2, lines 55-68, FIG. 2 and Example 1 of US Pat. No. 5,112,950; count 2, line 2 to col. 19, line 21 and tab. 2 US Pat. No. 4,868,112; count 2, line 1 to col. 3, line 19, col. 3, line 40 to count. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of US Pat. No. 7,041,635; or count. 4, lines 25-53, US Pat. No. 6,458,563. In certain embodiments of the present invention, factor VIII without the B domain has a deletion of most of the B domains, but still contains the amino-terminal sequences of the B domain, which are natural in the case of in vivo proteolytic processing a primary translation product into two polypeptide chains, as disclosed in WO 91/09122, which is incorporated herein by reference in its entirety. In certain embodiments of the present invention, factor VIII without the B domain with a deletion of amino acids 747-1638, i.e. virtually complete deletion of the B domain. Hoeben R.C., et al. J Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. Factor VIII without the B domain may also contain a deletion of factor VIII amino acids 771-1666 or amino acids 868-1562. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety. An additional deletion domain B that is part of the present invention contains: a deletion of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 59395942)), from 797 to 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 to 1646 (Kaufman (PCT Publication No. WO 87/04187)), 747-1560 (Sarver, et al., DNA ( 1987) 6:553-564)), 741 to 1648 (Pasek (PCT Application No. 88/00831)), or 816 to 1598 or 741 to 1648 (Lagner (Behring Inst. Mitt. (1988) No 82: 16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the previous deletions can be made in any sequence of factor VIII used in embodiments of the present invention.

Proteins that are involved in clotting include factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, protein C and tissue factor (together or independently coagulating protein(s)). The interactions of major clotting proteins on intrinsic and extrinsic pathways of clotting are shown in FIG. 2. Most coagulation proteins are present in zymogenic form, but when activated, exhibit procoagulative protease activity in which they activate other coagulation proteins that

- 30 041874 contribute to the internal or external coagulation pathway and thrombus formation. In the intrinsic pathway of the coagulation cascade, FVIII associates with an activated factor IX, factor X, calcium, and phospholipid complex. The factor VIII heterodimer has no enzymatic activity, but the heterodimer becomes active as a factor IXa cofactor after proteolytic activation with thrombin or factor Xa, with factor VIIIa activity, which is characterized by its ability to form a membrane binding site in the case of factors IXa and X in a conformation suitable to activate factor X with factor IXa. After cleavage with thrombin, activated FVIII (FVIIIa) dissociates from von Willebrand factor and binds to the negatively charged phospholipid PL, and the resulting complex participates as a factor IXa cofactor in the activation of the factor X complex (tenase). Within the C2 domain and amino acid residues 1649 to 1689 in the A3 domain are von Willebrand factor (vWF) binding sites that act in conjunction with von Willebrand factor, whereby the circulating complex protects FVIII from rapid degradation in the blood (Weiss HJ, et al. al. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest (1977) 60:390).

Activated factor VIII is a heterotrimer that consists of an A1 domain, an A2 domain, and a light chain that contains the A3-C1-C2 domains. Factor IX activation is accomplished by a two-step removal of the activation peptide (Ala 146-Arg 180) from the molecule (Bajaj et al., Human factor IX and factor IXa, in METHODS IN ENZYMOLOGY. 1993). The first cleavage is at the Arg 145-Ala 146 site with either factor Xia or factor VIIa/tissue factor. A second, and rate-limiting, cleavage is made at the Arg 180-Val 181 site. Activation removes 35 residues. Activated human factor IX exists as a heterodimer of a C-terminal heavy chain (28 kDa) and an N-terminal light chain (18 kDa), which are held together by a single disulfide bridge that anchors the enzyme to the Gla domain. Factor IXa in turn activates factor X in interaction with activated factor VIII. In addition, both factors IX and X can be activated by factor VIIa complexed with a lipidated tissue factor derived from an external pathway. Factor Xa then participates in the final common pathway whereby prothrombin is converted to thrombin, and thrombin in turn converts fibrinogen to fibrin to form a thrombus.

Defects in the clotting process can lead to bleeding disorders (coagulopathy) in which case the time required for the formation of a blood clot increases. Such defects can be congenital or acquired. For example, hemophilia A and B are inherited diseases characterized by deficiencies in FVIII and FIX, respectively. In other words, biologically active factor VIII corrects the coagulation defect in plasma obtained from individuals suffering from hemophilia A. Recombinant FVIII has been shown to be effective and approved in the treatment of hemophilia A in adults and children, and is also used to stop bleeding or prevent bleeding, associated with trauma and/or surgery. Such therapeutic applications of factor VIII can be problematic in the treatment of individuals who exhibit factor VIII deficiency, as well as individuals with von Willebrand's disease. In addition, individuals who receive factor VIII replacement therapy often develop antibodies to these proteins, which often reduce or eliminate the procoagulant activity of the associated FVIII. Continued treatment is extremely difficult due to the presence of these antibodies, which reduce or negate the effectiveness of the treatment.

In a separate aspect, the present invention provides for the content in the CFXTEN fusion protein composition of FVIII sequences that are identical to human FVIII, sequences that have homology with FVIII sequences, sequences that are natural, such as from humans, primates, mammals (including domestic animals ), or a processed FVIII variant; all of which retain at least a portion of the procoagulant activity of native FVIII, and which are useful in the prevention, treatment, mediation, or amelioration of hemophilia A or a bleeding event associated with trauma, surgery, or clotting factor VIII deficiency. Sequences with homology to FVIII can be found using standard homology search techniques such as NCBI BLAST or in public databases such as Chemical Abstracts Services Databases (e.g. CAS Registry), GenBank, The Universal Protein Resource (UniProt) and subscriptions. , providing access to databases such as GenSeq (for example, Derwent).

In a separate embodiment of the present invention, the FVIII incorporated into the subject's CFXTEN compositions is a recombinant polypeptide with a sequence corresponding to the FVIII protein found in nature. In a further embodiment of the present invention, the FVIII is a non-natural variant of the FVIII sequence, a fragment, homologue or mimetic of a naturally occurring sequence that retains at least a portion of the procoagulant activity of the corresponding native FVIII. In a further embodiment of the present invention, the FVIII is a processed variant with all or part of

- 31 041874 but with a deleted B domain (FVIII BDD), which may be in any heterodimeric form, or may remain in single stranded form (scFVIII), later described by Meulien et al., Protein Eng. (1988) 2(4):301-306. Non-limiting examples of FVIII BDDs are Factor VIII sequences in which amino acids are deleted between residue number 741 and residue number 1640 (numbering relative to native, mature FVIII), or between residue number 745 and residue number 1640, or between residue number 745 and remainder 1640, or between remainder 741 and remainder 1690, or between remainder 745 and remainder 1667, or between remainder 745 and remainder 1657, or between remainder 747 and remainder number 1642, or between remainder number 751 and remainder number 1667.

In a further embodiment of the present invention, FVIII contains heterologous sequences, which may contain XTEN, as described in more detail below. Tab. 1 provides a non-limiting list of FVIII amino acid sequences that are encompassed by the CFXTEN fusion proteins of the present invention. In certain embodiments of the present invention, the FVIIIs inserted into the CFXTEN fusion proteins comprise proteins that have at least about 70% sequence identity, or alternatively 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with an amino acid sequence of comparable length, selected from the table. 1.

Table 1

Amino acid sequences of FVIII

Title (source) Amino acid sequence SEQID NO: polypeptide tidpredscenary FVIII (human) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVD ARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLG PTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQT SQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSW HSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAE DYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTD ETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNV ILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGY VFD SLQLS VCLHEVAYW YILSIGAQTDFLS VFF SGYTFKHKMVYED TLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS SCD 1

- 32 041874

KNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNA TTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSL SDLQEAKYETFSDDPSPGAroSNNSLSEMTHFRPQLHHSGDMVFTP ESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGT DNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDS KLLESGLMNSQES SWGKNVS STESGRLFKGKRAHGPALLTKDNAL FKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTE FKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKE GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRN LFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGT KNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHT AHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRS KRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYN EKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRV LFQDNS SHLP AAS YRKKDSGVQES SHFLQGAKKNNLSLAILTLEMT GDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKV HIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKV PFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKT AFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLC SQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDED EN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVP QFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPT FKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIH FSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLI GEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWA PKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFS SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFN PPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDA QITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQV DFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQN GKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME VLGCEAQDLY mature FVIII (man) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR 2

- 33 041874

FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRT PMPKIQNVS S SDLLMLLRQSPTPHGLSLSDLQEAKYETF SDDP SPGA IDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATE LKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQL DTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKN VSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSAT NRKTHroGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKN ATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKM LFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLS EKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHN QEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNV EGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLG NQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELE KRIIVDDT STQW SKNMKHLTP STLTQ ID YNEKEKGAITQ SPLSDCLT RSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKK DSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSV TYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPG HLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLL DPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESN HAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRT TLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAW AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVY SNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAW STKEPF SWIKVDLLAPMIIHGIKTQGARQKF S SLYISQFIIMYSLDGK KWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATW SPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTP VVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII (dog) MQVELYTCCFLCLLPFSLSATRKYYLGAVELSWDYMQSDLLSALH ADTSFSSRVPGSLPLTTSVTYRKTVFVEFTDDLFNIAKPRPPWMGLL GPTIQAEVYDTVVIVLKNMASHPVSLHAVGVSYWKASEGAEYEDQ TSQKEKEDDNVIPGESHTYVWQVLKENGPMASDPPCLTYSYFSHV DLVKDLNSGLIGALLVCKEGSLAKERTQTLQEFVLLFAVFDEGKS WHSETNASLTQAEAQHELHTINGYVNRSLPGLTVCHKRSVYWHVI GMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTFLMDLGQ FLLFCHIPSHQHDGMEAYVKVDSCPEEPQLRMKNNEDKDYDDGLY DSDMDVVSFDDDSSSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYA PSGPTPNDRSHKNLYLNNGPQRIGKKYKKVRFVAYTDETFKTREAI QYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGINYVTPLHTGR 3

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LPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFI NLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENR SWYLTENMQRFLPNADVVQPHDPEFQLSNIMHSINGYVFDNLQLS VCLHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS GETVFMSMENPGLWVLGCHNSDFRNRGMTALLKVSSCNRNIDDY YEDTYEDIPTPLLNENNVIKPRSFSQNSRHPSTKEKQLKATTTPENDI EKIDLQ SGERTQLIK AQ S VS S SDLLMLLGQNPTPRGLFLSDLRE ATD RADDHSRGAIERNKGPPEVASLRPELRHSEDREFTPEPELQLRLNEN LGTNTTVELKKLDLKISSSSDSLMTSPTIPSDKLAAATEKTGSLGPP NMSVHFNSHLGTIVFGNNSSHLIQSGVPLELSEEDNDSKLLEAPLM NIQES SLRENVLSMESNRLFKEERIRGP ASLIKDNALFKVNIS S VKTN RAPVNLTTNRKTRVAIPTLLIENSTSVWQDIMLERNTEFKEVTSLIH NETFMDRNTTALGLNHVSNKTTLSKNVEMAHQKKEDPVPLRAEN PDLSSSKIPFLPDWIKTHGKNSLSSEQRPSPKQLTSLGSEKSVKDQN FLSEEKVVVGEDEFTKDTELQEIFPNNKSIFFANLANVQENDTYNQ EKKSPEEIERKEKLTQENVALPQAHTMIGTKNFLKNLFLLSTKQNV AGLEEQPYTPILQDTRSLNDSPHSEGIHMANFSKIREEANLEGLGNQ TNQMVERFPSTTRMSSNASQHVITQRGKRSLKQPRLSQGEIKFERK VIANDTSTQWSKNMNYLAQGTLTQIEYNEKEKRAITQSPLSDCSMR NHVTIQMNDSALPVAKESASPSVRHTDLTKIPSQHNSSHLPASACN YTFRERTSGVQEGSHFLQEAKRNNLSLAFVTLGITEGQGKFSS LGK SATNQPMYKKLENTVLLQPGLSETSDKVELLSQVHVDQEDSFPTKT SNDSPGHLDLMGKIFLQKTQGPVKMNKTNSPGKVPFLKWATESSE KIPSKLLGVLAWDNHYDTQIPSEEWKSQKKSQTNTAFKRKDTILPL GPCENNDSTAAINEGQDKPQREAMWAKQGEPGRLCSQNPPVSKH HQREITVTTLQPEEDKFEYDDTFSIEMKREDFDIYGDYENQGLRSFQ KKTRHYFIAAVERLWDYGMSRSPHILRNRAQSGDVQQFKKVVFQE FTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIVVTFKNQASRPYS FYSSLISYDEDEGQGAEPRRKFVNPNETKIYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLICRSNTLNPAHGRQVTVQE FALVFTIFDETKSWYFTENLERNCRAPCNVQKEDPTLKENFRFHAI NGYVKDTLPGLVMAQDQKVRWYLLSMGSNENIHSIHFSGHVFTVR KKEEYKMAVYNLYPGVFETVEMLPSQVGIWRIECLIGEHLQAGMS TLFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYS GSINAWSTKDPFSWIKVDLLAPMIIHGIMTQGARQKFSSLYVSQFII MYSLDGNKWHSYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIAQYI RLHPTHYSIRSTLRMELLGCDFNSC SMPLGMESKAISD AQITAS S YL SSMLATWSPSQARLHLQGRTNAWRPQANNPKEWLQVDFRKTMK VTGITTQGVKSLLISMYVKEFLIS S SQDGHNWTLFLQNGKVKVFQG NRDSSTPVRNRLEPPLVARYVR LHPQSWAHHIALRLEVLGCDTQQPA FVIII (pig) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD 4

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DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRT PMPKIQNVS S SDLLMLLRQSPTPHGLSLSDLQEAKYETF SDDPSPGA IDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATE LKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQL DTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKN VSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSAT NRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKN ATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKM LFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLS EKNKVWGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHN QEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNV EGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLG NQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELE KRIIVDDTST QWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLT RSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKK DSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSV TYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPG HLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLL DPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESN HAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRT TLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAW AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVY SNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAW STKEPF SWIKVDLLAPMIIHGIKTQGARQKF S SLYISQFIIMYSLDGK KWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI RSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTNMF ATW SPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTP VVNSLDPPLLTRYLRIH PQSWVHQIALRMEVLGCEAQDLY FVIII (Mouse) AIRRYYLGAVELSWNYIQSDLLSVLHTDSRFLPRMSTSFPFNTSIMY KKTVFVEYKDQLFNIAKPRPPWMGLLGPTIWTEVHDTVVITLKNM ASHPVSLHAVGVSYWKASEGDEYEDQTSQMEKEDDKVFPGESHT YVWQVLKENGPMASDPPCLTYSYMSHVDLVKDLNSGLIGALLVC KEGSLSKERTQMLYQFVLLFAVFDEGKSWHSETNDSYTQSMDSAS ARDWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEIHSI 5

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FLEGHTFFVRNHRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKH DGMEAYVKVDSCPEESQWQKKNNNEEMEDYDDDLYSEMDMFTL DYDSSPFIQIRSVAKKYPKTWIHYISAEEEDWDYAPSVPTSDNGSYK SQYLSNGPHRIGRKYKKVRFIAYTDETFKTRETIQHESGLLGPLLYG EVGDTLLIIFKNQASRPYNIYPHGITDVSPLHARRLPRGIKHVKDLPI HPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINPERDLASGLIGP LLICYKESVDQRGNQMMSDKRNVILFSIFDENQSWYITENMQRFLP NAAKTQPQDPGFQASNIMHSINGYVFDSLELTVCLHEVAYWHILSV GAQTDFLSIFF SGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLW VLGCHNSDFRKRGMTALLKVSSCDKSTSDYYEEIYEDIPTQLVNEN NVIDPRSFFQNTNHPNTRKKKFKDSTIPKNDMEKIEPQFEEIAEMLK VQSVSVSDMLMLLGQSHPTPHGLFLSDGQEAIYEAIHDDHSPNAID SNEGPSKVTQLRPESHHSEKIVFTPQPGLQLRSNKSLETTIEVKWKK LGLQ VS SLP SNLMTTTILSDNLK ATFEKTD S SGFPDMP VHS S SKLS T TAFGKKAYSLVGSHVPLNASEENSDSNILDSTLMYSQESLPRDNILS IENDRLLREKRFHGIALLTKDNTLFKDNVSLMKTNKTYNHSTTNEK LHTESPTSIENSTTDLQDAILKVNSEIQEVTALIHDGTLLGKNSTYLR LNHMLNRTT STKNKDIFHRKDEDPIPQDEENTIMPFSKMLFLSES SN WFKKTNGNNSLNSEQEHSPKQLVYLMFKKYVKNQSFLSEKNKVT VEQDGFTKNIGLKDMAFPHNMSIFLTTLSNVHENGRHNQEKNIQEE IEKEALIEEKVVLPQVHEATGSKNFLKDILILGTRQN ISLYEVHVPVL QNITSINNSTNTVQIHMEHFFKRRKDKETNSEGLVNKTREMVKNYP SQKNITTQRSKRALGQFRLSTQWLKTINCSTQCIIKQIDHSKEMKKF ITKSSLSDSSVIKSTTQTNSSDSHIVKTSAFPProLKRSPFQNKFSHVQ ASSYIYDFKTKSSRIQESNNFLKETKINNPSLAILPWNMFIDQGKFTS PGKSNTNSVTYKKRENIIFLKPTLPEESGKIELLPQVSIQEEEILPTET SHGSPGHLNLMKEVFLQKIQGPTKWNKAKRHGESIKGKTESSKNT RSKLLNHHAWDYHYAAQIPKDMWKSKEKSPEIISIKQEDTILSLRP HGNSHSIGANEKQNWPQRETTWVKQGQTQRTCSQIPPVLKRHQRE LSAFQSEQEATDYDDAITIETIEDFDIYSEDIKQGPRSFQQKTRHYFI AAVERLWDYGMSTSHVLRNRYQSDNVPQFKKVVFQEFTDGSFSQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISY KEDQRGEEPRRNFVKPNETKIYFWKVQHHMAPTEDEFDCKAWAY FSDVDLERDMHSGLIGPLLICHANTLNPAHGRQVSVQEFALLFTIFD ETKSWYFTENVKRNCKTPCNFQMEDPTLKENYRFHAINGYVMDTL PGLVMAQDQRIRWYLLSMGNNENIQSIHFSGHVFTVRKKEEYKMA VYNLYPGVFETLEMIPSRAGIWRVECLIGEHLQAGMSTLFLVYSKQ CQIPLGMASGSIRDFQITASGHYGQWAPNLARLHYSGSINAWSTKE PFSWIKVDLLAPMIVHGIKTQGARQKFSSLYISQFIIMYSLDGKKWL SYQGNSTGTLMVFFGNVDSSGIKHNSFNPPIIARYIRLHPTHSSIRST LRMELMGCDLNSCSIPLGMESKVISDTQITASSYFTNMFATWSPSQ ARLHLQGRTNAWRPQVNDPKQWLQVDLQKTMKVTGIITQG VKSL FTSMF VKEFLIS S SQDGHHWTQILYNGKVKVFQGNQDS STPMMNS LDPPLLTRYLRIHPQIWEHQIALRLEILGCEAQQQY FVIII BDD variant (US Patent No. MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVD ARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLG PTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQT SQREKEDDKVFPGGSHTLVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGWKILL 6

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7632921, SEQ GO

NO: 3)

BDD-2

FVIII

HSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAE DYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTD ETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNV ILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGY VFD SLQLS VCLHEVAYW YILSIGAQTDFLS VFF SGYTFKHKMVYED TLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS SCD KNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRT TLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAW AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVY SNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAW STKEPF SWIKV DLLAPMIIHGIKTQGARQKF S SLYISQFIIMYSLDGK KWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATW SPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTP VVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY______ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCD KNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEM KKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWD YGMS S SPHV LRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC

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RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSM PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP Q VNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ SWVHQIALRMEVLGCEAQDLY BDD-3 FVIII (G1648) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVE MKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPH VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPY IRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTV QEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSM PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP Q VNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ SWVHQIALRMEVLGCEAQDLY 8 BDD-4 FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH 9

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BDD-5

FVIII

DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSF SQQ SPRSFQKKTRHYFIA AVERLWD YGMS S SPHV LRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSM PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP Q VNNPKEWL Q VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ SWVHQIALRMEVLGCEAQDLY_______________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKQSPRSFQKKTRHYFIAA VERLWD YGMS S SPHVLRNRAQ SGS VPQFKKVVFQEFTDGSFTQPL YRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEE DQRQGAEPRKNFVKPNETKTYFWK VQHHMAPTKDEFDCKAWAY FSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAL YNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNK CQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTK EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKW

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QTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRS TLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSP SKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGV KSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPV VNGCSLDPPLLTRYLRIHPQSW BDD-6 FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDTISVEMKK EDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAE VEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNET KTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLL VCHTNTLNPAHGRQVTVQE FALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQI TASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHG IKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMP LGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQ VNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLY eleven BDD-7 FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG 12

-41 041874 BDD-8 FVIII predecessor (US Patent No. 6,818,439 SEQ ID NO: 47)

LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSF SQSPRSFQKKTRHYFIAAVERLWD YGMS S SPHVL RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIR AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGP LLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLL SMGSNENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQI TASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHG IKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMP LGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQ VNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLY________________________ MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVD ARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLG PTIQAEVYD TVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQT SQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSW HSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAE DYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTD ETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNV ILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGY VFD SLQLS VCLHEVAYW YILSIGAQTDFLS VFF SGYTFKHKMVYED TLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS SCD KNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRT TLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAW AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMG SNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVY SNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAW STKEPF SWIKVDLLAPMIIHGIKTQGARQKF S SLYISQFIIMYSLDGK KWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATW SPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG

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VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTP VVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY mature BDD-9 FVIII (US Patent No. 6818439) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSF SQNPP VLKRHQREITRTTLQ SDQEEID YDDTIS VEM KKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWD YGMS S SPHV LRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNP AHGRQVTVQEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSM PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP Q VNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ SWVHQIALRMEVLGCEAQDLY 14 BDD-10 FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP 15

- 43 041874

GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVE MKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPH VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPY IRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG N VD S SGIK HNIFNPPII AR YIRLHPTH YSIRSTLRMELMGCDLNSC SM PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP QVNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ __SWVHQIALRMEVLGCEAQDLY_______________________ BDD-11 ATRATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT FVIII SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITL

KNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGG SHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDA ASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEV HSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYVK VDS CPEEPQLRMKNNEEAED YDDDLTD SEMD V VRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPD DRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGIL GPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFK YKWT VT VEDGPTK SDPRCLTRYYS SF VNMERD LASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTE NIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVA YWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMS MENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDI SAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTI SVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLL GPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNF VKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHS GLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENM ERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSN ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHI RDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLN SCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLI SSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRI HPQ SWVHQIALRMEVLGCEAQDLY

- 44 041874

BDD-12 FVIII

BDD-13 FVIII

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEroYDDTISVE MKKEDFDIFDEDENQ SPRSFQKKTRHYFIAA VERLWD YGMS S SPH VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPY IRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHG RQVTVQEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVD S SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSM PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP QVNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ SWVHQIALRMEVLGCEAQDLY_______________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVV YKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIF LEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLY SRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEM

KKEDFDIFDEDENQ SPRSFQKKTRHYFIAAVERLWD YGMS S SPHVL RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIR AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGP LLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLL SMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQI TASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHG IKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMP LGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQ VNNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLY

The present invention also provides CFXTEN, which contains FVIII with various amino acid deletions, insertions and substitutions made in the FVIII sequences of Table. 1, which retain procoagulant activity. Examples of conservative amino acid substitutions in polypeptide sequences are shown in table. 2. In embodiments of CFXTEN in which the sequence identity of FVIII is less than 100% as compared to the specific sequence disclosed herein, the present invention provides for the replacement of any of the other 19 naturally occurring L-amino acids with a given amino acid residue of that FVIII, which may be at any position within the FVIII sequence that contains contiguous amino

- 45 041874 acid residues. If any substitution results in an undesirable change in procoagulant activity, then one of the alternative amino acids may be used and protein design evaluated using the methods described herein (e.g., assays in Table 49) or using any of the methods and principles of conservative and non-conservative mutations as set forth, for example, in US Pat. No. 5,364,934, the contents of which are incorporated by reference in their entirety, or using methods generally known in the art. In a preferred substitution, the FVIII component of embodiments of CFXTEN is modified by replacing the R1648 residue (numbered relative to the naturally mature FVIII form) with glycine or alanine to prevent proteolytic processing into the heterodimer form. In a further substitution, the FVIII component of the CFXTEN embodiments is modified by replacing the Y1680 residue (numbering relative to the natural mature form of FVIII) with phenylalanine. In a further embodiment of the present invention, the FVIII component of the CFXTEN embodiments is modified by replacing the Y1680 residue (numbering relative to the natural mature FVIII) with phenylalanine and the R1648 residue (numbering relative to the natural mature FVIII) with glycine or alanine.

In a separate embodiment of the present invention, the FVIII fusion protein composition has one or more amino acid substitutions designed to reduce the binding of FVIII factor inhibitors to epitopes recognized by the antibodies of Table 1. 9 which contain, but are not limited to, Lys(377), Lys(466), Lys(380), Ser(488), Arg(489), Arg(490), Leu(491), Lys(493) substitutions , Lys(496), His(497), Lys(499), Lys(512), Lys(523), Lys(556), Met(2199), Phe(2200), Leu(2252), Val(2223) and Lys(2227). In addition, variants may contain, for example, polypeptides, with one or more amino acid residues added or deleted at or near the N- or C-terminus of the full length native amino acid sequence or FVIII domain, as long as the variant retains a portion if not all of the procoagulant activity of the native peptide. The resulting FVIII sequences that retain at least a portion (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% or more) procoagulant activity, as compared to native circulating FVIII, is considered applicable to the fusion protein in the compositions of the present invention. Examples of FVIII variants known in the art include those described in US Pat. No. 6,316,226; 6818439; 7632921; 20080227691, which are incorporated herein by reference. In a particular embodiment of the present invention, the FVIII sequence variant has aspartic acid at amino acid position 75 replaced by valine (numbering relative to the naturally occurring mature form of FVIII).

table 2

Illustrative conservative amino acid substitutions

original balance Illustrative substitutions Ala (A) val; leu; ile Arg(R) lys; gin; asn Asn(N) gin; his; lys; arg Asp(D) Glu Cys(C) Ser Gln(Q) Asn Glu(E) asp Gly(G) Pro His(H) asn:gin:lys:arg He(I) leu; val; met; ala; phe: norleucine Leu(L) norleucine: ile: val; met; ala:phe Lys (K) arg:gin:asn Met(M) leu; phe; ile Phe(F) leu: val: ile; ala Pro (P) gly Ser(S) Thr Thr(T) Ser TRP(W) Tyr Tyr(Y) Trp:phe:thr:ser Wai (V) lie; leu; met; phe; ala; norleucine

- 46 041874

III) Expanded recombinant polypeptides.

In a separate aspect, the present invention provides XTEN polypeptide compositions that are useful as fusion protein partner(s) for binding to, and/or inserting within, a FVIII polypeptide resulting in a CFXTEN fusion protein. XTEN are generally non-naturally occurring, substantially non-repetitive sequence polypeptides that have little or no secondary or tertiary structure under physiological conditions. XTEN typically has about 36 to about 3000 amino acids, of which most or all of the amino acids are small hydrophilic amino acids. As used herein, XTEN specifically excludes whole antibodies or antibody fragments (eg, single chain antibodies and Fc fragments). XTEN polypeptides are useful as fusion protein partners in that they play a variety of roles that have certain desirable pharmacokinetic, physico-pharmacological and pharmaceutical properties when attached to the FVIII protein to create the CFXTEN fusion protein. Such CFXTEN fusion protein compositions have enhanced properties over the corresponding non-XTEN FVIII, making them useful in the treatment of certain conditions associated with FVIII deficiencies or bleeding disorders, as described in more detail below.

Selection criteria for XTEN fused to FVIII proteins are used to generate patentable fusion protein compositions that are typically related to physical/chemical properties and conformational structure attributes of Xten which in turn are used to confer enhanced pharmaceutical, pharmacological and pharmacokinetic properties FVIII fusion protein compositions. The unstructured characterization and physical/chemical properties of XTEN result in, inter alia, a total amino acid composition disproportionately limited to 4-6 hydrophilic amino acids, binding of amino acids in a quantitative non-repeating structure, and length of the XTEN polypeptide. In a preferred feature shared with XTEN but uncharacteristic of polypeptides, the XTEN properties disclosed herein are not associated with absolute basic amino acid sequences, as evidenced by the variety of illustrative sequences in Table 1. 4 which, in various length ranges, have similar properties, many of which are described in the examples. The XTENs of the present invention may exhibit one or more or all of the following properties: advantageous unstructured conformations, conformational flexibility, increased water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, a certain degree of charge, and an increase in hydrodynamic (or Stokes) radii; properties that may make them particularly useful as fusion protein partners. Non-limiting examples of the improved properties that XTEN confer on XTEN-fused FVIII-containing fusion proteins over non-XTEN FVIII include increased overall solubility and/or metabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, decreased absorption rate when administered subcutaneously or intramuscularly, decreased binding to factor VIII clearance receptors, decreased reactivity to anti-antibodies, applicable improvements in substrate interactions, and/or improved pharmacokinetic properties when administered to a subject. The improved pharmacokinetic properties of CFXTEN formulations compared to non-XTEN FVIII include longer terminal half-life (e.g., two-fold, three-fold, four-fold or greater), increased area under the curve (AUC) (e.g., 25%, 50%, 100% or more), lower volume of distribution and improved absorption after subcutaneous or intramuscular injection (an advantage over commercially available forms of FVIII that must be administered intravenously). In addition, CFXTEN compositions that contain cleaved sequences (described in more detail below) are believed to slow down the secretion of biologically active FVIII so that the administered CFXTEN acts as a depot. It is specifically contemplated that inventive CFXTEN fusion proteins may exhibit one or more, or any combination, of the improved properties disclosed herein. As a result of these improved properties, CFXTEN formulations are believed to allow for less frequent dosing compared to non-XTEN related FVIII when administered at similar dosages. Such CFXTEN fusion protein compositions may be used to treat certain factor VIII related diseases as described herein.

Many methods and assays are known in the art for determining the physical/chemical properties of proteins, such as CFXTEN compositions that contain XTEN. Such properties include, but are not limited to, secondary or tertiary structure, solubility, protein aggregation, stability, absolute and apparent molecular weight, purity and uniformity, melting properties, contamination, and water content. Methods for analyzing these properties include analytical centrifugation, EPR, ion exchange HPLC, size exclusion HPLC, reverse phase HPLC, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, ion exchange HPLC, size exclusion HPLC, IR, NMR, Raman scattering spectroscopy, refractometry and UV/visible spectroscopy. Additional spo

- 47 041874 are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.

The XTEN component(s) of CFXTEN are designed to behave under physiological conditions as denatured peptide sequences, regardless of polymer length. Denatured describes the state of the peptide in solution, which is characterized by a large conformational freedom of the peptide chain. Most peptides and proteins assume a denatured conformation in the presence of high concentrations of denaturants or at elevated temperatures. Peptides in the denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by the absence of long range interactions as determined by NMR. Denatured conformation and unstructured conformation are used herein as synonyms. In certain embodiments, the present invention provides XTEN sequences that, under physiological conditions, are largely devoid of secondary structure. In other cases, XTEN sequences are essentially devoid of secondary structure under physiological conditions, while XTEN may assume a random helix conformation. Significantly devoid, as used in this context, means that at least 50% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure formation, as measured or determined using those described herein. Substantially devoid, as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the amino acid residues of XTEN XTEN sequences do not contribute to the formation of secondary structure, as measured or determined using described in this document.

Many methods have been developed in the art to distinguish between the presence or absence of secondary and tertiary structures in a given polypeptide. In particular, the secondary structure can be measured spectrophotometrically, for example, using circular dichroism spectroscopy in the far ultraviolet region of the spectrum (190-250 nm). Each of the elements of the secondary structure, such as the alpha helix and the beta sheet, gives an increase in the characteristics of the shape and size of the CD spectra, as well as the absence of these structural elements. Secondary structure can also be predicted in the case of a polypeptide sequence using certain computer programs or algorithms such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe algorithm -Robson (GOR) (Gamier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553) as described in US Patent and Published Application No. 20030228309A1 . For a given sequence, algorithms can predict whether or not some secondary structure exists at all, expressed as the total number and/or percentage of sequence residues that form, for example, alpha helices or beta sheets, or the percentage of sequence residues that are predicted to lead to the formation of a random spiral (which needs a secondary structure).

In a specific embodiment of the present invention, the XTEN sequences that are used in the subject's fusion protein compositions have an alpha helix percentage ranging from 0% to less than about 5%, as determined using the Chou-Fasman algorithm. In a further embodiment of the present invention, the XTEN fusion protein composition sequences have a percentage of beta sheets that ranges from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In certain embodiments of the present invention, the XTEN fusion protein composition sequences have an alpha helix percentage that ranges from 0% to less than about 5% and a beta sheet percentage ranges from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In certain embodiments, the XTEN fusion protein composition sequences have an alpha helix percentage of less than about 2% and a beta sheet percentage of less than about 2%. The XTEN fusion protein composition sequences have a high percentage of random helix as determined by the GOR algorithm. In certain embodiments, the XTEN sequence is at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% , at least about 95%, at least about 96%, at least about 97%, at least about 98%, and more preferably at least about 99% random spiral as determined by the GOR algorithm. In certain embodiments, the XTEN fusion protein composition sequences have an alpha helix percentage that ranges from 0% to less than about 5% and a beta sheet percentage that ranges from 0% to less than about 5%, as determined by using the Chou-Fasman algorithm and at least about 90% random spiral, as determined using the GOR algorithm. In further embodiments of the present invention, the XTEN fusion protein composition sequences have an alpha helix percentage of less than about 2% and a beta sheet percentage of less than about 2%, at least about 90% random helix, as determined by the GOR algorithm.

1. Non-repeating sequences.

The XTEN sequences of the CFXTEN embodiments are expected to be

- 48 041874 essentially non-repetitive. In general, repeat amino acid sequences tend to aggregate or form higher order structures, as evidenced by naturally occurring repeat sequences such as collagens and leucine zippers. These repeating amino acids may also tend to form contacts, resulting in crystalline or pseudocrystalline structures. In contrast, the low tendency of non-repeating sequences to aggregate allows for a long sequence XTEN structure with a relatively low frequency of charged amino acids that would otherwise be more prone to aggregation if the sequences were repetitive. The non-repeatability of an XTEN subject can be observed by assessing one or more of the following features. In a particular embodiment of the present invention, the substantially non-repeating XTEN sequence has about 36, or at least 72, or at least 96, or at least 144, or at least 288, or at least 400, or at least 500 , or at least 600, or at least 700, or at least 800, or at least 864, or at least 900, or at least 1000, or at least 2000, up to about 3000 or more amino acid residues , or has a length that ranges from about 36 to about 3000, about 100 to about 500, about 500 to about 1000, about 1000 to about 3000 amino acids, and residues in which no three contiguous amino acids in the sequences are amino acids of the same type, provided that the amino acid is not a serine, in which case no more than three adjacent amino acids are serine residues. In a further embodiment of the present invention, as described in more detail below, the substantially non-repetitive XTEN sequence contains motifs of 9 to 14 amino acid residues, with motifs consisting of 4 to 6 amino acid types selected from glycine (G), alanine ( A), serine (S), threonine (T), glutamate (E) and proline (P), and provided that the sequence of any two adjacent amino acid residues in any motif in the sequence motif does not repeat more than twice.

The degree of repeatability of a polypeptide or gene can be measured using computer programs or algorithms, or by other methods known in the art. According to the present invention, the algorithms that will be used to calculate the degree of repeatability of a particular polypeptide, such as XTEN, are disclosed herein and provide examples of sequences that are analyzed using the algorithm (see examples, below). In a separate aspect, the repeatability of a given length polypeptide is calculated (hereinafter the number of subsequences) according to the formula given in Equation 1:

„ %Zi ^Count i t

Number of subsequences —=— ⁴ ------- I t

where m = (length of amino acids of the polypeptide) - (length of amino acids of the subsequence) + 1; and Counti = total number of occurrences of each unique subsequence in the sequence^

An algorithm called SegScore was developed to apply the above equation to quantify the repeatability of polypeptides such as XTEN, providing the number of subsequences given that amino acid sequences with a given length n per repeatability by determining the number (calculation) of unique sequences with length s, occurring in the given length divided by the absolute number of subsequences within the given sequence length. Fig. 27 illustrates a logical block diagram of the SegScore algorithm, while FIG. 28 illustrates a schematic of how the subsequence count is calculated using a dummy XTEN with 11 amino acids and a subsequence length of 3 amino acid residues. For example, a given polypeptide length of 200 amino acid residues has 192 overlapping 9 amino acid subsequences and 198 3 amino acid subsequences, but the subsequence score of any given polypeptide will depend on the absolute number of unique subsequences and how often each unique subsequence (meaning different amino acid sequences) appear in a given sequence length.

In the context of the present invention, the number of subsequences means the sum of the matches of each unique 3-dimensional frame within 200 consecutive amino acids of the total XTEN polypeptide divided by the absolute number of unique 3-dimensional subsequences within the 200 amino acid sequence. Examples of such subsequence numbers derived from 200 consecutive amino acids, repetitive and non-repeating polypeptides are shown in Example 45. In a separate embodiment, the present invention provides a CFXTEN that contains a single XTEN, wherein the XTEN has a subsequence count of less than 12, more preferably less than 10, even more preferably less than 9, most preferably less than 8, even more preferably less than 7, even more preferably less than 6, and even more preferably less than 5. In a further embodiment, the present invention provides CFXTEN which contains at least two to about six XTENs in which 200 XTEN amino acids have less than 10 subsequences, more preferably

- 49 041874 less than 9, even more preferably less than 8, most preferably less than 7, even more preferably less than 6, and even more preferably less than 5. In embodiments of the CFXTEN fusion protein compositions described herein, the XTEN component of the fusion protein with the number of subsequences of 10 or less (ie 9, 8, 7, etc.) is also essentially non-repeating.

It is believed that the non-repeating characterization of the XTEN of the present invention, together with the specific amino acid types that predominate in XTEN rather than the absolute primary sequence, is believed to provide many of the enhanced physicochemical and biological properties of the CFXTEN fusion protein. These improved properties include higher expression of the fusion protein in the host cell, greater genetic stability of the gene encoding Xten, higher solubility, less tendency to aggregate, and improved pharmacokinetics of the resulting CFXTEN compared to fusion proteins containing polypeptides having repeat sequences. . These improved properties allow for more efficient production, lower cost of goods, and facilitate the development of XTEN-containing pharmaceutical formulations that contain extremely high protein concentrations, in some cases exceeding 100 mg/mL. In addition, the XTEN polypeptide sequences of embodiments of the present invention are designed to have a low degree of internal repeatability in order to reduce or substantially eliminate immunogenicity when administered to a mammal. Polypeptide sequences consisting of short repetitive motifs largely limited to only three amino acids, such as glycine, serine, and glutamate, when administered to a mammal, can result in relatively high antibody titers despite the absence of predicted T-cell epitopes in these sequences. This may be due to the repetitive nature of polypeptides, as it has been shown that repetitive epitope immunogens that contain protein aggregates, cross-linked immunogens, and repetitive carbohydrates are highly immunogenic and can, for example, lead to cross-linking of B-cell receptors that result in B-cell activation. -cells. (Johansson, J., et al. (2007) Vaccine, 25:1676-82; Yankai, Z., et al. (2006) Biochem Biophys Res Commun, 345:1365-71; Hsu, C. T., et al. ( 2000) Cancer Res, 60:3701-5); Bachmann M.F., et al. Eur J Immunol. (1995) 25(12):3445-3451).

2. Illustrative sequence motifs.

The present invention encompasses an XTEN used as fusion partners that contains multiple short sequence units, or motifs, in which the amino acid sequences of the motifs are non-repetitive. The non-repeating property is encountered despite using a modular design approach using a library of sequence motifs that are multimerized to create an XTEN sequence. Thus, while an XTEN sequence can be composed of multiple units from as few as four different types of sequence motifs, because the motifs themselves typically consist of non-repeating amino acid sequences, the overall XTEN sequence is designed to visualize a substantially non-repeating sequence.

In a particular embodiment of the present invention, XTEN has a substantially non-repeating sequence of more than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues, or even longer, with at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs , and despite the fact that each of the motifs has from about 9 to 36 amino acid residues. In additional embodiments of the present invention, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the sequences XTEN consists of non-overlapping sequence motifs, with each motif having 9 to 14 amino acid residues. In additional embodiments of the present invention, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the sequence XTEN consists of non-overlapping sequence motifs, with each motif having 12 amino acid residues. In these embodiments, it is preferred that the sequence motifs consist largely (eg, 90% or more), or entirely, of small hydrophilic amino acids such that, in general, the sequence has an unstructured, conformationally labile characteristic. Examples of amino acids that are included in XTEN are, for example, arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. As a result of testing variables such as codon optimization, junction polynucleotides encoding sequence motifs, protein expression, charge distribution and solubility of the expressed protein, and secondary and tertiary structures, it was found that the performance-enhanced XTEN compositions disclosed herein generally or mostly contain residues of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P),

- 50 041874 while the sequences are intended to be essentially non-repetitive. In a particular embodiment of the present invention, the XTEN sequences have predominantly four to six amino acid types selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P), which are located in a substantially non-repetitive sequence that is greater than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues in length. In a particular embodiment of the present invention, the XTEN sequence is made up of 4, 5, or 6 amino acid types selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In certain embodiments, the XTEN has sequences of more than about 36 to about 1000, or about 100 to about 2000, or about 400 to about 3000 amino acid residues, with at least about 80% of the sequence consisting of non-overlapping sequence motifs, with each motif having 9 to 36 amino acid residues, and with at least 90%, or at least 91%, or at least 92%, or at least 93% , or at least 94%, or at least 95%, or at least 96%, or at least 97%, or 100% of each of the motifs are composed of 4 to 6 types of amino acids selected from glycine (G) , alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and no more than 30% of any one type of amino acid in raw XTEN. In additional embodiments of the present invention, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs, with each of the motifs having 9 to 36 amino acid residues, with the motifs consisting of 4 to 6 amino acid types selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and that the content of any one type of amino acid in whole-grain XTEN does not exceed 40%, or about 30%, or 25%, or about 17%. In additional embodiments of the present invention, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs, with each of the motifs having 12 amino acid residues consisting of 4 to 6 amino acid types selected from glycine (G), alanine ( A), serine (S), threonine (T), glutamate (E), and proline (P) and, provided that the content of any one type of amino acid in whole-grain XTEN does not exceed 40%, or 30%, or about 25% . In other embodiments of the present invention, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the XTEN sequence consists of non-overlapping sequence motifs, with each motif having 12 amino acid residues consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P).

In additional embodiments of the present invention, XTEN contains substantially non-repeating sequences of more than about 36 to about 3000 amino acid residues, with at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the sequence consists of non-overlapping sequence motifs from 0 to 14 amino acid residues, while the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and that in any motif the sequence of any two adjacent amino acid residues is not repeated more than twice in the sequence motif. In additional embodiments of the present invention, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98% or about 99% of the XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues, with the motifs consisting of four to six amino acid types selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and provided that the sequence of any two adjacent amino acid residues in any sequence motif in the sequence motif does not repeat more than twice. In additional embodiments of the present invention, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98% or about 99% of the XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues, with the motifs consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and provided that in any sequence motif the sequence of any two adjacent amino acid residues is not repeated more than twice in the sequence motif. In other embodiments, the XTEN is composed of 12 amino acid sequence motifs, with the amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P ), and provided that the sequence of any two adjacent amino acid residues in any sequence motif does not repeat more than two times, and that the content of any one type of amino acid in full-length XTEN does not exceed 30%. The previous embodiments of the present invention are examples of essentially non-repeating XTEN sequences. Additional examples are detailed below.

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In certain embodiments, the present invention provides CFXTEN compositions that contain one, or two, or three, or four, five, six, or more non-repeating XTEN sequence(s) of from about 36 to about 1000 amino acid residues, or a total of about 100 up to about 3000 amino acid residues, with at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of several units of four or more non-overlapping sequence motifs selected from the amino acid sequences from table. 3, while, in general, the sequence remains essentially non-repetitive. In certain embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% of the sequence consists of several units of non-overlapping sequences selected from a single family of motifs selected from Table. 3, which leads to a family of sequences. As used herein, family means that an XTEN has motifs selected only from a single category of motifs from Table. 3; those. AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in XTEN not from the motif family are chosen in order to achieve the desired property, for example, to allow the inclusion of a restriction site by encoding nucleic acids, inclusion of a cleavage sequence, or achieve better binding to the FVIII CFXTEN clotting factor component. In certain embodiments of the XTEN families, the XTEN sequence contains multiple units of non-overlapping sequence motifs of the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the motifs BD, with the resulting XTEN, which shows the range of homology described above. In additional embodiments, the implementation of the present invention XTEN contains several units of sequence motifs from two or more families of motifs from table. 3. These sequences can be selected to achieve the desired physical/chemical properties, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repeatability, which are conferred by the amino acid composition of the motifs described more fully below. In the embodiments of the present invention described above in this paragraph, XTEN-embedded motifs can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.

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Table 3

XTEN sequence motifs of 12 amino acids and a family of motifs

Family of motifs SEQUENCE OF MOTIVE SEQID NO: AD GESPGGSSGSES 19 AD GSEGSSGPGESS 20 AD GSSESGSSEGGP 21 AD GSGGEPSESGSS 22 AE, AM GSPAGSPTSTEE 23 AE, AM, AQ GSEPATSGSETP 24 AE, AM, AQ GTSESATPESGP 25 AE, AM, AQ GTSTEPSEGSAP 26 AF, AM GSTSESPSGTAP 27 AF, AM GTSTPESGSASP 28 AF, AM GTSPSGESTAP 29 AF, AM GSTSSTAESPGP thirty AG, AM GTPGSGTASSSP 31 AG, AM GSSTPSGATGSP 32 AG, AM GSSPSASTGTGP 33 AG, AM GASPGTSSTGSP 34 AQ GEPAGSPTSTSE 35 AQ GTGEPSSTPASE 36 AQ GSGPSTESAPTE 37 AQ GSETPSGPSETA 38 AQ GPSETSTSEPGA 39 AQ GSPSEPTEGTSA 40 BC GSGASEPTSTEP 41 BC GSEPATSGTEPS 42 BC GTSEPSTSEPGA 43 BC GTSTEPSEPGSA 44 BD GSTAGSETSTEA 45 BD GSETATSGSETA 46 BD GTSESATSESGA 47 BD GTSTEASEGSAS 48

Denotes individual motif sequences which, when used together in different permutations, result in a family of sequences.

In certain embodiments of the XTEN families, the XTEN sequence contains multiple units of non-overlapping sequence motifs of the AD motif family, the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family , with the resulting XTEN, which exhibits the range of homology described above. In additional embodiments, the implementation of the present invention XTEN contains several units of sequences of the motif from two or more families of motifs from table. 3 selected to achieve the desired physicochemical characteristics, which include properties such as net charge, lack of secondary structure, or lack of repeatability that can be attributed to the amino acid composition of the motifs described more fully below. In the embodiments of the present invention described above in this paragraph, motifs or motif regions embedded in XTEN can be selected and assembled using the methods described herein in order to achieve an XTEN of from about 36, from about 42, from about 72, about 144, about 288, about 576, about 864, about 1000, about 2000 to about 3000 amino acid residues, or any length in between. Non-limiting examples of sequences of the XTEN family, used to include the subject of CFXTEN, are presented in table. 4. It is assumed that the specified sequence, which is mentioned in relation to the table. 4 has the sequence shown in table. 4, while the generalized reference to the AE144 sequence, for example, covers any AE sequence having 144 amino acid residues; e.g., AE144_1 A, AE144_2A, etc., or a generalized reference to an AG144 sequence, e.g., is intended to encompass any AG sequence that has 144 amino acid residues, e.g., AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, etc.

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Table 4 XTEN polypeptides

Title XTEN Amino acid sequence SEQID NO: AE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS 49 AE42 1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 50 AE42 2 PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG 51 AE42 3 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP 52 AG42 1 GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATTGSPGPSGP 53 AG42 2 GPGTPGSGTASSSPGSSTPSGATTGSPGSSPSASTGTGPGASP 54 AG42 3 SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA 55 AG424 SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG 56 AE48 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSST GS 57 AM48 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGAT GS 58 AE144 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTE EGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSE 59 AE144_1 A SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APG 60 AE144_2 A TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPG 61 AE144_2 B TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPG 62 AE144_3 A SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS APG 63 AE144_3 B SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS APG 64 AE144_4 A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APG 65 AE144_4 B TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APG 66 AE144_5 A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEG 67 AE144_6 B TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA 68

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PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APG AF144 GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPG PGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSA 69 AG144_1 SGTASS SPGS STPSGATGSPGTPGSGTAS S SPGS STPSGATGSPGS S TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTASS SPGS STPSGATGSPGS SPSASTGTGPGSSPSASTGTGP GASP 70 AG144_2 PGS SPS ASTGTGPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGAT GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSS 71 AG144_A GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA S S SPGS STP SGATGSPGTPGSGT AS S SPGASPGT S STGSPGASPGT S STGSP 72 AG144B GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSP 73 AG144_C GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTS STGSP 74 AG144_F GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTS STGSP 75 AG144_3 GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT GPGS SP S ASTGTGPGASPGT S S TGSPGTPGSGT AS S SPGS STP SGA TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGT S STGSP 76 AG144_4 GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAST GTGPGASPGT S STGSPGS SP S ASTGTGPGTPGSGTAS S SPGS S TP S GATGSP 77 AE288_1 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAP 78 AE288_2 GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG 79

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SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP AG288_1 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGT AS S SPGS STP SGATGSPGTPGSGT AS S SPGS STP SG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG T SSTGSPGTPGSGT ASS SPGS STPSGATGSPGS SPS ASTGTGPGS SP SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GSSTPSGATGS 80 AG288_2 GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG SPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT AS S SPGSSTPSGATGSPGASPGTS STGSPGTPGSGT AS S SPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSST PSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSP 81 AF504 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASS SPGSSTPSGATGSPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTA S S SPGS STP SGATGSPGTPGSGT AS S SPGASPGT S STGSPGASPGT S STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSS TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GASPGTS STGSPGS SP S ASTGTGPGTPGSGT AS S SPGASPGT S STG SPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP 82 AF540 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPG PGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG TAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPES GSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPE SGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSE SPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTST PESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTS TPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT SPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPG TSTPESGSASPGSTSESPSGTAP 83 AD576 GS SESGS SEGGPGSGGEPSESGS SGS SESGS SEGGPGS SESGS SEG GPGS SESGS SEGGPGS SESGS SEGGPGS SESGS SEGGPGESPGGS S GSESGSEGS SGPGES SGS SESGS SEGGPGS SESGS SEGGPGS SESGS SEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGE PSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEG SSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGS GGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSES GESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEG GPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSE 84

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SGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGG SSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGG EPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSS ESGSSEGGPGSEGSSGPGPGESS AE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAP 85 AF576 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPG PGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG TAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPES GSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPE SGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSE SPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTST PESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTS TPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT SPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPG TSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASP GTSTPESGSASP 86 AG576 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSS TGSPGASPGT S STGSPGTPGSGT AS S SPGASPGT S STGSPGASPGT SSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GASPGTS STGSPGTPGSGTAS S SPGASPGTS STGSPGASPGTS STG SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGT AS S SPGS STP SGATGSPGTPGSGT AS S SPGS STP SG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG T SSTGSPGTPGSGT ASS SPGS STPSGATGSPGS SPS ASTGTGPGS SP SASTGTGPGASPGTSSTGS 87 AE624 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSST GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT 88

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ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAP AD836 GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESG SSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSE GGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGS SGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESG SSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESP GGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGS SESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGP GSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESG SSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGP GESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEP SESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGG EPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGS SEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGS SGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGESP GGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGS SESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSS GSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEG GPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGSGGEPSE SGSS 89 AE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAP 90 AF864 GSTSESPSSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTA PGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGT APGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESS 91

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TAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAE SPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPS GTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESP SGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPE SGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSS TAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTST PESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGS TSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPG TSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGP GTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTA PGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSA SPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSG TAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGES STAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGE SSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSA STGTGPGSSTPSGATGSPGSSTPSGATGSP AG864_2 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA S S SPGS STP SGATGSPGTPGSGT AS S SPGASPGT S STGSPGASPGT S STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS GT AS S SPGS STP SGATGSPGS SP S ASTGTGPGS SP S ASTGTGPGS ST PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTASS SPGS STPSGATGSPGS SPS ASTGTGPGS SPS ASTGTGP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSP 92 AM875 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPG PGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA SPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTP SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTS TEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGT STEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAES PGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSE 93

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GSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPS AE912 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSST GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAP 94 AM923 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGAT GSPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAE SPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPES GSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTST EEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESS TAPGTPGSGTAS S SPGS STPSGATGSPGS SPS ASTGTGPGSEP ATS GSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSST AESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTE PSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSS PSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP 95 AM1318 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPG PGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA SPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE 96

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GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTP SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTS TEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGT STEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGP GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTA PGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGES STAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPS GESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGA SPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPG TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAP GSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSE TPGSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSG TAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSS TGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAP VS 864 GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTE PGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTST EPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGT EPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSG TEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPST SEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEP SEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPA TSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGA SEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTS TEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGS EPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSG SGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSA GSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGS AGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEP GAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEP GSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATS GTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPAT SGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGAS EPTSTEPGTSTEPSEPGSA 97 BD864 EGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTA GSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGT SESATSESGAGTSTEASEGSASGSETATSGSETAGATSGSETA 98

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GTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGS ASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATS GSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSES ATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGT STEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGA GSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGS ASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSE TSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETA TSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTS ESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASG STAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESG AGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSG SETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETAT SGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA AE948 GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSE TPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEG SAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPS EGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPAT SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSET PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE TPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGP 99 ΑΕΙ 044 GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPE SGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSE GSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTE PSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSE 100

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TPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESA TPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP AGSPTSTEEGTSESATPESGPGTSESATPESGPGTST ΑΕΙ 140 GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSA PGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GS APGSEP AT SGSETPGT SES ATPESGPGT STEP SEGS APGSEP AT S GSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEP SEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSES ATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA 101 ΑΕ1236 GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTS TEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSP TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGS EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSESATPES GPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESA TPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTE 102

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PSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP AE1332 GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSG SETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPG SEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESG PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPS EGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESA TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPA GSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGTS ESATPESGPGTSESATPESGPGTSTEPSEGSAPGTST 103 AE1428 GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPT STEEGSP AGSPT STEEGT STEP SEGS APGSEP AT SGSETPGSP A GSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GTSESATPESGPGTSESATPESGPGTSESATPESGPGTSESATPESG PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSE GSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESA TPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPA TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSESATPESG PGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSPA 104 AE1524 GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE 105

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TPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTE PSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPA GSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGT SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPG TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGS ETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPS EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPA AE1620 GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS APGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST EEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPT STEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST 106 AE1716 GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEG SAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESA TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAG 107

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SPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSE TPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT STEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSE AE1812 GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEP ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTS E S ATPES GPGSP AGSPT S TEEGT S TEP SEGS APGSEP AT S GSETPGT SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEG SAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSES ATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGTSTEPSEGSAPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEP 108 AE1908 GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST EEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPA TSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSE SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGS EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG SEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP 109

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GSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESG PGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP AE2004A GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTE PSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTE EGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE 110 AG948 GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAST GTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASP GTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSS PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTAS S SPGTPGSGT AS S SPGSSTPSGATGSPGS STPSGATGSP GSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSST PSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGS SPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST 111

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GTGPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSG T AS S SPGTPGSGT AS S SPGS STP SGATGSPGTPGSGT AS S SPGASP GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSP AG1044 GTPGSGT AS S SPGTPGSGT AS S SPGS SPS ASTGTGPGTPGSGT AS S SPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT GSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGS GTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTP GSGT AS S SPGASPGTS STGSPGTPGSGT AS S SPGS SP S ASTGTGPG SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSP GSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATG SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG TGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPS GATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG SGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSST GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTS STGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSSPSASTGTGPGSSPSASTGTGP GTPGSGTASSSPGS SPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSP GSST 112 AG1140 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST GSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGTPGSGT ASS SPGS STPSGATGSPGS STPSGATGSPGS SPS ASTGTGPGS SPS ASTGTGPGASPGTS STGSPGTPGSGT AS S SPGAS PGTS STGSPGS STPSGATGSPGTPGSGT AS S SPGS SPS ASTGTGPGS STPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSP GSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATG SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTA SSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAS TGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPS ASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGTPGSGTASS SPGS SPS ASTGTGPGTPGSGT ASS SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA S S SPGASPGTS STGSPGS STPSGATGSPGS STPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSA STGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS ASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTP GSGT ASS SPGASPGTS STGSPGS SPS ASTGTGPGASPGTS STGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP 113

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GSST AG1236 GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGT GPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTA SSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAS TGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS ASTGTGPGTPGSGT AS S SPGTPGSGTAS S SPGS STPSGATGSPGAS PGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPG ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATG SPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGASPGTS STGSPGS STPSGATGSPGTPGSGT AS S SPGSSPS ASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGT S STGSPGS SP S ASTGTGPGTPGSGT AS S SPGTPGSGTAS S SP GASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASS SPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTG TGPGTPGSGT AS S SPGTPGSGTAS S SPGS SP S ASTGTGPGTPGSGT AS S SPGASPGTS STGSPGS STPSGATTGSPGTPGSGT AS S TGSPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GASP 114 AG1332 GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGT GPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSS TGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGT SSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP SGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG TPGSGTAS S SPGASPGTS STGSPGASPGTS STGSPGTPGSGT AS S SP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTG TGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSG ATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPG TSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPG SGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS PSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPG SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST GSPGASPGTS STGSPGTPGSGT AS S SPGTPGSGTAS S SPGS SP S AST GTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSG TASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGS GT AS SSPGS SPS ASTGTGPGS SPSASTGTG PGASPGTS STGSPGS SP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGT PG 115 AG1428 GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATG SPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT 116

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GSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTS STGSPGS SPS ASTGTGPGTPGSGTAS S SPGASPGTS STGSPGASPG T SSTGSPGTPGSGT ASS SPGTPGSGTAS S SPGASPGTS STGSPGASP GT S STGSPGTPGSGT AS S SPGTPGSGTAS S SPGS SP S ASTGTGPGS S PSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSP GSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTG SPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPG T SSTGSPGSSTPSGATGSPGTPGSGT AS S SPGTPGSGTAS S SPGS SP SASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGS STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSP GTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASS SPGTPGSGTAS S SPGS STPSGATGSPGTPGSGT AS S SPGASPGS STPSGATGSPGTPGSGT AS S SPGSPS A STGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTP SGATGSPGTPGSGT AS P AG1524 GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTG SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTG TGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAST GTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGT SSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP GTPGSGT AS S SPGS SP S ASTGTGPGASPGTS STGSPGASPGT S STG SPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT AS S SPGTPGSGTAS S SPGASPGTS STGSPGS STPSGATGSPGTPGS GTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSST PSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGS STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSP GSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS SPGASPGTS STGSPGASPGTS STGSPGTPGSGT AS SSPGTPGSGT A S S SPGS STPSGATGSPGTPGSGTASS SPGASPGTS STGSPGS STPSG ATGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPS ASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGAS PGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGASPGTSSTGSPGS STPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSP GTPG 117 AG1620 GSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGT GPGTPGSGT AS S SPGASPGT S S TGSPGS STP 118

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TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGT AS S SPGS SPS ASTGTGPGSSPS ASTGTGPGASPGTS ST GSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSG ATGSPGASPGTS STGSPGS STPSGATGSPGTPGSGT AS S SPGSSPS ASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGS STPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGSSTPS GATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPG SGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTP GSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGT ASS SPGTPGSGTAS S SPGTPGSGT ASS SPGS STPSGATG SPGSST AG1716 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGT GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST GTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPS GATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPG SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPG SSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSP GSSTPSGATGSPGTPGSGTASS SPGS SPS ASTGTGPGASPGTS STG SPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSST GSPGTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGASPGTS STGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPG TSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASS SPGTPGSGT AS S SPGS SPS ASTGTGPGS SPS ASTGTGPGS S PSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG ASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGP GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASS SPGASPGTS STGSPGTPGSGT AS S SPGTPGSGTAS S SPGASPGT S ST GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSG ATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPG SGTASSSPGSSPSASTGTGPGSSTPS GATGSPGASPGTSSTGSPGA SPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG TPG 119 AG1812 GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTAS S SPGS SPS ASTGTGPGASPGTS STGSPGS STPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGT ASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPS GATGSPGS SP S ASTGTGPGTPGSGT AS S SPGASPGT S STGSPGS ST PSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGT PGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG 120

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SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGT ASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPS GATGSPGS SP S ASTGTGPGS SP S ASTGTGPGTPGSGT AS S SPGASP GTSSTGSPGASPGTS STGSPGTPGSGT AS S SPGASPGTS STGSPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT GSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSG ATGSPGTPGSGT ASS SPGS STPSGATGSPGS STPSGATGSPGS STPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGA SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG ASP AG1908 GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASS SPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGTPGSGTASS SPGASPGTS STGSPGTPGSGT ASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSA STGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTP GSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPG ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP GSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS SPGASPGTS STGSPGASPGTS STGSPGTPGSGT AS SSPGTPGSGT A S S SPGS STPSGATGSPGTPGSGTASS SPGASPGTS STGSPGS STPSG ATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS GATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSS TGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGT SSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTP SGATGSPGASPGTS STGSPGS SPS ASTGTGPGTPG SGT AS S SPGS S TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPG SSP 121 AG2004A GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG ATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS PSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGT AS S SPGASPGTS STGSPGTPGSGT AS S SPGTPGSGT A S S SPGS STPSGATGSPGTPGSGTAS S SPGASPGTS STGSPGS STPSG 122

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ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSPGASPGT S STGSPGS SP S ASTGTGPGTPGSGT AS S SPGASP GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGT GPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS TGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSG TASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPS ASTGTGPGS SPS ASTGTGPGASPGTS STGSPGTPGSGT AS S SPGS S TPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPG ASP AE72B SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPG 123 AE72C TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPG 124 AE108A TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTS 125 AE108B GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSTEPSEGSAP 126 ΑΕΙ 44 A STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGS 127 ΑΕΙ 44V SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APG 128 ΑΕ180Α TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES 129 ΑΕ216Α PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGSEPATSGPGTSESAT 130 ΑΕ252Α ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES 131 ΑΕ288Α TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT 132

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SESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESA AE324A PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT SESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS 133 AE360A PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGTSESAT 134 AE396A PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS 135 AE432A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATS 136 AE468A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT 137 AE504A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST 138

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EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSP AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE540A TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEP 139 AE576A TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESA 140 AE612A GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA T 141 AE648A PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA 142

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GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE684A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATS 143 AE720A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAT PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTE 144 AE756A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS 145

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PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAT PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPG TSES AE792A EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT STEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS 146 AE828A PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSESAT 147 AG72A GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGASPGTSSTGSPGTPGSGTASS 148 AG72B GSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT 149

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GPGASPGTSSTGSPGTPGSGTASSSP AG72C SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGA 150 AG108A S ASTGTGPGS SPS ASTGTGPGTPGSGT AS S SPGS STPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GTPGSGT AS S SPGASP 151 AG108B PGTPGSGT AS S SPGS STP SGATGSPGTPGSGT AS S SPGS STP SGAT GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSS 152 AG144A PGS SPS ASTGTGPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGAT GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSS 153 AG144B PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASP 154 AG180A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS 155 AG216A TGTGPGS SP S ASTGTGPGTPGSGT AS S SPGS STP SGATGSPGS SP S ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGSSTPSG 156 AG252A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSS 157 AG288A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGS 158 AG324A T SSTGSPGTPGSGT ASS SPGASPGTS STGSPGASPGTS STGSPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAST GTGPGASPGT S STGSPGS SP S ASTGTGPGTPGSGT AS S SPGS S TP 159 AG360A TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG 160

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GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA TGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG396A GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTAS S SPGS STPSGATGSPGTPGSGT AS S SPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAST GTGPGASPGT S STGSPGS SP S ASTGTGPGTPGSGT AS S SPGS S TP S GATGSPGSSTPSGATGSPGASPGT 161 AG432A GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST PSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGTPGSGTASS SPGS STPSGATGSPGSSPS AST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPG SGTASSSPGSSTPS 162 AG468A TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA S S SPGS STPSGATGSPGTPGSGTASS SPGS STPSGATGSPGS STPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG ASPG 163 AG504A TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG S STPSGATGSPGS SPS ASTGTGPGASPGTS STGSPGASPGTS STGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA S S SPGS STPSGATGSPGTPGSGTASS SPGS STPSGATGSPGS STPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP 164 AG540A T SSTGSPGASPGTS STGSPGS SPS ASTGTGPGTPGSGT AS S SPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG 165

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S STPSGATGSPGS STPSGATGSPGS SPS ASTGTGPGASPGTS STGSP GASPGTS STGSPGTPGSGTAS S SPGASPGTS STGSPGASPGTS STG SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGT AS S SPGS STP SGATGSPGTPGSGT AS S SPGS STP SG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG T SSTGSPGTPGSGT ASS SPGS STPSGATGSPGS SPS ASTGTGPGS SP SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGASPG AG576A T SSTGSPGTPGSGT ASS SPGASPGTS STGSPGASPGTS STGSPGASP GTSSTGSPGS SPS ASTGTGPGTPGSGT AS S SPGASPGTS STGSPGA SPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGT AS S SPGASPGTS STGSPGASPGTS STGSPGASPGTS ST GSPGASPGTS STGSPGTPGSGTAS S SPGS STPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGASPG 166 AG612A STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS GT AS S SPGS STP SGATGSPGS SP S ASTGTGPGS SP S ASTGTGPGS ST PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP GASPGTS 167 AG648A GT ASS SPGS STPSGATGSPGS SPS ASTGTGPGS SPS ASTGTGPGS ST PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTASS SPGS STPSGATGSPGS SPS ASTGTGPGS SPS ASTGTGP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGT 168

- 80 041874

GPGASPGT S STGSPGS SP S ASTGTGPGTPGSGT AS S SPGS STP AG684A TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG T SSTGSPGASPGTS STGSPGASPGTS STGSPGTPGSGT AS S SPGS ST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGS STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA TGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG 169 AG720A TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSS TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GASPGTS STGSPGS SP S ASTGTGPGTPGSGT AS S SPGASPGT S STG SPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG T SSTGSPGTPGSGT ASS SPGASPGTS STGSPGASPGTS STGSPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAST GTGPGASPGTS STGSPGS SP S ASTGTGPGTPGSGT AS S SPGS S TP S GATGSPGSSTPSGATGSPGASPG 170 AG756A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG S STPSGATGSPGS SPSASTGTGPGASPGTS STGSPGASPGTS STGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTS STGSPGTPGSGTASS SPGS STPSGATGSPGTPGSGTA S S SPGS STPSGATGSPGTPGSGTASS SPGS STPSGATGSPGS STPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGASPG 171

-81 041874

AG792A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG S STPSGATGSPGS SPS ASTGTGPGASPGTS STGSPGASPGTS STGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA S S SPGS STPSGATGSPGTPGSGTASS SPGS STPSGATGSPGS STPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG ASPG 172 AG828A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG S STPSGATGSPGS SPS ASTGTGPGASPGTS STGSPGASPGTS STGSP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA S S SPGS STPSGATGSPGTPGSGTASS SPGS STPSGATGSPGS STPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP 173 AG288 D E GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATG SPGS SPS ASTGTGPGASPGTS STGSPGASPGTS STGSPGTPGSGT A SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS GATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSP 1699

In additional embodiments of the present invention, the CFXTEN composition comprises one or more non-repeating XTEN sequences with lengths that range from about 36 to about 3000 amino acid residues, with at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non-overlapping sequence motifs of 36 amino acids selected from one or more polypeptide sequences from table. 13-17, either as a family of sequences, or provided that the motifs are selected from two or more families of motifs.

In those embodiments of the present invention where the XTEN component of the CFXTEN fusion protein has less than 100% of its amino acids consisting of 4, 5, or 6 amino acid types selected from glycine (G), alanine (A), serine (S), threonine ( T), glutamate (E) and proline (P), or less than 100% of the sequence consists of sequence motifs from table. 3 or sequence XTEN from table. 4 and 13-17, the other XTEN amino acid residues are selected from any of the other 14 naturally occurring L-amino acids, but are preferably selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90, 91, 92, 93, 94, 95, 96, 97 , 98% or at least about 99% hydrophilic amino acids. XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), or are interspersed throughout an XTEN sequence that is located within or between sequence motifs , or concentrated in one or more short stretch of the XTEN sequence, for example, to create a linker between XTEN and FVIII components. In such cases where the CFXTEN XTEN component contains amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), preferably less than about 2% or less than about 1% of the amino acids are hydrophobic residues, such that the resulting sequences typically have no secondary structure, e.g., no more than 2% alpha helix or 2% beta sheets, as determined using the methods disclosed herein . Hydrophobic residues that are

- 82 041874 are less acceptable in the construction of XTEN, contain tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine and methionine. Additionally, it is possible to construct XTEN sequences to contain less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%. Or did not contain any of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid deamination). Thus, in certain embodiments of the present invention, the XTEN component of the CFXTEN fusion protein that contains other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline ( P) has a sequence with less than 5% of residues involved in alpha helices and beta sheets, as measured by the Chou-Fasman algorithm, and has at least 90%, or at least about 95%, or more random helix formation, as measured by the GOR algorithm.

3. Sequence length.

In additional aspects, the present invention provides XTENs of various lengths for inclusion in CFXTEN compositions, with the length of the XTEN sequence(s) being chosen based on a property or function obtained in the fusion protein. Depending on the intended property or function, CFXTEN compositions contain short or medium length XTEN located within the FVIII sequence or between the domains of FVIII and/or longer XTEN sequences that can serve as carriers located in fusion proteins as described herein. . Although not intended to be limiting, XTEN or XTEN fragments contain short segments of about 6 to about 99 amino acid residues, medium length of about 100 to about 399 amino acid residues, and longer segments of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, an XTEN for inclusion in a subject CFXTEN encompasses XTENs or XTEN fragments with a length of about 6, or about 12, or about 36, or about 40, or about 42, or about 72, or about 96, or about 144, or about 288, or about 400 or about 500 or about 576 or about 600 or about 700 or about 800 or about 864 or about 900 or about 1000 or about 1500 or about 2000 or about 2500 or up to about 3000 amino acid residues in length. In addition, the XTEN sequences can be from about 6 to about 50, from about 50 to about 100, from about 100 to 150, from about 150 to 250, from about 250 to 400, from about 400 to about 500, from about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The exact length of the XTEN inserted into a CFXTEN subject may vary without adversely affecting the activity of the CFXTEN composition. In a particular embodiment of the present invention, the one or more XTENs that are used in the CFXTEN disclosed herein are 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from one of the sequences in the family XTEN; those. AD, AE, AF, AG, AM, AQ, BC or BD. In a further embodiment of the present invention, the two or more XTENs that are used in the CFXTEN disclosed herein are 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from two sequences of the family XTEN; those. AD, AE, AF, AG, AM, AQ, BC, or BD, with preferred sequence combinations from the AE and AG families. In certain embodiments, the implementation of the present invention CFXTEN, which contains one or more XTEN used in this document, contains XTEN, selected from any of the sequences from table. 4, which can be linked to the FVIII component directly or via the spacer sequences disclosed herein.

In particular, CFXTEN conformation structures, where XTEN serves as a conformationally labile linker, or are introduced into outer loops or random regions of the FVIII sequence in order to increase the mass, flexibility, or hydrophilicity of the region, or which are intended to interfere with receptor clearance in the case of FVIII for to enhance pharmacokinetic properties, or to interfere with binding to FVIII inhibitors or other anti-FVIII antibodies, or provided that short or medium length XTEN is used to facilitate tissue penetration or to alter the strength of interactions of the CFXTEN fusion protein with its target, or provided that it is desired to distribute the total length of XTEN in short or medium length segments at multiple locations within the FVIII sequence, the present invention provides CFXTEN compositions with one, two, three, four, five or more short or medium length XTEN sequences administered between, or in pre cases, one or more FVIII domains or within the outer loops, or at other sites in the FVIII sequences, such as, but not limited to, localization at or close to the insertion sites as defined in Table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or as illustrated in FIG. 8-9. In a particular embodiment of the above, the CFXTEN fusion protein comprises multiple XTEN segments, e.g., at least two, or at least three, or at least four, or at least five or more XTEN segments, in which the XTEN segments may be identical. or they can be different and yet CFXTEN retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the procoagulant activity of natural FVIII, provided that it is analyzed using one of the analyses.

- 83 041874 calls disclosed in this document. In other specific conformation structures of CFXTEN, provided that XTEN serves as a carrier, in order to increase the mass of the fusion protein, or to change the strength of interactions of the CFXTEN fusion protein with its target, or to increase the pharmacokinetic properties of the fusion protein, the present invention provides compositions of CFXTEN with one or more medium or long length XTEN sequences introduced at the C-terminus, in the B domain (or the remainder of the BDD sequence) between, or within one or more FVIII domains, within outer loops, or at other sites in the FVIII sequences such as, but not limited to, insertion sites as defined in Table. 5, tab. 6, tab. 7, tab. 8, and table. 9 or as illustrated in FIG. 8-9. Despite this, the incorporation of several short to medium length XTENs in a CFXTEN formulation is thought to confer improved properties on the CFXTEN fusion proteins compared to CFXTEN fusion proteins with the same number of amino acids with fewer but longer XTENs, and still result in compositions with procoagulant activity and extended half-life; the rationale for which is described in this document in relation to the derived radii of several XTENs.

In embodiments of the present invention where the CFXTEN fusion proteins comprise multiple XTEN sequences, the total length of the total volume of residues in the XTEN sequences is greater than about 100 to about 3000, or about 200 to about 2000, or about 400 to about 1000 amino acid residues, and The XTENs may be identical, or they may be different in sequence, net charge, or length. In a particular embodiment of CFXTEN that contains multiple XTENs, each of the individual XTEN sequences exhibits at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with the motif or XTEN selected from the table. 3, 4 and 13-17, or a fragment thereof, when optimally aligned with a sequence of comparable length.

As described in more detail below, methods in which CFXTEN are disclosed are designed by choosing the length of XTEN and its inclusion site within CFXTEN to confer a target half-life, maintain procoagulant activity, reduce binding to FVIII inhibitors, or improve physicochemical properties (e.g. , stability, or solubility) of the CFXTEN fusion protein, construct and express a coding construct, and isolate and regenerate recombinant CFXTEN fusion proteins. In general, total XTEN lengths greater than about 400 residues incorporated into CFXTEN formulations result in longer half-life compared to shorter total lengths, eg shorter than about 280 residues. In a particular embodiment of the present invention, the CFXTEN fusion protein structures are expected to contain at least a single XTEN as carrier, with a sequence length of at least about 400, or at least about 600, or at least about 800, or at least at least about 900, or at least about 1000 or more amino acids. In a further embodiment of the present invention, multiple XTENs are incorporated into fusion proteins to achieve a total length of at least about 400, or at least about 600, or at least about 800, or at least about 900, or at least at least about 1000 or more amino acids, while the XTENs may be identical or different in sequence or length. As used herein, the total length is intended to cover the total length, in amino acid residues, provided that more than one XTEN is inserted into the CFXTEN fusion protein. Both of the above embodiments of the present invention are designed to confer increased bioavailability and/or increased terminal half-life after administration to the subject compared to CFXTEN which contains shorter cumulative XTEN lengths and still results in procoagulant activity and hemostasis effect. When administered subcutaneously or intramuscularly, _Cmax is reduced, but the area under the curve (AUC) is increased, compared with a similar dose of CFXTEN with a shorter total length of XTEN or FVIII not bound to XTEN, thereby contributing to the ability to maintain effective levels of the CFXTEN formulation in over a longer period of time and allows for periods of up to 2, 4, 7, 10, 14 or 21 days between doses, as described in more detail below. Thus, XTEN imparts a depot property to the introduced CFXTEN, in addition to the other physicochemical properties described herein.

If XTEN is used as a carrier, the present invention takes advantage of the discovery that increasing the length of non-repetitive, unstructured polypeptides enhances the unstructured nature of XTEN and thus enhances the physical/chemical and pharmacokinetic properties of fusion proteins that contain the XTEN carrier. As described in more detail in the examples, a proportional increase in the length of XTEN, even if generated by a repeating order of motifs of single family sequences (e.g., four AE motifs from Table 3), results in a sequence with a high percentage (e.g., 90% or more) of formation random helix, as determined by the GOR algorithm, or reduced content of alpha helices or beta sheets (for example, less than 2%), as determined by the Chou-Fasman algorithm, compared

- 84 041874 XTEN with shorter lengths. In addition, increasing the length of the unstructured polypeptide fusion partner, as described in the examples, results in a fusion protein with a disproportionate increase in terminal half-life (e.g., up to 50, 100, 200 or more hours), compared to fusion proteins with unstructured polypeptide partners with shorter sequence lengths. The improved pharmacokinetic properties of CFXTEN compared to non-XTEN FVIII are described in more detail below.

In additional aspects, the present invention provides methods for generating short or medium length XTEN from longer XTEN donor sequences, wherein the longer XTEN donor sequence is N-terminally or C-terminally truncated, or a fragment is generated from the interior of the donor sequence such that, thus resulting in a short or medium length XTEN. In non-limiting examples, as schematically illustrated in FIG. 16A-C, the 864 amino acid residue AG sequence can be processed to produce a 144 residue AG sequence, a 288 residue AG sequence, a 576 residue AG sequence, or other intermediate lengths if the 864 residue AE sequence (as illustrated in FIG. 16D , E) can be processed to produce multiple 144 residue AE sequences, 288 or 576 residue AE sequences, or other shorter or medium lengths. It is separately contemplated that such an approach may be used with any of the XTEN embodiments described herein or with any of the sequences shown in Table 1. 4 or 13-17 in order to result in an XTEN of the required length. In preferred embodiments of the present invention, a CFXTEN that contains multiple XTENs has an XTEN that exhibits at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least мере около 99% или 100% идентичности последовательности с последовательностями, выбранными из AE42_1, AE42_2, AE42_3, AG42_1, AG42_2, AG42_3, AG42_4, AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, AG144_F, AG144_3, AG144_4, AE288_1, AE288_2, AG288_1, AG288_2 and AG288_DE.

4. Total charge.

In additional embodiments of the present invention, the unstructured characterization of an XTEN polypeptide can be improved by including amino acid residues with a net charge and/or reducing the overall percentage (e.g., less than 5%, or 4%, or 3%, or 2%, or 1%) hydrophobic amino acids in XTEN sequences. In general, net charge and net charge density are controlled by altering the content of charged amino acids in XTEN sequences, either positive or negative, with net charge typically represented as the percentage of amino acids in the polypeptide that create a charged state, in addition to those residues that have been annulled. residue with the opposite charge. In certain embodiments of the present invention, the net charge density of XTEN compositions may be above +0.1 or below -0.1 charge/residue. By net charge density of a protein or peptide, as used herein, is meant the net charge divided by the total number of amino acids in the protein or propeptide. In additional embodiments of the present invention, the total charge of XTEN may be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% , about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more. Based on net charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 , 5.5, 6.0 or even 6.5. In preferred embodiments of the present invention, XTEN will have an isoelectric point between 1.5 and 4.5 and carry a negative net charge under physiological conditions.

Since most tissues and surfaces in a human or animal are negatively charged, in some embodiments, XTEN sequences are designed to be negatively charged in order to minimize non-specific interactions between the XTEN that contains the compositions and various surfaces such as blood vessels, healthy tissues or various receptors. Without wishing to be bound by any theory, XTEN may adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that are individually negatively charged and that are distributed along the sequence of the XTEN polypeptide. In certain embodiments of the present invention, the XTEN sequence is constructed with at least 90% or 95% charged residues separated by other residues such as serine, alanine, threonine, proline, or glycine, resulting in a more even charge distribution, better expression, or a purified state. . This distribution of net negative charge across the extended lengths of the XTEN sequence can lead to an unstructured conformation, which in turn can lead to an effective increase in hydrodi

- 85 041874 namic radius. In preferred embodiments of the present invention, the negative charge of a particular XTEN is provided by the inclusion of glutamic acid residues. As a rule, glutamic acid residues are evenly spaced throughout the XTEN sequence. In some cases, XTEN may contain about 10-80, or about 15-60, or about 20-50 glutamic acid residues per 20kDa XTEN, which can result in XTENs with charged residues that have very similar pKas, which can increase charge homogeneity. product and reduces its isoelectric point, improves the physico-chemical properties of the resulting hybrid protein CFXTEN in case, as a result, simplification of the purification procedure. For example, when a negatively charged XTEN is desired, the XTEN may be selected only from the AE sequence family, which has approximately 17% of the total charge due to the incorporated glutamic acid, or may include varying amounts of the glutamic acid-containing motifs from Table 1. 3 to provide the desired degree of net charge. Non-limiting examples of AE XTEN include, but are not limited to, 36, 42, 144, 288, 576, 624, 864, and 912 sequences of the AE family from Table. 4 and 14, or fragments thereof. In a separate embodiment of the present invention, the XTEN sequence from Table. 4 or 13-17 may be modified to include additional glutamic acid residues to achieve the desired negative charge. Accordingly, in a specific embodiment, the present invention provides XTEN wherein the XTEN sequences contain about 1, 2, 4, 8, 10, 15, 17, 20, 25% or even about 30% glutamic acid. In a separate embodiment, the present invention provides for the inclusion of up to 5% aspartic acid residues in XTEN to achieve a negative charge in addition to glutamic acid.

In additional embodiments of the present invention in which no net charge is desired, the XTEN may be selected from, for example, AG components of XTEN, such as the AG motifs of Table 1. 3, or motives AM from table. 3, which do not have a net charge. Non-limiting examples of AG XTEN contain, but are not limited to, 36, 42, 144, 288, 576, and 864 sequences of the AG family from Table. 4 and 16, or fragments thereof. In a further embodiment of the present invention, XTEN may contain varying amounts of AE and AG motifs (in order to have a total charge that is considered optimal for a given use or to maintain a given physicochemical property.

Without wishing to be bound by any theory, the higher net charge XTEN formulations of CFXTEN are expected to have fewer non-specific interactions with various negatively charged surfaces such as blood vessels, tissues or various receptors, which would further reduce active clearance. On the other hand, it is believed that CFXTEN XTEN formulations with low (or no) net charge will have a higher degree of interaction with surfaces that can enhance the activity of the corresponding blood clotting factor, given the known contribution of the cell (for example, platelets) and vascular surfaces in coagulation process and activation intensity of clotting factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850-9858 ).

The XTEN compositions of the present invention generally have no or low levels of positively charged amino acids. In certain embodiments, XTENs may have less than about 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2%, or less than about 1% positively charged amino acid residues. Despite this, the present invention provides constructs in which a limited number of positively charged amino acids, such as lysine, are included in XTEN to allow conjugation between the lysine epsilon-amine and a functional group on a peptide, a linker bridge, or a functional group on a drug or small molecule that conjugates to the XTEN backbone. In a particular embodiment of the above, the XTEN of a particular CFXTEN has from about 1 to about 100 lysine residues, or from about 1 to about 70 lysine residues, or from about 1 to about 50 lysine residues, or from about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to about 10 lysine residues, or about 1 to about 5 lysine residues, or alternatively, only one lysine residue. Using the above lysine-containing XTEN, fusion proteins can be constructed that make up XTEN, the FVIII clotting factor, plus a chemotherapeutic agent or other clotting factor or cofactor used in the treatment of coagulopathic conditions, with the maximum number of agent molecules incorporated into the component XTEN is determined by the amount of lysine or other amino acids with reactive side chains (eg, cysteine) embedded in XTEN.

As hydrophobic amino acids provide structure to a polypeptide, the present invention ensures that the hydrophobic amino acid content of XTEN is typically less than 5%, or less than 2%, or less than 1% of the hydrophobic amino acid content. In a particular embodiment of the present invention, the content of the amino acids methionine and tryptophan in the XTEN component of the CFXTEN fusion protein is typically less than 5%, or less than 2%, and more preferably,

- 86 041874 less than 1%. In a further embodiment of the present invention, the XTEN of specific CFXTEN compositions will have a sequence that has less than 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2% positively charged amino acid residues, the sum of the residues methionine and tryptophan is less than 2%, and the sum of asparagine and glutamine residues is less than 5% of the total XTEN sequence.

5. Low immunogenicity.

In additional aspects, the XTEN sequences provided herein have a low degree of immunogenicity or are essentially non-immunogenic. A number of factors may contribute to low immunogenicity of XTEN, such as non-repeating sequence, non-structured conformation, high degree of solubility, low or no self-aggregation, low or no proteolytic sites within the sequence, and low or no epitopes in XTEN sequences.

Conformational epitopes are formed by a protein surface region that consists of several discontinuous amino acid sequences of the protein's antigen. Precise protein folding translates these sequences into well-defined, stable spatial conformations, or epitopes, that can be recognized as foreign by the host's humoral immune system, resulting in an antibody to the protein or activation of an immune cellular response. In the latter case, the immune response to the human protein is highly dependent on T cell epitope recognition, which is a function of the binding specificity of that individual's HLA-DR allotype peptide. The involvement of the MHC Class II peptide complex via the cognate T cell receptor on the surface of the T cell, as well as the cross-linking of some other co-receptors such as CD4 molecules, can induce activated states within the T cell. Activation results in the release of cytokines that additionally activate other lymphocytes, such as B cells, to produce antibodies or activate killer T cells as a complete cellular immune response.

The ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) depends on a number of factors; especially from its primary sequence. In a separate embodiment of the present invention, a lower degree of immunogenicity is achieved by designing XTEN sequences that resist antigen processing in antigen presenting cells and/or selecting sequences that also do not bind to MHC receptors. The present invention provides fusion proteins of CFXTEN with substantially non-repetitive XTEN polypeptides designed to reduce binding to MHC II receptors as well as preventing formation of T cell receptor epitopes or antibody binding resulting in a low degree of immunogenicity. The avoidance of immunogenicity can be attributed, at least in part, to the result of the conformational flexibility of the XTEN sequences; those. the absence of a secondary structure due to the choice of order and amino acid residues. For example, of particular interest are sequences having, in aqueous solution or under physiological conditions, a low propensity to adopt compactly folded conformations, which can lead to conformational epitopes. Administration of fusion proteins that contain XTEN using conventional therapeutic methods and dosing generally does not result in the formation of neutralizing antibodies to the XTEN sequence and also reduces the immunogenicity of the FVIII fusion partner in CFXTEN formulations.

In a particular embodiment of the present invention, the XTEN sequences that are used in the subject's fusion proteins may be substantially free of epitopes recognized by a human T cell. The elimination of such epitopes in order to generate less immunogenic proteins has been previously disclosed; see for example WO 98/52976, WO 02/079232 and WO 00/3317, which are incorporated herein by reference. Human T cell epitope assays have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T-cell epitopes or non-human sequences. This is achieved by testing direct repeats of these sequences for T-cell epitopes and for the occurrence of 6- to 15-mer and in particular 9-mer sequences that are not human, and then redesigning the XTEN sequence to eliminate or disrupt the sequence epitope. In certain embodiments of the present invention, the XTEN sequences are substantially non-immunogenic in terms of restriction amounts of XTEN epitopes predictably binding MHC receptors. With a decrease in the number of epitopes capable of binding to MHC receptors, there is a concomitant decrease in T cell activation potential as well as T cell helper function, reduced B cell activation or upregulation, and reduced antibody production. The low rate of predictive T cell epitopes can be determined using epitope prediction algorithms such as, for example, TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17:555-61) as shown in Example 46. The result of TEPITOPE in the case of a given peptide frame within a protein is the log K _d (dissociation constant, affinity, dissociation rate) of binding of that peptide frame to several of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555). The result varies in the range of at least 20 units, from about 10 to about 10 (corresponding to binding limits of 10e ¹⁰ Kd to 10e ^-10 Kd), and can be reduced by avoiding hydrophobic amino acids that serve as anchor residues during display a peptide in an MHC such as M, I, L, V, F. In certain embodiments of the present invention, the XTEN component incorporated into CFXTEN does not have a predictive T-cell epitope at a TEPITOPE threshold of about -5, or -6 , or -7, or -8, or -9, or if TEPITOPE results in -10. As used in this document, the -9 result is a more stringent TEPITOPE result than the -5 result.

In a further embodiment of the present invention, inventive XTEN sequences, including those containing subject-introduced CFXTEN fusion proteins, appear to substantially non-immunogenically restrict known proteolytic sites from the XTEN sequence, reducing XTEN processing into small peptides that can bind to MHC II receptors. In a further embodiment of the present invention, the XTEN sequence is substantially non-immunogenic when using a sequence that is substantially devoid of secondary structure conferring resistance to multiple proteases consistent with high structure entropy. Accordingly, reduced TEPITOPE output and elimination of known proteolytic sites from XTEN reproduce XTEN formulations that contain XTEN of CFXTEN fusion protein formulations substantially incapable of binding to mammalian receptors, including the immune system, or the active clearance receptor of the target FVIII. In a specific embodiment of the present invention, the CFXTEN fusion protein XTEN may have >100 nmol Kd of binding to mammalian receptors, or greater than 500 nmol kDa, or greater than 1 μmol Kd to a mammalian cell surface receptor or a circulating polypeptide receptor.

Additionally, the non-repetitive sequence and corresponding absence of XTEN epitopes limits the ability of B cells to bind to, or become activated, XTEN. The repeat sequence recognizes and can form multivalent contacts even with some B cells and, as a result, crosslinking of multiple independent T cell receptors can stimulate B cell proliferation and antibody production. In contrast, while XTEN can make contacts with many different B cells, compared to its extended sequence, each individual B cell can only make one or a small number of contacts with an individual XTEN, due to the lack of sequence repetitiveness. Without being bound by any theory, XTEN generally has a much lower tendency to stimulate B cell proliferation and thus an immune response. In a specific embodiment of the present invention, CFXTEN reduces immunogenicity compared to the corresponding FVIII that is not fused to XTEN. In a specific embodiment of the present invention, administration of up to three parenteral doses of CFXTEN to a mammal results in detectable anti-CFXTEN IgG at a 1:100 serum titer, but not at a 1:1000 dilution. In a further embodiment of the present invention, administration of up to three parenteral doses of CFXTEN to a mammal results in detectable anti-FVIII IgG at a 1:100 serum titer but not at a 1:1000 dilution. In a further embodiment of the present invention, administration of up to three parenteral doses of CFXTEN to a mammal results in detectable anti-XTEN IgG at a 1:100 serum titer but not at a 1:1000 dilution. In the above embodiments, the mammal may be a mouse, rat, rabbit, or cynomolgus monkey.

An additional feature of XTEN with non-repetitive sequences, in relation to sequences with a high degree of repetitiveness, is a non-repetitive form of XTEN with weaker contacts with the antibody. Antibodies are multivalent molecules. For example, IgG has two identical binding sites and IgM has 10 identical binding sites. Thus, antibodies against repeat sequences can form multivalent contacts with such high avidity repeat sequences, which can affect the specific activity and/or elimination of such repeat sequences. In contrast, antibodies against non-repeating XTEN can produce monovalent interactions, resulting in less chance of an immune response, so that the CFXTEN compositions of the composition can remain in circulation for an extended period of time. In addition, it is believed, as schematically illustrated in FIG. 6, the conformationally labile unstructured nature of XTEN provides steric hindrance to the FVIII region close to the XTEN insertion site and provides steric hindrance for binding by FVIII inhibitors.

In additional aspects, the XTEN subject used as a hybrid partner has a large hydrodynamic radius; this property in certain embodiments of the present invention confers a corresponding increase in apparent molecular weight to the CFXTEN fusion protein containing XTEN, while in additional embodiments of the present invention it increases steric hindrance to FVIII inhibitors and anti-FVIII antibodies, reduces

- 88 041874 their ability to communicate with CFXTEN. As described in Example 26, binding of XTEN to therapeutic protein sequences results in a CFXTEN composition that can have increased hydrodynamic radii, an increased average molecular weight, and an increased average molecular weight ratio compared to a non-XTEN therapeutic protein. For example, in therapeutic applications in which a prolonged half-life is desired, compositions in which XTEN with a large hydrodynamic radius is provided as a fusion protein containing a therapeutic protein, in addition to a glomerular pore size of approximately 3-5 nmol, can effectively increase the hydrodynamic radius formulations (corresponding to an apparent molecular weight of about 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55:1261-1277), resulting in reduced renal clearance of circulating proteins with a corresponding increase in terminal half-life and other improved pharmacokinetic properties. The hydrodynamic radius of a protein is provided by its molecular weight, as well as by its structure, including shape or compactness. Without being bound by any theory, XTEN may adopt open conformations, consistent with electrostatic repulsion between individual peptide charges or the inherent flexibility imparted by particular amino acids in sequences that are unable to adopt a secondary structure. The open, extended, and unstructured conformation of an XTEN polypeptide may have a larger proportional hydrodynamic radius compared to sequence polypeptides of similar length and/or molecular weight that have a secondary and/or tertiary structure, such as conventional globular proteins. Methods for determining hydrodynamic radius are well known in the art, for example using gel permeation chromatography (SEC) as described in US Pat. Nos. 6,406,632 and 7,294,513. molecular weight and/or average molecular weight ratio, and thus allows cross-linking of CFXTEN to the desired limit values of average molecular weights or hydrodynamic radii. Accordingly, in certain embodiments of the present invention, the CFXTEN fusion protein may be configured with XTEN such that the fusion protein may have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or about 12 nm, or about 15 nm, or about 20 nm, or about 30 nmol or more. In previous embodiments of the present invention, the large hydrodynamic radius afforded by XTEN in the CFXTEN fusion protein may result in reduced clearance of the resulting fusion protein, increased terminal half-life, and increased mean retention time.

Typically, the actual molecular weight of the mature form of the FVIII component is about 265 kDa, while in the case of FVIII BDD, it is about 165 kDa. The actual molecular weight of the CFXTEN fusion protein, when containing FVIII BDD plus one or more XTENs, ranges from about 200 to about 270 kDa, depending on the length of the XTEN components. As described in the examples, when the molecular weights of the CFXTEN fusion proteins are obtained from gel permeation chromatography assays, the open conformation of XTEN, due to the low degree of secondary structure, results in an increase in the apparent molecular weight of the fusion proteins in which they are inserted. In certain embodiments of the present invention, a CFXTEN that contains FVIII and at least one or more XTENs exhibits an apparent molecular weight of at least about 400 kDa, or at least about 500 kDa, or at least about 700 kDa, or at least at least about 1000 kDa, or at least about 1400 kDa, or at least about 1600 kDa, or at least about 1800 kDa, or at least about 2000 kDa. Accordingly, CFXTEN fusion proteins that contain one or more XTENs exhibit an apparent molecular weight that is about 1.3 times greater, or about 2 times greater, or about 3 times greater, or about 4 times greater, or about 8 times, or about 10 times, or about 12 times, or about 15 times, the actual molecular weight of the fusion protein. In a particular embodiment of the present invention, the isolated CFXTEN fusion protein of any of the embodiments of the present invention disclosed herein exhibits an average molecular weight ratio under physiological conditions that is higher by about 1.3, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or about 15 times higher. In a further embodiment of the present invention, the CFXTEN fusion protein has, under physiological conditions, an average molecular weight ratio that, relative to the actual molecular weight of the fusion protein, is greater by about 3 to about 20, or about 5 to about 15, or about 8 to about 12, or about 9 to about 10 times. The increased apparent molecular weight of the claimed CFXTEN compositions is believed to enhance the pharmacokinetic properties of the fusion proteins by combining factors that include reduced active clearance, reduced binding to FVIII inhibitors, and reduced capillary and venous bleeding losses.

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Compositions CFXTEN

The present invention provides compositions that contain fusion proteins having factor VIII linked to one or more XTEN sequences, wherein the fusion protein has the effect, when administered to a subject, to replace or supplement the amount of FVIII present in the internal or contact activated coagulation pathway. The present invention satisfies a long felt need to increase the final half-life of exogenously administered to a subject in need of it, factor VIII. One way to increase the circulating half-life of a therapeutic protein is to ensure that renal clearance or metabolism of the protein is reduced. Another way to increase the terminal half-life is to reduce the active excretion of the therapeutic protein, provided that the receptors mediate active protein metabolism or other endogenous mechanisms. Both methods can be carried out by conjugating the protein to a polymer which, on the one hand, is able to provide an increased molecular size (or hydrodynamic radius) of the protein and hence reduced renal clearance, and, on the other hand, prevents the binding of the protein to clear receptors or other proteins that contribute to metabolism or clearance. Thus, certain objectives of the present invention include, but are not limited to, providing improved FVIII molecules with longer circulating half-life or finite half-life, reducing the number or frequency of administrations of the FVIII composition required while retaining at least some of the activity compared to natural coagulation factor VIII and/or increasing the ability to treat clotting defects and uncontrolled bleeding more successfully, more effectively, more economically and/or with a higher level of safety, compared with the currently available formulations of factor VIII.

Accordingly, the present invention provides recombinant factor VIII fusion protein compositions that comprise FVIII covalently linked to one or more extended recombinant polypeptides (XTEN), resulting in a CFXTEN fusion protein composition. The term CFXTEN as used herein is meant to encompass hybrid polypeptides that constitute at least one payload region that contains FVIII, or a portion of FVIII capable of binding with procoagulant activity to clotting factor FVIII and at least one other a region that contains one or more XTEN polypeptides that can be dispersed within the payload region and/or end-tethered. In a separate embodiment of the present invention, the FVIII is a natural FVIII. In a further embodiment of the present invention, FVIII is a sequence variant, fragment, homologue, or mimetic of a native sequence that retains at least a portion of the procoagulant activity of native FVIII, as disclosed herein. Non-limiting examples of FVIII, suitable for inclusion in the composition, contain the sequence of the table. 1 or sequences that have at least 80%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the sequence from the table. 1. In a preferred embodiment of the present invention, FVIII is a variant FVIII sequence without B-domains (BDD), such as the BDD sequences from Table 1. 1, or other similar sequences known in the art. In a further preferred embodiment of the present invention, CFXTEN comprises a B-domain-free (BDD) FVIII sequence variant expressed with a native 19 amino acid signal sequence that is cleaved during protein maturation.

The compositions of the present invention contain fusion proteins that are useful when administered to a subject in need thereof in the event of promoting or preventing or neutralizing a disease associated with factor VIII deficiencies or defects in endogenously obtained FVIII, or clotting disorders associated with trauma, surgery, factor VIII deficiencies or defects. Of particular interest are CFXTEN fusion protein formulations in which the pharmacokinetic parameter is increased, seeks increased solubility, increased stability, or some other improved pharmaceutical property over native FVIII, or in which an increase in terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and/or improved patient care. The CFXTEN fusion proteins of embodiments of the present invention disclosed herein exhibit one or more, or any combination, improved feature and/or embodiments of the present invention as described herein. In certain embodiments of the present invention, the CFXTEN fusion composition remains above the cut-off by at least 0.01-0.05, or 0.05 to 0.1, or 0.1 to 0.4 IU/mL at administered to a subject over a longer period of time than non-XTEN FVIII, and administered at a similar dose to a subject in need thereof (e.g., a subject such as a human, mouse, or monkey with hemophilia A).

- 90 041874

FVIII of the claimed compositions, in particular those disclosed in table. 1, along with their respective nucleotide and amino acid sequences, are available from public databases such as Chemical Abstracts Services Databases (e.g. CAS Registry), GenBank, The Universal Protein Resource (UniProt), in a subscription that provides databases such as GenSeq (for example, Derwent), as well as in the patent and source literature. Polynucleotide sequences useful for expressing sequences from a subject's CFXTEN may be a wild-type polynucleotide sequence that encodes a given FVIII (e.g., either full-length or mature), or, in some cases, the sequence may be a variant wild-type polynucleotide sequence (e.g., a polynucleotide, which encodes a biologically active wild-type protein, while the DNA sequence of the polynucleotide is optimized, for example, for expression in specific species, or the polynucleotide encodes a wild-type protein variant, such as a site-specific mutant or an allelic variant. in the art to use a consensus or wild-type cDNA sequence or FVIII codon optimizing variant to generate the CFXTEN construct of the present invention using methods known in the art and/or in combination with manual guidance and methods presented in this document and described in more detail in the examples.

In a separate embodiment of the present invention, the CFXTEN fusion protein contains a unique FVIII molecule that exhibits at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98% or at least about 99% or 100% sequence identity with the sequence from the table. 1 associated with a unique XTEN (eg, XTEN as described above) that contains, but is not limited to, 42, 144, 288, 576, or 864 amino acid AE or AG family sequences, as illustrated in Table 1. 4. In a further embodiment of the present invention, CFXTEN contains a unique FVIII associated with two XTENs, while the XTEN may be the same or they may be different. In a further embodiment of the present invention, the CFXTEN fusion protein comprises a unique FVIII molecule linked to one, two, three, four, five, six or more XTEN sequences, wherein FVIII is a sequence that has at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98%, or at least about 99% or 100% sequence identity, compared with the protein sequence selected from the table. 1 when optimally aligned and each one or more XTENs have at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98% or at least about 99% or 100% sequence identity, compared with one or more sequences selected from any of the table. 3, 4 and 13-17, with optimal alignment. In the above embodiment of the present invention, where CFXTEN has two or more XTENs, the XTENs may be the same or they may be different sequences. In yet another further embodiment of the present invention, the CFXTEN fusion protein contains a unique FVIII that exhibits at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98% at least about 99% or 100% sequence identity, compared with sequences of comparable length, selected from the table. 1, in optimal alignment, provided that three, four, five, six, or more XTEN sequences are scattered and linked, which may be identical or may be different, and each having at least about 80% sequence identity, or , alternatively, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98%, or at least about 99% or 100% identity sequence, compared with sequences selected from any of the table. 3, 4 and 13-17, or fragments thereof, with optimal alignment. In another further embodiment of the present invention, the present invention provides a CFXTEN fusion protein that contains a sequence with at least about 80% sequence identity, or alternatively, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99 or 100% sequence identity with the sequence from the table. 21, with optimal alignment.

1. Conformations of the fusion protein CFXTEN.

The present invention provides CFXTEN fusion protein compositions with CF and XTEN components linked in specific N- to C-terminal conformations.

In a separate embodiment of the CFXTEN composition, the present invention provides a fusion protein of Formula I:

(XTEN) _x -CF-(XTEN) _y I where CF is Factor VIII as defined herein, which contains sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or

- 91 041874 at least about 98%, or at least about 99% or 100% sequence identity with sequences from the table. 1; x is either 0 or 1 and y is either 0 or 1, where x+y>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. Accordingly, the CFXTEN fusion composition may have the XTEN-CF, XTEN-CF-XTEN, or CF-XTEN conformations.

In a further embodiment of the CFXTEN composition, the present invention provides a Formula II fusion protein:

(XTEN)x-(S) _x -(CF)-(XTEN) _y II where CF is factor VIII as defined herein, which contains sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequences, given in table. 1; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain a cleavage sequence or amino acids compatible with restriction sites; x is either 0 or 1 and y is either 0 or 1, where x+y>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein, wherein the fusion protein has the formula III (XTEN)x-(S)x-(CF)-(S) _y -(XTEN) _y III where CF is factor VIII, as defined herein, which contains sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96% , or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequence presented in table. 1; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain a cleavage sequence or amino acids compatible with restriction sites; x is either 0 or 1 and y is either 0 or 1, where x+y>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula IV (Al)-(XTEN)u-(A2)-(XTEN) _v -(B)-(XTEN) _w -(A3)- (XTEN)x-(Cl)-(XTEN) _y -(C2)-(XTEN)z IV where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; A3 is the A3 domain of FVIII; B is the B domain of FVIII, which may be a fragment or a splice variant of the B domain; C1 is the C1 domain of FVIII; C2 is the C2 domain of FVIII; v is either 0 or 1; w is either 0 or 1; x is either 0 or 1; y is either 0 or 1; y is either 0 or 1, provided that u + v + x + y + z >1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula V (XTEN)t-(S) _a -(Al)-(S)b-(XTEN) _u -(S)b-(A2 )-(S)c-(XTEN)v-(S)c-(B)-(S)d-(XTEN)w-(S)d-(A3)(S)e-(XTEN)x-( S)e-(Cl)-(S)f-(XTEN) _y -(S)f-(C2)-(S) _g -(XTEN)z V where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; A3 is the A3 domain of FVIII; B is a FVIII B domain, which may be a fragment or a splice variant of the B domain; C1 is the C1 domain of FVIII; C2 is a C2 domain

- 92 041874

FVIII; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain a cleavage sequence or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that t + u + v + w + x + y + z>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In an additional embodiment of formula V, the spacer region is a glycine or a sequence selected from the table. 11 and 12.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII formula VI fusion protein (XTEN)u-(S)a-(Al)-(S)b-(XTEN)v-(S)b-(A2)- (S)c-(XTEN) _w -(S)c-(A3)-(S)d-(XTEN)x(S)d-(Cl)-(S)e-(XTEN) _y -(S) e-(C2)-(S)f-(XTEN) _z VI where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; A3 is the A3 domain of FVIII; C1 is the C1 domain of FVIII; C2 is the C2 domain of FVIII; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain cleavage sequence(s) or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that u + v + w + x + y + z>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In an additional embodiment of formula V, the spacer region is a glycine or a sequence selected from the table. 11 and 12.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula VII (SP)-(XTEN)x-(CS)x-(S) _x -(FVIII_l-745)-(S) _y -(XTEN) _y -(S) _y -(FVIII_1635-2332)-(S)z-(CS)z-(XTEN)z VII where SP is a signal sequence, preferably with the sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is is the splitting sequence shown in table. 12, S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain amino acids compatible with restriction sites, FVIII_1-745 is residues 1-745 of FVIII and FVIII_1635-2332 is residues 1635 -2332 FVIII, x is either 0 or 1, y is either 0 or 1, and z is either 0 or 1, where x+y+z>2; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity of the sequences shown in table. 4. In a separate embodiment of Formula VII, the spacer region is GPEGPS (SEQ ID NO: 1612). In an additional embodiment of Formula V, the spacer region is a glycine or a sequence selected from Table 1. 11 and 12.

In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII fusion protein of formula VIII (Al)-(S)a-(XTEN)v-(S)a-(A2)-(Bl)-(S)b-(XTEN ) _w -(S)b-(B2)-(A3)-(S)c-(XTEN)x-(S)c(Cl)-(S)d-(XTEN) _y -(S)d-( C2)-(S)e-(XTEN) _z VIII where A1 is the A1 domain of FVIII; A2 is the A2 domain of FVIII; B1 is a fragment of the B domain, which may be from residue 741 to 743-750 FVIII, or alternatively from about residue 741 to about residue 745 FVIII; B2 is a fragment of the B domain, which may have FVIII residues 1635-1686 to 1689, or alternatively from about residue 1640 to about

- 93 041874 residue 1689 FVIII; A3 is the A3 domain of FVIII; C1 is the C1 domain of FVIII; C2 is the C2 domain of FVIII; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain a cleavage sequence or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that u + v + w + x + y + z>1; and XTEN is an expanded recombinant polypeptide as described herein that contains, but is not limited to, sequences that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the sequences shown in table. 4. In a separate embodiment of formula VIII, the spacer region is GPEGPS (SEQ ID NO: 1612). In an additional embodiment of formula V, the spacer region is a glycine or a sequence selected from Table. 11 and 12. In a further embodiment of the CFXTEN composition, the present invention provides a recombinant factor VIII formula IX fusion protein (Al _N )-(S)a-(XTEN) _t -(S)b-(Al _c )-(A2 _N ) -(S) _c -(XTEN) _u -(S) _d -(A2c)-(B _N )-(S)e-(XTEN)v-(S)f-(Bc)(A3 _N )-(S )g-(XTEN)w-(S)h-(A3c)-(ClN)-(S)i-(XTEN) _x -(S) _j -(Clc)-(C2N)-(S) _k -( XTEN) _y -(S)i(C2c)-(S)m-(XTEN) _z IX where A1N is a fragment of the A1 domain from residue number at least 1 (numbering relative to native, mature FVIII) to residue number not greater than 371, A1 _c is a fragment of the A1 domain from residue numbered at least 2 to residue number not greater than 372; A2N is a fragment of the A2 domain from a residue with a number of at least 373 to a residue with a number of no more than 739, A2 _c is a fragment of the A2 domain from a residue with a number of at least 374 to a residue with a number of no more than 740; BN is a fragment of domain B from a residue number of at least 741 to a residue of number no more than 1647, B _c is a fragment of domain B from a residue of at least 742 to a residue of number no more than 1648; A3N is a fragment of the A3 domain from a residue with a number of at least 1649 to a residue with a number of no more than 2019, A3 _c is a fragment of the A3 domain from a residue with a number of at least 1650 to a residue with a number of no more than 2019; C1N is a C1 domain fragment from at least 2020 to at most 2171, C1 _c is a C1 domain fragment from at least 2021 to at most 2172; C2N is a fragment of the C2 domain from a residue with a number of at least 2173 to a residue with a number of no more than 2331, C2c is a fragment of the C2 domain from a residue with a number of at least 2174 to a residue with a number of no more than 2332; S is a spacer region that has from 1 to about 50 amino acid residues, which may optionally contain a cleavage sequence or amino acids compatible with restriction sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; l is either 0 or 1; m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1, provided that t + u + v + w + x + y + z>1; and XTEN is an extended recombinant polypeptide as described herein, which contains, but is not limited to, sequences that share at least 90% identity with the sequences shown in Table. 4. In a separate embodiment of formula IX, the spacer region is GPEGPS (SEQ ID NO: 1612). In an additional embodiment of formula IX, the spacer region is a glycine or a sequence selected from Table. 11 and 12.

Embodiments of Formulas IV-VIII encompass conformations of CFXTEN, with one or more XTENs having lengths that range from about 6 amino acids to >1000 amino acids (e.g., sequences selected from any of Tables 3, 4, and 13- 17, or fragments or sequences thereof that also exhibit at least about 90-99% or more sequence identity), are introduced and linked between adjacent Factor VIII domains or linked to the N- or C-terminus of FVIII. In additional embodiments of Formulas V-VIII, the present invention further provides conformations wherein the XTENs are linked to the FVIII domains via a spacer sequence, which may optionally be amino acids compatible with restriction sites or may contain cleaved sequences.

- 94 041874 (e.g., sequences from Tables 11 and 12, described more fully below), such that the XTEN encoding sequence may be, in the case of a restriction site, integrated into the CFXTEN construct and, in the case of a cleavage sequence, XTEN may be freed from the fusion protein by the action of a protease if the sequence is cleaved. Embodiments of formulas VI-VIII differ from formula V in which the FVIII component of formulas VI-VIII are only forms of factor VIII without B domains (FVIII BDD) that retain short residual sequences of the B domain, non-limiting example sequences of which are provided in Table . 1, wherein one or more XTENs, or XTEN fragments with lengths that range from about 6 amino acids to >1000 amino acids (e.g., sequences selected from any of Tables 3, 4, and 13-17), are administered and bind between adjacent domains of factor VIII and/or between the remaining residues of domain B, such as from table. 8. An embodiment of Formula IX generally differs from those of other formulas in that one or more XTENs are administered within FVIII domains rather than between domains, and/or has an XTEN linked to the C-terminus of FVIII (or linked via a spacer region to the C-terminus of FVIII).

In certain embodiments of CFXTEN, the fusion protein comprises a form of FVIII without V-domains, with the B-domain deletion beginning at position one near amino acid residue 745 and ending at position two, at amino acid residue number 1635 to about 1690 relative to to the full length human factor VIII sequence and XTEN links the first position and the second position of the B domain deletion. In a specific embodiment of the foregoing, the first position and the second position of the B domain deletion are selected from the positions in Table. 8. In a further embodiment of the above, at least one XTEN binds the first and second position, while at least one XTEN binds in factor VIII amino acid residue 745 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1690, or amino acid residue 745 and amino acid residue 1667, or amino acid residue 745 and amino acid residue 1657, or amino acid residue 745 and amino acid residue 1657, or amino acid residue 747 and amino acid residue 1642, or amino acid residue 751 and amino acid residue 1667. In a particular embodiment of CFXTEN, in which factor VIII contains XTEN, linking the first position and the second position of the B domain deletion described in the embodiments of the present invention under this paragraph, XTEN is a sequence that has at least 80%, or at least ok about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity, compared with a sequence of comparable length, selected from any of the table. 4, tab. 13, tab. 14, tab. 15, tab. 16 and tab. 17, at optimal alignment, with CFXTEN retaining at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% , or at least about 80%, or at least about 90% of the procoagulant activity of natural FVIII.

The present invention contemplates all possible permutations of XTEN inserts between, or within, the FVIII domains or at or close to the insertion points of Table 1. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or those illustrated in FIG. 8-9, with optional binding of additional XTEN to the N- or C-terminus of FVIII, optionally linked through an additional cleavage sequence selected from Table 8-9. 12, which leads to the composition CFXTEN; non-limiting examples of which are illustrated in FIG. 5 and 12. In a separate embodiment of the present invention, CFXTEN contains the FVIII BDD sequence from Table. 1, in which one or more XTEN, with each having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or more sequence identity, compared with the sequence from any of the table. 3, 4, and 13-17, or fragments thereof, are inserted between any two residual amino acids of the B domain of the FVIII BDD sequence, resulting in a single-stranded FVIII fusion protein, with CFXTEN retaining at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the natural FVIII procoagulant activity. In the above embodiment of the present invention, CFXTEN may have an additional XTEN sequence from any of the table. 4 and 13-17 linked to the N- or C-terminus of the fusion protein. In an additional embodiment of the present invention, CFXTEN contains at least the first XTEN, administered within the site shown in table. 8, with CFXTEN retaining at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least at least about 80%, or at least about 90%, of the procoagulant activity of native FVIII. In a separate embodiment of the Formula VII fusion protein, CFXTEN contains the FVIII BDD sequence from Table 1. 1, in which two or more XTENs are each

- 95 041874 which has at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity, compared with the sequence from any of the table. 3, 4, and 13-17, or fragments thereof, are linked to a FVIII-BDD sequence into which at least one XTEN is inserted from about 3 to about 20 amino acid residues to the C-terminal side of the R740 amino acid of the FVIII cleavage site, and from about 3 to about 20 amino acid residues to the N-terminal side of the R1689 amino acid cleavage site of the residual amino acid domain B FVIII of the FVIII BDD sequence, resulting in a single-stranded FVIII fusion protein, and one or two XTENs are linked by a cleavage sequence to N- and/or C -terminus of the FVIII-BDD sequence, with CFXTEN exhibiting at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80 %, or at least about 90% of the procoagulant activity of natural FVIII after XTEN secretion by cleavage of cleaved sequences.

In a separate embodiment of the present invention, the A3 domain contains an a3 acidic region or a portion thereof. In a further embodiment of the present invention, at least one XTEN is administered within the a3 acid region or part thereof, the N-terminus of the a3 acid region or part thereof, the C-terminus of the a3 acid region or part thereof, or combinations thereof. In certain embodiments of the present invention, at least one XTEN is introduced into the C2 domain, the N-terminus of the C2 domain, the C-terminus of the C2 domain, or a combination thereof. In additional embodiments of the present invention, Factor VIII comprises all or part of the B domain. In other embodiments of the present invention, at least one XTEN is administered within all or part of the B domain, the N-terminus of the B domain, the C-terminus domain B, or combinations thereof.

2. Conformations of the fusion protein CFXTEN with internal XTEN.

In additional aspects, the present invention provides a CFXTEN configured with one or more XTEN sequences that are internal to the FVIII sequence. In a specific embodiment, the present invention provides CFXTEN configured with one or more XTEN sequences that are internal to the FVIII sequence to confer properties such as, but not limited to, increased stability, increased protease resistance, increased resistance to clearance mechanisms, which contain, but are not limited to, interaction with excretion of FVIII receptors or inhibitors, and increased hydrophilicity, compared to FVIII without embedded XTEN.

The present invention contemplates that various conformations or sequence variants of FVIII may be used as a platform into which one or more XTENs are inserted. These conformations include, but are not limited to, natural FVIII, FVIII BDD, single chain FVIII (scFVIII), and conformational variants thereof. In the case of scFVIII, the present invention provides CFXTEN, which can be constructed by replacing one or more amino acids of the FVIII processing site. In a separate embodiment of the present invention, the scFVIII used in CFXTEN is created by replacing R1648 in the FVIII RHQREITR sequences (SEQ ID NO: 1698) with glycine or alanine to prevent proteolytic processing into a heterodimeric form. It is specifically contemplated that any embodiments of CFXTEN disclosed herein with FVIII residue 1648 may have a glycine or alanine substitution for arginine at position 1648. that surround the R1648 processing site are replaced with XTEN as illustrated in FIG. 10A and 10B. In a particular embodiment of the present invention, at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% or more of the B domain is replaced with the XTEN sequence disclosed herein. a document that contains one or more of the R740, R1648, or R1689 cleavage sites. In a further embodiment of the present invention, CFXTEN has a B domain FVIII sequence between the FXIa cleavage sites R740 and R1689 (also retain at least 1-5 adjacent B domain amino acids between the cut site and the start of XTEN to allow the protease to access the cut site ) are replaced by XTEN. In a further embodiment of the present invention, CFXTEN has a B domain FVIII sequence between the FXIa N745 and P1640 cleavage site, which is substituted with XTEN. In additional embodiments, the present invention provides sequence variants of the FVIII BDD CFXTEN in which portions of the B domain are deleted but only one of the FXI activation sites, R740 or R1689 (and 1-5 contiguous B domain amino acids) is left within the construct, wherein that XTEN, after FXIa cleavage, remains linked at one end to either the light or heavy chain, as illustrated in FIG. 5B and 5D. In a specific embodiment of the above, CFXTEN comprises the BDD FVIII sequence, wherein the amino acids N745 to P1640, or S743 to Q1638, or P747 to V1642, or between N745 and Q1656, or between N745 and S1657, or between N745 and T1667, or between N745 and Q1686, or

- 96 041874 between R747 and V1642, or between T751 and T1667 is deleted, and the XTEN sequence is linked between these amino acids connecting the heavy and light chains, and may additionally contain additional XTEN, introduced either within the outer loops, between the FVIII domains, or on N or C-terminal sequence BDD FVIII, such as one or more insertion sites from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or those illustrated in FIG. 8-9. In a further embodiment of the above, CFXTEN contains the FVIII BDD sequence in which the amino acids between K713 and Q1686, or between residues 741 and 1648, are deleted, and XTEN is linked between the two amino acids, and additional XTEN is inserted either in the outer loops, between the FVIII domains, or at the N- or C-terminus of the FVIII BDD sequence, which contains, but is not limited to, one or more insertion sites from Table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or those illustrated in FIG. 8-9. In certain embodiments of the present invention, such CFXTEN sequences may have one or more XTENs that exhibit at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with the XTEN sequence from any of the tables. 4 and 13-17.

The present invention provides other CFXTEN with internal XTEN in various conformations; illustrative conformation schemes are illustrated in FIG. 5 and 10. Regions suitable for XTEN insertion sites include known FVIII domain boundaries, exon boundaries, known outer loops, and solvent accessible surface region sites as determined by X-ray diffraction analysis, and structure models derived from molecular dynamics simulations of FVIII, regions of low order (assessed using the programs described in the caption to Fig. 7), regions of low homology/lack of conservatism across species, and hydrophilic regions. In a further embodiment of the present invention, the XTEN insertion sites are selected based on the binding sites of the putative FVIII excretion receptor. In a further embodiment of the present invention, CFXTEN comprises XTEN administered at locations not in close proximity to mutations involved in hemophilia A listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database eliminate (Kembal-Cook G, et al., The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res. (1998) 26(1):216-219). In a further embodiment of the present invention, potential XTEN insertion sites comprise residues within FVIII epitopes that are binding anti-FVIII antibodies seen in sensitized hemophiliacs and that do not otherwise serve as protein interactive sites. Regions and/or sites that are considered, as an exception, XTEN insertion sites, contain residues/regions of factor VIII that are important in various interactions, contain other folding proteins, residues that surround each arginine activation/cleavage inactivation site acting in according to protease thrombin, factor Xa, activated protein C, residues that surround the signal processing site (residue 1) if the construct contains a signal sequence, areas known to interact with other proteins such as FIXa, FX/FXa, thrombin, activated protein C, cofactor of protein S to protein C, von Willebrand factor, sites known to interact with phospholipid cofactors in coagulation, residues that are involved in domain interactions, residues coordinating Ca++ or Cu ⁺⁺ ions, cysteine residues that are involved in SS intramolecular bonds, fixed amino acid FVIII mutation point site and site derived from haemophilia A subjects that affect procoagulant activity, and FVIII mutation sites made in a research laboratory that affect procoagulant activity. The sites considered for either insertion (to extend half-life) or exclusion (needed to remove used FVIIIa or FXa) contain regions known to interact with heparin sulfate proteoglycan (HSPG) or low density lipoprotein receptor (LPR)-related protein.

As a result of the analysis of the above criteria, as described in example 34, throughout the FVIII BDD sequence, various insertion sites or insertion site regions are identified and/or confirmed as candidates for XTEN insertion, non-limiting examples of which are given in table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 and shown schematically in FIG. 8 and 9. In a separate embodiment of the present invention, CFXTEN contains XTEN inserts between separate FVIII domains, ie. between A1 and A2, or between A2 and B, or between B and A3, or between A3 and C1, or between C1 and C2 domains. In a further embodiment of the present invention, the CFXTEN contains an XTEN introduced into the B domain or between the remaining residues of the BDD sequence. In a further embodiment of the present invention, CFXTEN contains XTEN, introduced within known exon boundaries of the gene encoding FVIII, represents as exons elements of an evolutionarily conserved sequence that has a high probability of functioning in the context of other protein sequences. In an additional embodiment, the present

- 97 041874 th invention CFXTEN contains XTEN, introduced within the outer loops defined by the x-ray structure of FVIII. In a further embodiment of the present invention, CFXTEN contains XTEN administered within a low order region, defined as having low electron density or no electron density detected by X-ray diffraction analysis. In a further embodiment of the present invention, CFXTEN comprises XTEN administered within a low order region predicted by structure prediction algorithms such as, but not limited to, the FoldIndex, RONN, and Kyte & Doolitlle algorithms. In a further embodiment of the present invention, CFXTEN contains XTEN administered within sequence regions with a high frequency of hydrophilic amino acids. In a further embodiment of the present invention, CFXTEN comprises XTEN administered within epitopes capable of being bound by naturally occurring anti-FVIII antibodies in sensitized hemophilia patients. In a further embodiment of the present invention, CFXTEN comprises XTEN administered within sequence regions with low sequence conservation and/or differences in sequence segment length across the sequence of FVIII of different species. In a further embodiment of the present invention, CFXTEN contains XTEN linked to the N-terminus and/or C-terminus. In a further embodiment, the present invention provides CFXTEN conformations with an administered XTEN selected according to two or more criteria from the embodiments listed above. In a further embodiment, the present invention provides CFXTEN conformations with at least one, alternatively at least two, alternatively at least three, alternatively at least four, alternatively at least five, or more XTENs introduced into the sequence factor VIII, while the insertion points are on, or close to, the N- or C-terminal side on at least one, two, three, four or five, or six or more amino acids selected from the amino acids of the residue of the insert from table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or those illustrated in FIG. 8-9, or, alternatively, within one, or within two, or within three, or within four, or within five, or within six amino acids of the amino acids of the residue of the insert from table. 5, tab. 6, tab. 7, tab. 8 and tab. 9, or within various amino acid ranges of the insertion residue schematically depicted in the case of the exemplary FVIII BDD sequence in FIG. 9.

As described above, one or more intra-XTENs, or an XTEN fragment, may have a sequence length of 6 to 1000 or more amino acid residues. In certain embodiments of the present invention, while CFXTEN has one or two or three or four or five or more XTEN sequences internal to FVIII, the XTEN sequences may be identical or may be different. In a particular embodiment of the present invention, each XTEN located within has at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with the same length or XTEN fragments or motifs selected from any of the table. 3, 4 and 13-17, with optimal alignment. In a further embodiment, the present invention provides a CFXTEN configured with one or more XTENs administered internally to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with the sequence from the table. 1, despite the fact that the inserts are placed on the insertion points or the range of insertion points indicated in the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9, fig. 8 or within the range of inserts as illustrated in FIG. 9. It should be clear to those skilled in the art that XTEN administered within the FVIII sequence at the insertion point of Table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 is linked via its N- and C-terminus to the flanking amino acids of FVIII (or via spacer binding or cleavage sequences as described above) if the XTEN linked to the N- or C-terminus of FVIII can only be linked to a unique amino acid FVIII (or with a binding spacer or amino acid cleaved sequence as described above). By way of example only, CFXTEN variants with three internal XTENs may have: XTEN (as described herein) inserted between BDD FVIII residues 741 and 1640, residues 18 and 19, and residues 1656 and 1657; or XTEN inserted between BDD FVIII residues 741 and 1640, residues 1900 and 1901, and at the C-terminus at residue 2332; or XTEN inserted between BDD FVIII residues 26 and 27, residues 1656 and 1657, and residues 1900 and 1901; or XTEN inserted between BDD FVIII residues 741 and 1640, residues 1900 and 1901, and at the C-terminus at residue 2332.

When evaluating the hybrid proteins CFXTEN with XTEN from table. 5 introduced at the localization, it was found that inserts in certain regions of the FVIII sequence resulted in CFXTEN with good expression and retention of procoagulant activity. Accordingly, in preferred embodiments, the present invention provides CFXTEN fusion proteins configured with one, or two, or three, or four, or five, or six or more XTENs, each of which has at least about 80% sequence identity, or, alternatively, 81,

- 98 041874

82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity, compared to XTEN selected from any of tab. 4 and 13-17 introduced into, or associated with, a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with the sequence from the table. 1, despite the fact that the insert is placed on the insertion point within one, or two, or three, or four, or five, or six or more of the ranges shown in table. 7. In previous embodiments of the present invention, CFXTEN fusion proteins with XTEN inserts retain at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of procoagulant activity, compared to the corresponding unbound FVIII. with XTEN.

When evaluating hybrid proteins CFXTEN with XTEN, introduced into one or more localizations from the table. 5, it was unexpectedly found that a high percentage of fusion proteins with XTEN inserts retained procoagulant activity as described in Example 25. Accordingly, the present invention provides CFXTEN fusion proteins configured with one, two, three, four, five, six, or more XTENs, wherein the resulting fusion protein exhibits at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or more procoagulant activity , compared with the corresponding FVIII, not associated with XTEN, provided that it is analyzed using the analysis of coagulant activity described in this document. In a preferred embodiment, the present invention provides CFXTEN fusion proteins that contain one, or two, or three, or four, or five, or six or more XTENs, each of which has at least about 80% sequence identity, or alternatively, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity compared to XTEN selected from any of the tables. 4 and 13-17 linked to an FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with the sequence from the table. 1, despite the fact that the inserts are placed on one or more insertion points selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 and wherein the resulting fusion protein exhibits at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% or more procoagulant activity than the corresponding non-XTEN FVIII when assayed in vitro using the assay described herein (eg, chromogenic assay). As a subject of CFXTEN fusion proteins generally exhibit an increased terminal half-life compared to native FVIII, one skilled in the art will appreciate that CFXTEN with lower procoagulant activity relative to an equimolar amount of native FVIII is nonetheless acceptable at administration as a therapeutic composition to a subject in need thereof. In a further embodiment of the present invention, CFXTEN fusion proteins that contain one, or two, or three, or four, or five or more XTENs, each of which has at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared to XTEN selected from any of the table. 4 and 13-17 linked to an FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99 or 100% sequence identity, compared with the sequence from the table. 1, despite the fact that the insert is placed on one or more insertion points or a range of insertion points selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9, while the resulting fusion protein exhibits an FVIII activity of at least about 0.5 IU/ml, or at least about 0.75 IU/ml, or at least about 1.0 IU/ml, or at least at least about 1.5 IU/mL, or at least about 2.0 IU/mL, or at least about 2.5 IU/mL, or at least about 3 IU/mL, or at least about 4 IU /ml, or at least about 5 IU/ml, or at least about 7 IU/ml, or at least about 10 IU/ml, or at least about 20 IU/ml, or at least about 30 IU /ml when expressed in a cell culture medium and analyzed using chromogenic analysis, in which the culture and expression are consistent with the methods described in this document; for example, the methods from example 25.

It is believed that the opening of insertion sites, while FVIII retains at least some of its procoagulant activity, may also allow the insertion of other peptides and polypeptides with either unstructured or structured characteristics that are associated with extended half-life if fused to the FVIII protein in one or more of the same sites. Non-limiting examples that contain albumin, albumin fragments, immunoglobulin Fc fragments, human chorionic gonadotropin C-terminal peptide (CTP) β subunit, HAP sequence, transferrin, PAS polypeptides of U.S. Patent Application No. 20100292130, polyglycine linkers, polyserine linkers, peptides, and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and

- 99 041874 prolines (P) with varying degrees of secondary structure from less than 50% to more than 50%, among other things, would be suitable for insertion into certain active sites of the FVIII insert.

In embodiments of the fusion protein described herein, the CFXTEN fusion protein may further comprise one or more cleavage sequences from Table 1. 12, or other sequences known in the art, the cleavage sequence is located between, or within, 6 amino acid residues, at the intersection of FVIII and XTEN sequences, which may contain two cleavage sequences in this internal XTEN sequence. In a particular embodiment of the present invention, a CFXTEN that contains cleaved sequences has two identical cleaved sequences, each located at or near the respective ends of one or more internal XTENs, such that the XTENs are released from the fusion protein when cleaved by a protease, which binds to and cleaves this sequence. Sequences that can be cleaved are described more fully below, and illustrative sequences are shown in table. 12.

Table 5

XTEN insert localizations associated with the BDD FVIII sequence

No. Insertion point XTEN Paste Remainder FVIII BDD forward sequence FVIII domain 1 0 (N-terminus) ATR Al 2 3 R RYY Al 3 17 M QSD Al 4 18 Q SDL Al 5 22 G ELP Al 6 24 L PVD Al 7 26 V DAR Al 8 28 A RFP Al 9 32 R RVP Al 10 38 F PFN Al AND 40 F NTS Al 12 41 N TSV Al 13 60 N IAK Al 14 61 I ACRE Al 15 65 R PPW Al 16 81 Y DTV Al 17 111 G AEY Al 18 116 D QTS Al 19 119 S QRE Al 20 120 Q REK Al

- 100 041874

21 128 V FPG Al 22 129 F PGG Al 23 130 P GGS Al 24 182 G SLA Al 25 185 A KEK Al 26 188 TO TQT Al 27 205 G KSW Al 28 210 S ETK Al 29 211 E TKN Al thirty 216 L MQD Al 31 220 R DAA Al 32 222 A ASA Al 33 223 A SAR Al 34 224 S ARA Al 35 230 TO MHT Al 36 243 P GLI Al 37 244 G LIG Al 38 250 R KSV Al 39 318 D GME Al 40 333 P QLR Al 42 334 Q LRM Al 43 336 R MKN al 44 339 N NEE al 45 345 D YDD al 46 357 V VRF al 47 367 s FIQ al 48 370 s RPY al 49 375 A KKH A2 50 376 TO PRC A2 51 378 H PKT A2

- 101 041874

52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2 55 409 S QYL A2 56 416 P QRI A2 57 434 E TFK A2 58 438 T REA A2 59 441 A IQH A2 60 442 I QHE A2 61 463 I IFK A2 62 487 Y SRR A2 63 490 R LPK A2 64 492 P KGV A2 65 493 TO GVK A2 66 494 G VKH A2 67 500 D FPI A2 68 506 G EIF A2 69 518 E DGP A2 70 556 TO ESV A2 71 565 Q IMS A2 72 566 I MSD A2 73 598 P AGV A2 74 599 A GVQ A2 75 603 L EDP A2 76 616 S ING A2 77 686 G LWI A2 78 713 TO NTG A2 79 719 Y EDS A2 80 730 L LSK A2 81 733 TO NNA A2 82 745 N PPV IN 83 1640 P PVL IN 84 1652 R TTL IN 85 1656 Q SDQ AZ 86 1685 N QSP AZ 87 1711 M sss AZ 88 1713 s SPH AZ 89 1720 N RAQ AZ 90 1724 S GSV AZ 91 1725 G SVP AZ 92 1726 S VPQ AZ 93 1741 G SFT AZ 94 1744 T QPL AZ 95 1749 R GEL AZ 96 1773 V TFR AZ 97 1792 Y EED AZ

- 102 041874

98 1793 E EDQ A3 99 1796 Q RQG A3 100 1798 Q GAE A3 101 1799 G AEP A3 102 1802 R RKN A3 103 1803 R KNF A3 104 1807 V KPN A3 105 1808 TO PNE A3 106 1827 To DEF A3 107 1844 E KDV A3 108 1861 N TLN A3 109 1863 L NPA A3 110 1896 E RNC A3 111 1900 R ARS A3 112 1904 N IQM A3 FROM 1905 I QME A3 114 1910 R TFK A3 115 1920 A ING A3 116 1937 D QRI A3 117 1981 G VFE A3 118 2019 N KCQ A3 119 2020 TO CQT Cl 120 2044 G QWA Cl 121 2068 F SWI Cl 122 2073 V DLL Cl 123 2090 R QKF Cl 124 2092 TO FSS Cl 125 2093 F SSL Cl 126 2111 TO WQT Cl 127 2115 Y RGN Cl 128 2120 T GTL Cl 129 2125 V FFG Cl 130 2171 L NSC Cl 131 2173 S CSM C2 132 2188 A QIT C2 133 2223 V NNP C2 134 2224 N NPK C2 135 2227 TO EWL C2 136 2268 G HQW C2 137 2277 N GKV C2 138 2278 G KVK C2 139 2290 F TPV C2 140 2332 Y From the end of FVIII CT

Designates an XTEN insertion point based on the amino acid number of mature full-length human FVIII, with the insertion being either N-terminal or C-terminal of the indicated amino acid. Forward sequence in FVIII BDD with deletion 746-1639.

Table 6

Exemplary XTEN Insert Locations Associated with FVIII Polypeptide

No. Insertion point XTEN Paste Remainder FVIII BDD forward sequence FVIII domain Distance from the remainder of the insert 9 32 P RVP Al -3,+6 31 220 R DAA Al - 34 224 S ARA Al +5 43 336 R MKN al -1,+6 44 339 N NEE al -4, +5 52 399 V LAP A2 -6, +3 56 416 P QRI A2 +6 75 603 L EDP A2 6, +6 85 1656 Q SDQ IN -3,+6 87 1711 M sss A3 -6,+1 91 1725 G SVP A3 +6 113 1905 I QME A3 +6 114 1910 P TFK A3 -5,+6

The distance from the insertion residue corresponds to the relative number of amino acids from the N-terminus (negative numbers) or C-terminus (positive numbers) designated as the insertion residue (residue 0) where an insertion can be made. The -x designation corresponds to an insertion site that is x amino acids further from the N-terminal side of the designated insert residue. Similarly, the designation +x corresponds to an insertion site that is x amino acids further from the C-terminal side of the designated insert residue. For example, -1, +2 indicate that the insertion is made at the N-terminus or C-terminus of the amino acid residues designated -1.0, +1, or +2.

Table 7

Additional Illustrative Locations of the XTEN Insert Associated with the FVIII Polypeptide

No. Insertion point range XTEN First remainder of the insert FVIII domain 3 18-32 Q Al 8 40 F Al 18 211-224 E Al 27 336-403 R Al, A2 43 599 A A2 47 745-1640 N IN 50 1656-1728 Q B, AZ 57 1796-1804 R AZ 65 1900-1912 R AZ 81 2171-2332 L C1,C2

denote the insertion site numbering range relative to the amino acid number of mature human FVIII.

Table 8

Exemplary localizations of the XTEN insert within FVIII polypeptide variants without B domains

Insertion point range XTEN First remainder of the insert The second remainder of the insert 740-1640 R R 740-1690 R S 741-1648 S R 743-1638 S Q 745-1638 N Q 745-1640 N R 745-1656 N Q 745-1657 N S 745-1667 N T 745-1686 N Q 747-1642 R V 751-1667 T T

designate amino acids linked within the variant without B domains and adjacent A3 domain to amino acids numbered relative to the amino acid number of mature human FVIII;

denote amino acids bound by XTEN administered by BDD-FVIII.

- 104 041874

Table 9

Exemplary FVIII Polypeptide-Associated XTEN Insertion Locations That Lead to Procoagulant Activity

No. Insertion point XTEN Paste Remainder FVIII BDD forward sequence FVIII domain 2 3 R RYY Al 4 18 Q SDL Al 5 22 G ELP Al 7 26 V DAR Al 9 32 R RVP Al AND 40 F NTS Al 18 116 D QTS Al 19 119 S QRE Al 26 188 TO TQT Al 29 211 E TKN Al thirty 216 L MQD Al 31 220 R DAA Al 34 224 S ARA Al 35 230 TO ΜΗΤ Al 40 333 R QLR Al 43 336 R MKN al 44 339 N NEE al 52 399 V LAP A2 53 403 D DRS A2 55 409 S QYL A2 56 416 R QRI A2 60 442 I QHE A2 62 487 Y SRR A2 63 490 R LPK A2 66 494 G VKH A2 69 518 E DGP A2 74 599 A GVQ A2 75 603 L EDP A2 78 713 TO NTG A2 82 745 N PPV IN 85 1656 Q SDQ A3 87 1711 m sss A3 89 1720 N RAQ A3 91 1725 G SVP A3 99 1796 Q RQG A3 102 1802 R RKN A3 110 1896 E RNC A3 111 1900 R APC A3 112 1904 N IQM A3 FROM 1905 I QME A3 114 1910 R TFK A3 121 2068 F SWI Cl 130 2171 L NSC Cl 135 2227 TO EWL C2 137 2277 N GKV C2 140 2332 Y From the end of FVIII C2

forward sequence in FVIII BDD with deletion 746-1639.

In additional aspects, the present invention provides component libraries and methods for generating libraries derived from nucleic acids encoding FVIII, XTEN segments, and XTEN-related FVIII segments that are useful in generating genes encoding a CFXTEN subject. As a first step, a gene library encoding FVIII and XTEN introduced at various unique sites at or within 1-6 amino acids of the insertion site defined in Table 1. 5 or illustrated in FIG. 8-9, CFXTEN activity and pharmacokinetic properties are generated, expressed, and reconstituted, as illustrated in FIG. 15. Those CFXTENs that show improved properties are then used to generate genes encoding the FVIII segment and insertion site, plus XTEN, with components from each improved insert present in the library, as illustrated in FIG. 11. In a specific embodiment of the present invention, library components are assembled using standard recombinant techniques, in a combinatorial manner as illustrated in FIG. 11, resulting in permutations of CFXTEN with multiple internal and N- and C-terminal XTENs that may contain an insert

- 105 041874 sites from, or close to those, tab. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or, as illustrated in FIG. 8-9. The resulting constructs are then evaluated for potency and improved pharmacokinetic properties, and those candidates that result in CFXTEN with improved properties, such as reduced active clearance, protease resistance, reduced immunogenicity, and increased pharmacokinetic properties, compared to non-XTEN-bound FVIII, evaluate additionally.

3. XTEN hinge available for embedding.

As described in detail elsewhere herein and as illustrated in FIG. 33-36, researchers recognize that each FVIII A domain contains at least two XTEN loops available for insertion into which XTEN sequences are introduced without abolishing the procoagulant activity of the recombinant protein or the ability of the recombinant proteins to be expressed in vivo or in vitro in the host cell. Researchers define insertable XTEN loops as regions with, among other attributes, a smooth surface or solvent exposure and high conformational flexibility. Domain A1 contains the XTEN loop region available for embedding-1 (A1-1) and the XTEN loop region available for embedding-2 (A1-2), domain A2 contains the XTEN loop region available for embedding-1 (A2-1 ), and the XTEN loop region available for embedding-2 (A2-2), the A3 domain contains the XTEN loop region available for embedding-1 (A3-1) and the XTEN loop region available for embedding-2 (A3-2 ).

In certain aspects, the recombinant FVIII protein, as described above, comprises at least one XTEN sequence introduced into at least one XTEN loop available for insertion, A1-1, A1-2, A2-1, A2-2, A3- 1 or A3-2, while the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell. In certain aspects, the recombinant FVIII protein as described above comprises at least two XTEN sequences introduced into FVIII, e.g., into two different XTEN loops available for insertion, A1-1, A1-2, A2-1, A2-2 , A3-1 or A3-2, while the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell. In addition, the recombinant FVIII protein as described above may contain two or more XTEN sequences inserted into a unique XTEN loop available for insertion with or without XTEN sequences inserted into other XTEN loops available for insertion, provided that the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell. In certain aspects, the recombinant FVIII protein as described above may comprise at least one XTEN sequence inserted into at least one XTEN loop accessible for insertion as described above and may further comprise one or more XTEN sequences inserted into a3 , while the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell. In certain aspects, the recombinant FVIII protein of the present invention may comprise three, four, five, six or more XTEN sequences inserted into one or more insertable XTEN loops or a3, wherein the recombinant FVIII protein has procoagulant activity and may be expressed in vivo or in vitro in the host cell.

In certain aspects, the recombinant FVIII protein, as described above, contains at least one XTEN sequence introduced into a3, while the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. In certain aspects, the recombinant FVIII protein of the present invention comprises at least one XTEN sequence inserted into a3 and further comprises one or more XTEN sequences inserted into one or more XTEN loops available for insertion as described above, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell.

Researchers recognize that the recombinant FVIII protein of the present invention contains at least two XTEN loops available for insertion in each of the FVIII domain A region, which results in the insertion of an XTEN sequence that has procoagulant activity and still remains capable of being expressed in vivo or in vitro in the host cell. Determine the different crystal structures of FVIII, different degrees of resolution. These FVIII and FVIIIa structures, determined by X-ray diffraction crystallography and molecular dynamics simulation, are used to generate a model of the accessible surface area and conformational flexibility of FVIII. For example, the crystal structure of human FVIII is determined by Shen et al. Blood 111: 1240-1247 (2008) and Ngo et al. Structure 16: 597-606 (2008). Data for these structures is available from the Protein Data Bank (pdb.org) under accession numbers 2R7E and 3CDZ, respectively.

The crystal structure of the predicted secondary structure of human FVIII heavy and light chains according to Shen et al. is illustrated in FIG. 37A and 37B. The crystal structures of the various beta strands predicted by Shen et al. are numbered sequentially in FIG. 8A and 8B. In certain embodiments of the present invention, insertable XTEN loops A1-1, A1-2, A2-1, A2-2, A3-1 and A3-2 are contained within conformationally labile loops.

- 106 041874 structures with an open surface in the FVIII domains. A1-1 is located between beta strand 1 and beta strand 2, A1-2 is located between beta strand 11 and beta strand 12, A2-1 is located between beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-1 is located between beta strand 38 and beta strand 39, and A3-2 is located between beta strand 45 and beta strand 46, in accordance with the secondary structure of mature FVIII, which is preserved with account number 2R7E in the PDB database (PDB:2R7E) and as shown in FIG. 8A and 8B. The secondary structure of account number PDB 2R7E illustrated in FIG. 8A and 8B corresponds to the purpose of the standardized secondary structure according to the DSSP program (Kabsch and Sander, Biopolymers, 22:2577-2637 (1983)). The mature FVIII DSSP secondary structure, which is stored in the PDB with accession number 2R7E, can be accessed from the DSSP database available from the web site swift.cmbi.ru.nl/gv/dssp/ (last accessed February 9, 2012) (Joosten et al., 39 (Suppl. 1): D411-D419 (2010)).

In certain aspects, the conformationally labile open-surface loop structure that contains A1-1 corresponds to the region of native mature human FVIII from about amino acid 15 to about amino acid 45 in FIG. 30. In certain aspects, A1-1 corresponds to the region of natural mature human FVIII from about amino acid 18 to about amino acid 41 in FIG. 30. In certain aspects, the conformationally labile open-surface loop structure that contains A1-2 corresponds to the region of native mature human FVIII from about amino acid 201 to about amino acid 232 in FIG. 30. In certain aspects, A1-2 corresponds to the region of naturally occurring mature human FVIII from about amino acid 218 to about amino acid 229 in FIG. 30. In certain aspects, the conformationally labile open-surface loop structure that contains A2-1 corresponds to the region of native mature human FVIII from about amino acid 395 to about amino acid 421 in FIG. 30. In certain aspects, A2-1 corresponds to the region of natural mature human FVIII from about amino acid 397 to about amino acid 418 in FIG. 30. In certain aspects, the conformationally labile, open-surface loop structure that contains A2-2 corresponds to the region of native mature human FVIII from about amino acid 577 to about amino acid 635 in FIG. 30. In certain aspects, A2-2 corresponds to the region of natural mature human FVIII from about amino acid 595 to about amino acid 607 in FIG. 30. In certain aspects, the conformationally labile open-surface loop structure that contains A3-1 corresponds to the region of native mature human FVIII from about amino acid 1705 to about amino acid 1732 in FIG. 30. In certain aspects, A3-1 corresponds to the region of natural mature human FVIII from about amino acid 1711 to about amino acid 1725 in FIG. 30. In certain aspects, the conformationally labile open-surface loop structure that contains A3-2 corresponds to the region of native mature human FVIII from about amino acid 1884 to about amino acid 1917 in FIG. 3. In certain aspects, A3-2 corresponds to the region of natural mature human FVIII from about amino acid 1899 to about amino acid 1911 in FIG. thirty.

In certain aspects, the recombinant FVIII protein of the present invention comprises one or more XTEN sequences inserted into one or more insertable XTEN loops, FVIII, or the a3 region, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell. The XTEN sequences introduced contain those that increase the in vivo half-life or the in vivo or in vitro stability of FVIII.

In certain aspects, the recombinant FVIII protein of the present invention comprises XTEN sequences, introduced directly in the forward direction, of one or more amino acids corresponding to one or more amino acids in mature natural human FVIII, which contains, but is not limited to: amino acid 18 in FIG. 30, amino acid 26 in FIG. 30, amino acid 40 in FIG. 30, amino acid 220 in FIG. 30, amino acid 224 in FIG. 30, amino acid 399 in FIG. 30, amino acid 403 in FIG. 30, amino acid 599 in FIG. 30, amino acid 603 in FIG. 30, amino acid 1711 in FIG. 30, amino acid 1720 in FIG. 30, amino acid 1725 in FIG. 30, amino acid 1900 in FIG. 30, amino acid 1905 in FIG. 30, amino acid 1910 in FIG. 30 or any combination thereof that contains the appropriate inserts in the FVIII BDD variants described herein.

In certain aspects, the recombinant FVIII protein of the present invention comprises at least one XTEN sequence inserted into the a3 region of FVIII, either alone or in combination with one or more XTEN sequences inserted into insertionable XTEN loops of the A domains (e.g., A1-1, A1-2, A2-1, A2-2, A3-1 or A3-2 as described above), while the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in the host cell . In certain aspects, at least one XTEN sequence is inserted into the a3 region directly downstream of the amino acids that correspond to amino acid 1656 in FIG. 30. In certain aspects, the recombinant FVIII protein of the present invention comprises an XTEN sequence inserted into the a3 region as described, and further comprises one or more XTEN sequences inserted directly into

- 107 041874 direct direction from one or more amino acids corresponding to one or more amino acids of mature natural human FVIII, which contain, but are not limited to: amino acid 18 in FIG. 30, amino acid 26 in FIG. 30, amino acid 40 in FIG. 30, amino acid 220 in FIG. 30, amino acid 224 in FIG. 30, amino acid 399 in FIG. 30, amino acid 403 in FIG. 30, amino acid 599 in FIG. 30, amino acid 603 in FIG. 30, amino acid 1711 in FIG. 30, amino acid 1720 in FIG. 30, amino acid 1725 in FIG. 30, amino acid 1900 in FIG. 30, amino acid 1905 in FIG. 30, amino acid 1910 in FIG. 30 or any combination of them.

Any person skilled in the art will appreciate that the previous aspects of the FVIII loop available for insertion of a natural protein into which a heterologous protein is introduced are also applicable to FVIII variants without B domains described herein; for example, the sequences given in table. 1. In the practical implementation of the present invention, it should be understood that the sequence of BDD-FVIII from the table. 1 can be substituted for the recombinant FVIII protein of the various embodiments of the present invention described above, and the resulting constructs are expected to similarly retain procoagulant activity.

4. Violation associated with agents that bind FVIII.

Another object of the present invention is to provide CFXTEN fusion protein procoagulant compositions for use in human patients suffering from a coagulopathy such as hemophilia A who have natural or acquired antibodies, inhibitors or other FVIII binding proteins or molecules that affect the activity or the half-life of the CFXTEN fusion protein, with CFXTEN retaining a higher amount of procoagulant activity than the corresponding non-XTEN FVIII. As used herein, FVIII binding agent means any molecule capable of binding to natural FVIII or to a recombinant factor VIII fusion protein of the present invention that contains factor VIII or a fragment thereof, either native, derived, or recombinantly produced. Separately, it is contemplated that the FVIII binding agent contains anti-FVIII antibodies and FVIII inhibitors, among other proteins capable of binding to FVIII in particular. In a separate aspect, the present invention provides CFXTEN procoagulant fusion proteins that exhibit reduced binding to an anti-FVIII antibody or FVIII inhibitor that interferes with the procoagulant activity of FVIII. As used herein, an anti-FVIII antibody or an anti-factor VIII antibody means an antibody capable of binding FVIII or the FVIII component of CFXTEN of the present invention, said antibody which includes, but is not limited to, the antibodies of Table. 10 or a polyclonal antibody from a hemophilia A patient with FVIII inhibitors. The term antibody includes monoclonal antibodies, polyclonal antibodies, antibody fragments, and antibody fragment clones. As used herein, a FVIII inhibitor or anti-FVIII inhibitory antibody means an antibody capable of binding FVIII or the FVIII component of CFXTEN of the present invention and which reduces, by any means, the procoagulant activity of FVIII or the FVIII component of CFXTEN. In additional aspects, the present invention provides CFXTEN fusion proteins that retain procoagulant activity in the presence of an FVIII inhibitor. In additional aspects, the present invention provides CFXTEN fusion proteins that contain FVIII that exhibits an increased terminal half-life in the presence of an FVIII binding agent compared to FVIII not bound to XTEN.

Most inhibitory antibodies to human factor VIII act by binding to epitopes located in the A2 domain or C2 domain of factor VIII, interfering with specific functions associated with these domains (US patent No. 6770744; Fulcher et al. Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments Proc Natl Acad Sci USA (1985) 82:7728-7732 Scandella et al Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of fVIII fragments expressed in Escherichia coli Proc Natl. Acad. Sci. USA (1988) 85:6152-6156). While 68% percent of inhibitory antibodies are reported to be directed against the A2 and/or C2 domain, 3% act against the A1 domain and 46% against the a3 acidic region (Lavigne-Lissalde, G., et al. Characteristics, mechanisms of action, and epitope mapping of anti-factor VIII antibodies. Clin Rev Allergy Immunol (2009) 37:67-79). For example, certain heavy chain-specific inhibitors react with the 18.3 kD amino-terminal segment of the A2 domain (Scandella D, et al. 1988); Lollar P et al. Inhibition of human factor VIIIa by anti-A2 subunit antibodies. J Clin Invest 1994;93:2497). FVIII contains a phospholipid binding site in the C2 domain between amino acids 2302 and 2332, and there is also a von Willebrand factor binding site in the C2 domain that acts in combination with amino acids 1649-1689 in the A3 domain. The C2 domain also has epitopes that, when bound by inhibitors, block FVIII activation by thrombin or factor Xa. Inhibitors that bind specifically to the light chain recognize epitopes in the A3 domain or the main antigenic region in the C2 domain, and can lead to reduced procoagulant activity by preventing FVIII from binding to the phospholipid or by reducing the rate of dissociation of FVIII and von Willebrand factor (Gilles JG, et al. Antifactor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do

- 108 041874 not exhibit isotypic restriction. Blood (1993) 82:2452; Shima M, et al. A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost (1993) 69:240). Non-limiting examples of monoclonal FVIII inhibitors are given in table. 9. In patients with high inhibitor titers, there is an increased risk of developing rebleeding in some joints, which may eventually lead to reduced quality of life, disability, or death from excessive blood loss (U.S. Patent Application No. 20120065077; Zhang et al ., Clinic Rev. Allerg. Immunol., 37:114124 (2009); Gouw and van den Berg, Semin. Thromb. Hemost., 35:723-734 (2009)).

While not wishing to be bound by any particular theory, it is believed that the unstructured characteristic of XTEN incorporated into CFXTEN fusion proteins allows XTEN to adopt conformations that result in steric hindrance of inhibitors that might otherwise bind to FVIII epitopes, as illustrated in FIG. 6, because inserted XTEN adopts various random helix conformations, it spatially spans regions of the FVIII component of the fusion protein and sterically interferes with the ability of the inhibitor to bind to the FVIII epitope.

In a separate embodiment, the present invention provides CFXTEN that exhibits procoagulant activity and reduced binding in the presence of an antibody that binds to the C2 domain of Factor VIII compared to the corresponding factor VIII not bound to XTEN and/or native FVIII. In a further embodiment, the present invention provides CFXTEN that exhibits procoagulant activity and reduced binding in the presence of an antibody that binds to the A2 domain of Factor VIII compared to the corresponding factor VIII not bound to XTEN or native FVIII. In a further embodiment, the present invention provides CFXTEN that exhibits procoagulant activity and reduced binding in the presence of antibodies binding to the A2 and C2 domains of Factor VIII compared to the corresponding factor VIII not bound to XTEN or native FVIII. In a separate embodiment, the present invention provides CFXTEN that exhibits procoagulant activity and reduced binding, as compared to the corresponding FVIII not bound to XTEN, in the presence of an antibody selected from the group consisting of the antibodies in Table. 10. In a separate embodiment of the present invention, the CFXTEN fusion protein exhibits reduced binding to the GMA8021 antibody. In a further embodiment of the present invention, the CFXTEN fusion protein exhibits reduced binding to the GMA8008 antibody. In a further embodiment of the present invention, the CFXTEN fusion protein exhibits reduced binding to the ESH4 antibody. In a further embodiment of the present invention, the CFXTEN fusion protein exhibits reduced binding to the ESH8 antibody. In a further embodiment of the present invention, the CFXTEN fusion protein exhibits reduced binding to the B02C11 antibody. In a further embodiment of the present invention, the CFXTEN fusion protein exhibits reduced binding and a higher degree of procoagulant activity compared to the corresponding non-XTEN FVIII in the presence of hemophilia A subject plasma with inhibitors of the FVIII polyclonal antibody, while the higher degree procoagulant activity is determined using an in vitro assay such as the Bethesda assay or other assay described herein.

CFXTEN that exhibits reduced binding to FVIII inhibitors may have one, or two, or three, or four, or five, or six or more separate XTENs, embodiments of which are disclosed herein. In the previous embodiments of the present invention according to this paragraph, CFXTEN exhibits at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or less antibody binding when assessed in vitro in an assay capable of assaying antibody binding to FVIII, such as those described herein below or known in the art. In addition, reduced binding of a subject's CFXTEN to FVIII-binding antibodies can be assessed by retaining a greater degree of procoagulant activity in the presence of the antibody, compared to FVIII not bound to XTEN, as described in the examples. Thus, in embodiments of the present invention that relate to reduced binding by the FVIII inhibitors described herein, CFXTEN, when reacted with an anti-FVIII antibody, exhibits at least 5%, or 10%, or 15%, or 20%, or 30% or 40% or 50% or 60% or 70% or 80% or 100% or 200% or 300% or 400% or 500% or more of activity in the coagulation activity assay ( such as described herein below), compared with the corresponding FVIII, not associated with XTEN, and when reacted with an antibody. In the above, the anti-FVIII antibody may be an antibody from table. 9 or a circulating anti-FVIII antibody from a hemophilia A subject. 10, or a polyclonal anti-FVIII antibody preparation, such as, but not limited to, hemophilia A subject plasma with FVIII inhibitors that results in a Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15 , 20, 30, 40,

- 109 041874

50, 60, 70, 80, 100, or 200 Bethesda units is less than non-XTEN FVIII and analyzed under similar conditions. In a further embodiment, the present invention provides a CFXTEN wherein the tested fusion protein results in less than 50%, or less than 40%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 14%, or less than 13%, or less than 12%, or less than 11%, or less than 10% Bethesda units, compared to non-XTEN FVIII when analyzed under similar conditions using a Bethesda assay and a polyclonal anti-FVIII antibody preparation, such as, but not limited to, plasma from a hemophilia A subject with FVIII inhibitors.

Table 10 Anti-factor VIII antibodies

Antibody designation epitope Inhibitor titer BU/mg Link BO2C11 Domain C2 Met2199/Phe2200 20000 U.S. 6770744 Blood (2007) 110:4234-4242 NMC VIII-5 Domain C2 Glu2181-Val2243 U.S. 6770744 ESH2 light chain ADI ESH4 Light chain 2303-2332 39 U.S. 6770744 Blood (2007) 110:4234-4242 ESH8 Domain C2 2248-2285 10000 U.S. 6770744 Blood (2007) 110:4234-4242 RHD5 (LMBP 6165CB) Domain C1 WO 2005/016455 US patent application 20090263380 LE2E9 Domain C1 US Patent Application 20090263380 Blood (2000) 95:156-163 154 Domain C2 1300 Blood (2007) 110:4234-4242 F85 Domain C2 6 Blood (2007) 110:4234-4242 F100 Domain C2 5 Blood (2007) 110:4234-4242 F137 Domain C2 6 Blood (2007) 110:4234-4242 189 Domain C2 1900 Blood (2007) 110:4234-4242 1117 Domain C2 1800 Blood (2007) 110:4234-4242 1109 Domain C2 1500 Blood (2007) 110:4234-4242

- 110 041874

Met2199/Phe2200 1B5 Domain C2 930 Blood (2007) 110:4234-4242 ZS6 Domain C2 71 Blood (2007) 110:4234-4242 3D12 Domain C2 Phe2196 2600 Blood (2007) 110:4234-4242 D102 Domain C2 3800 Blood (2007) 110:4234-4242 3G6 Domain C2 25000 Blood (2007) 110:4234-4242 2-77 Domain C2 25000 Blood (2007) 110:4234-4242 B45 Domain C2 21000 Blood (2007) 110:4234-4242 AT 9 Domain C2 31000 Blood (2007) 110:4234-4242 IN AND Domain C2 3300 Blood (2007) 110:4234-4242 B75 Domain C2 unknown Blood (2007) 110:4234-4242 D105 Domain C2 Val2223/Lys2227 0.8 Blood (2007) 110:4234-4242 F77 Domain C2 26000 Blood (2007) 110:4234-4242 F178 Domain C2 18000 Blood (2007) 110:4234-4242 F67 Domain C2 21000 Blood (2007) 110:4234-4242 G99 Domain C2 Val2223/Lys2227 15000 Blood (2007) 110:4234-4242 G86 Domain C2 4300 Blood (2007) 110:4234-4242 114 Domain C2 44000 Blood (2007) 110:4234-4242 155 Domain C2 10000 Blood (2007) 110:4234-4242 2-117 Domain C2 >0.4 Blood (2007) 110:4234-4242 GMA012 Domain A2 497-510; 584-593 GMA GMA8001 Domain AZ 156 GMA GMA8002 Domain A1 <1 GMA GMA8003 Domain C2 GMA GMA8004 Domain A1 GMA GMA8005 Domain Al AZ/A1 GMA GMA8006 Domain C2 GMA GMA8008 Domain C2 1047 GMA GMA8009 Domain A2 7923 GMA GMA8010 LC domain GMA GMA8011 Domain C1 97 GMA GMA8012 Domain A1 AZ 204 GMA GMA8013 Domain AZS2 thirty GMA GMA8014 Domain C2 7799 GMA GMA8015 Domain A2 17079 GMA GMA8016 Domain A2 <1 GMA GMA8017 Domain A2 334 GMA GMA8018 LC domain 242 GMA GMA8019 CR-LC domain GMA GMA8020 Domain A1 AZ 196 GMA GMA8021 Domain A2 33928 GMA 4A4 Domain A2 40000 J Thromb Haemost (2009) 7:658-664 3E6 Domain C2 41 Blood (2007) 110:4234-4242

American Diagnostica Inc. website, URL located on the Internet, americandiagnostica.com/html/Product_Detail.asp?idCategory=5&idSubCategory=104&idpro=ESH-8 accessed January 12, 2012.

Green Mountain Antibodies website, web URL, greenmoab.com/product_details/16316/21582.html accessed 12 January 2012.

Inhibitor and antibody binding assays.

The fusion proteins of the present invention can be analyzed to confirm reduced binding to FVIII inhibitors using methods known in the art. The assays that may be used include, but are not limited to, competitive and non-competitive assay systems using methods such as Western blots, radioimmunoassays, ELISAs, sandwich immunoassays, immunoprecipitation assays, precipitation assays, diffuse gel precipitation assays, immunodiffusion assays, agglutination tests, quantitative radioimmunoassays, fluorescence immunoassays, coagulation activity assays, factor VIII inhibitor assays, to name but a few. Such assays are common and well known in the art (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is presented in this reference document in its entirety). Illustratively described briefly below, but are not intended to be limiting.

Bethesda assay and Nijmegen modification Bethesda assay are inhibitor assays

- 111 041874 factor VIII, widely known as methods for determining FVIII inhibitors (Kasper CK, et al. Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. (1975) 34(2):612). Despite this, assays can be modified in order to analyze the binding of inhibitors to FVIII compositions using inhibitors such as polyclonal or monoclonal anti-FVIII antibodies that contain the antibodies of Table. 10, and methods such as those described in Example 52. Briefly, the modified Bethesda assay comprises mixing standardized volumes of test sample with an equivalent volume of inhibitor at a given concentration. The mixtures are incubated for 2 hours at 37° C. prior to analysis of the factor concentration using a coagulation activity assay, such as a chromogenic assay. Similarly, appropriate plasma is incubated with natural factor VIII levels, which is then analyzed as a positive control. The expected result is a titer that results in 50% positive control FVIII activity, described as Bethesda units. The Nijimegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost. (1995) 73(2):247-251).

Western blotting typically involves prepared protein samples, polyacrylamide gel electrophoresis of protein samples (e.g. 8%-20% SDS-PAGE, depending on antigen molecular weight), transfer of protein sample from polyacrylamide gel to a membrane such as nitrocellulose , PVDF or nylon, block membrane in blocking solution (e.g. PBS with 3% BSA or skim milk), wash membrane in wash buffer (e.g. PBSTween 20), block membrane with first antibody (antibody of interest), dilute in blocking buffer, washing the membrane in wash buffer, blocking the membrane with a secondary antibody (which recognizes the first antibody, such as an anti-human antibody) conjugated to an enzymatic substrate (such as horseradish peroxidase or alkaline phosphatase), or a radioactive molecule (such as 32 P or 125 I) diluted in blocking buffer, washing the membrane in washing buffer and detecting the presence view of the antigen. The person skilled in the art is aware of the parameters that can be changed to increase the detected signal and reduce the background noise. For further discussion regarding Western blotting protocols, see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISA assays can detect anti-FVIII antibodies regardless of their ability to block the procoagulant activity of FVIII, and are used to detect the development of anti-FVIII in hemophilia A patients. In a sample of 131 hemophilia A patients with inhibitors, the ELISA method resulted in 97.7% sensitivity and 78.8% specificity and has a high negative predictive value (98.6%) [Martin, P. G., et al. Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs. Clin Lab Haematol (1999) 21:125-128]. Other investigators have found a highly significant correlation between Bethesda titer and ELISA absorbance values for anti-FVIII optical density detection (Towfighi, F., et al. Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity Acta Haematol (2005) 114:84-90), with the added benefit of being able to detect non-inhibitory anti-FVIII antibodies. Assay protocols include preparation of bound ligand, which may contain a sample containing either a factor VIII polypeptide or CFXTEN fusion protein, plated into a well of a 96-well antibody microtiter plate, sample ligand for testing is added and incubated, then identifying antibody is added and incubated before washing and a secondary antibody conjugated to alkaline phosphatase or peroxidase is added and incubated for an additional period of time before the addition of TMB substrate and processed for reading with a 450 nm spectrophotometer. In ELISA assays, the antibody or inhibitor of interest must not be conjugated to the compound to be detected; instead, a second antibody (which recognizes the antibody or inhibitor of interest) conjugated to the compound of interest may be added to the well. Additionally, instead of applying an antibody to the well, a ligand can be applied to the well. The person skilled in the art will be aware of the parameters that can be changed in order to increase the detectable signal, as well as other variants of ELISA assays known in the art (See, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1).

Standard or modified coagulation activity assays are used to measure reduced binding of FVIII binding agents. In a separate exemplary method (described further in Example 28), the optimal concentration of a given FVIII inhibitor to use in the assay is first determined by a titration experiment using various amounts of inhibitory antibody incubated at 37° C. for 2 hours with a core vector expressing wild-type FVIII , which contains the double label His/Myc. FVIII activity is measured using the Coatest assay method described herein. In asset analysis

- 112 041874 The lowest concentration of FVIII that results in optimal inhibition is used. In the assay, FVIII inhibitor antibody at optimal concentration is mixed with individual test specimens and incubated at 37°C for 2 h. and baseline FVIII activity for each test sample. The present invention provides methods for producing CFXTEN that exhibit reduced binding to FVIII binding agents that contain FVIII inhibitors and retained procoagulant activity. In a specific embodiment of the present invention, a method for generating CFXTEN with reduced binding to FVIII inhibitors comprises the steps of selecting an FVIII sequence with at least 90% sequence identity to that of Table 1. 1 by selecting one, two, three, four, five, or six or more XTENs, each with at least 70%, or at least 80%, or at least 90%, or at least 95-99% sequence identity , compared with XTEN sequences of comparable length from Table. 4, creating expression constructs designed to detect the specified XTEN at or close to the localization selected from Table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 by expressing and extracting the resulting CFXTENs and analyzing the resulting assay fusion proteins described herein to confirm the reduced binding of the CFXTEN fusion protein. Using a patentable method, CFXTEN exhibits at least 5% reduction, or at least 10% reduction, or at least 15% reduction, or at least 20% reduction, or at least 25% reduction, or reduced by at least 40%, or reduced by at least 50%, or reduced by at least 60%, or reduced by at least 70%, or reduced by at least 80% binding to the FVIII binding agent, which contains, but is not limited to, antibodies from table. 10 or anti-FVIII antibodies from a subject with hemophilia A, and retains at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% procoagulant activity, compared to the corresponding non-XTEN FVIII.

Up to 8-10% of patients with hemophilia A have antibodies that bind FVIII without affecting its procoagulant properties; they are thus not classified as FVIII inhibitors. Despite this, antibody binding to FVIII is thought to result in immune complexes that are cleared by the innate immune response or are more susceptible to proteolytic degradation (Kazatchkine MD. Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A. Clin Exp Immunol (1980) 39(2):315-320). Accordingly, it is an object of the present invention to provide CFXTEN fusion proteins that contain one or more XTENs that exhibit reduced antibody binding to FVIII that are non-inhibitors, while degradation or clearance of CFXTENs is reduced by at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or less, compared to the corresponding FVIII not bound to XTEN, or natural FVIII bound by similar antibodies. The reduced binding of an antibody to CFXTEN, compared to non-XTEN-bound FVIII or natural FVIII, can be analyzed using in vitro and in vivo methods. In vitro methods include the aforementioned ELISA and Western blot methods. Reduced degradation or clearance of CFXTEN can be assessed in vivo using animal models or in human clinical trials. In a separate type of trial, factor VIII or CFXTEN is administered alone, preferably by intravenous infusion, to groups of patients deficient in factor VIII that has antibodies that degrade or clear human therapeutic factor VIII. The dosage of the test article administered is between 5 and 50 IU/kg body weight, preferably 10-45 IU/kg, and more preferably 40 IU/kg body weight. Approximately 1 hour after each administration, the recovery of factor VIII or CFXTEN from blood samples is measured in a functional one-step or chromogenic coagulation activity assay to assess activity and using ELISA, HPLC or similar assay to assess the amount of intact factor VIII equivalent. Samples are taken again approximately 5-10 hours after infusion and recovery is measured. The complete recovery and rate of disappearance of factor VIII from samples is the predicted antibody titer, and comparison of factor VIII and CFXTEN results indicates the degree of reduced clearance and/or degradation of CFXTEN. In a separate embodiment of the present invention, the CFXTEN fusion protein exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 20% reduced at least 25%, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to anti -FVIII antibody that promotes clearance but, on the other hand, does not inhibit the procoagulant activity of intact natural FVIII. In a further embodiment of the present invention, the CFXTEN fusion protein exhibits a reduced by at least 5%, or a reduced by

- 113 041874 at least 10%, or reduced by at least 15%, or reduced by at least 20%, or reduced by at least 25%, or reduced by at least 40%, or reduced by at least 50%, or reduced by at least 60%, or reduced by at least 70%, or at least 80% reduced binding to an anti-FVIII antibody that promotes degradation of FVIII. In the previous embodiments of the present invention according to this paragraph, the reduced binding of an anti-FVIII antibody is alternatively determined by an increased K _D value of the FVIII antibody with a fusion protein, compared to FVIII by at least two times, or three times, or four times, or five times, or 10 times, or 33 times, or 100 times, or 330 times, or at least 1000 times, compared with binding to the corresponding FVIII, not associated with XTEN. In a particular embodiment of the present invention, CFXTEN fusion proteins that comprise one or more XTENs that exhibit reduced reactivity to an anti-FVIII antibody exhibit an increased terminal half-life when administered to a subject with anti-FVIII antibodies by at least 48 hours, or at least 72 hours, or at least 96 hours, or at least 120 hours, or at least 144 hours, or at least 14 days, or at least 21 days, compared to non-XTEN FVIII. In the above embodiment of the present invention, the subject may be a hemophilia A human subject, or it may be a hemophilia A mouse subject with circulating anti-FVIII antibodies.

Another aspect of the present invention is the use of the CFXTEN fusion protein in the case of specific therapy for coagulopathy in a subject with an FVIII inhibitor. The present invention provides a method of treating a subject with circulating FVIII inhibitor(s) which comprises the step of administering to the subject a clotting effective amount of a CFXTEN fusion protein, wherein the fusion protein exhibits higher procoagulant activity and/or a clotting effective concentration for a longer duration, compared to either the corresponding factor VIII not associated with XTEN, or compared to natural factor VIII, is administered to the subject using a similar amount and route of administration. In a separate embodiment of the method, the FVIII inhibitor in the subject is an anti-FVIII antibody. In a further embodiment of the present invention, the FVIII inhibitor is a neutralizing anti-FVIII antibody. In a separate embodiment of the present invention, the FVIII inhibitor is an anti-FVIII antibody that is linked to the A1 domain of FVIII. In a further embodiment of the present invention, the FVIII inhibitor is an anti-FVIII antibody that is linked to the A2 domain of FVIII. In a further embodiment of the present invention, the FVIII inhibitor is an anti-FVIII antibody that is linked to the A3 domain of FVIII. In a further embodiment of the present invention, the FVIII inhibitor is an anti-FVIII antibody that is linked to the C1 domain of FVIII. In a further embodiment of the present invention, the FVIII inhibitor is an anti-FVIII antibody that is linked to the C2 domain of FVIII. In a further embodiment of the present invention, the FVIII inhibitor is an anti-FVIII antibody that is associated with both the C2 domain and the A2 domain of FVIII. In a further embodiment of the present invention, the FVIII inhibitor is linked to an FVIII epitope capable of being bound by one or more of the antibodies from Table 1. 10. In a further embodiment of the present invention, the FVIII inhibitor is a polyclonal antibody from a subject with hemophilia A with FVIII inhibitor antibodies.

It is an object of the present invention to design CFXTEN with XTEN administered in order to maximize the steric interference of FVIII binding agents that otherwise bind to FVIII and neutralize procoagulant activity or result in FVIII clearance or degradation. Accordingly, in a separate approach, the present invention provides a CFXTEN that contains one or more XTENs, with the XTEN administered close to the binding site of the FVIII inhibitor or anti-FVIII antibody. In a separate embodiment of the present invention, XTEN is associated with FVIII in the localization selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9, which is in the range of about 50, or about 100, or about 150, or about 200, or about 250, or about 300 amino acids of the FVIII epitope that is associated with the antibody of table. 10. In a further embodiment of the present invention, XTEN is linked to FVIII within about 50 or about 100 or about 150 or about 200 or about 250 or about 300 amino acids of the FVIII epitope in the A2 or C2 domain that is associated with an antibody from tab. 10. Accordingly, the present invention provides CFXTEN fusion proteins that contain one or more XTENs, while binding of FVIII inhibitors to the FVIII component of the fusion protein is reduced compared to the corresponding non-XTEN FVIII or natural FVIII and CFXTEN, maintaining procoagulant activity. In the previous embodiments of the present invention described above in this paragraph, the fusion proteins can be analyzed using the assays described herein below, the assays of the examples, or other assays known in the art, and the inhibitors can be an antibody from table. 10 may be polyclonal anti-FVIII, or may be blood or plasma from a subject with hemophilia A with FVIII inhibitors.

In additional aspects, CFXTEN are designed to maximize areas where

- 114 041874 XTEN can adopt random helical conformations that coat the fusion protein, thus leading to steric hindrance in the case of anti-FVIII antibodies that otherwise bind epitopes in the FVIII components of the fusion protein. The incorporation of several XTENs into CFXTEN is believed to provide a larger overall hydrodynamic radius of the XTEN component, compared to CFXTEN with a smaller XTEN, which also has approximately the same total XTEN amino acids. Empirically, the hydrodynamic radius in the case of a protein is calculated based on gel permeation chromatography, and the results of some fusion proteins using similar methods are described in the examples. In addition, the radius of XTEN polypeptides, such as those included in the embodiments of the present invention disclosed herein, can be approximated using mathematical formulas, since the limited types of amino acids used have known characteristics that can be quantified. In a specific embodiment of the present invention, the maximum radius of an individual XTEN polypeptide is calculated (hereinafter XTEN radius) according to the formula given in Equation II:

Radius XTEN = (Uddina XTEN 0.2037) + 3.4627 II

In a further embodiment of the present invention, the sum of the maximum XTEN radii of all XTEN segments in CFXTEN is calculated (hereinafter, the sum of XTEN radii) according to the formula given in Equation III: η

Radius; XTEN

Sum of XTEN radii = ¹⁼¹ III where n = number of XTEN segments and i is an iterator.

In a further embodiment of the present invention, the ratio of the sum of the XTEN radii of a CFXTEN that contains multiple XTENs in which the XTEN radius in the case of unique XTENs is of equivalent length (in the total number of amino acid residues as in CFXTEN) is calculated (hereinafter the XTEN radii ratio) according to with the formula given in Equation IV:

ΣΡ ₌ . Radius; XTEN η vtc\i India XTEN * θ 2037) + 3.4627

Radius ratio XTEN = ^1-1 long scarlet/jy where n = number of XTEN segments and i is an iterator.

When applying the equations to XTEN, one of skill in the art will appreciate that the calculated values are maximum values that may vary or decrease depending on the host cell used to express the XTEN polypeptide. It is believed that while expression of E. coli can result in XTEN that reaches the calculated values, expression in eukaryotic host cells in which XTEN can be glycosylated can result in a polypeptide radius less than the maximum calculated value. Such differences can be quantified using methods such as gel permeation chromatography, the methods of which are detailed in the examples.

In order to construct a CFTEN that maximizes the area within which XTEN can assume random helical conformations, CFXTEN structures with an XTEN radius ratio greater than 2 were found to provide greater fusion protein coverage than structures with values <2. Accordingly, in a particular embodiment, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6 or 2.7 or 2.8 or 2.9 or 3.0 or 3.1 or 3.2 or 3.3 or 3.4 or 3.5 or more . In certain embodiments, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or higher, contains at least three XTENs, with each XTEN having at least 42 to about 288 amino acids. , wherein at least two XTENs are linked to the fusion protein with a distance between the two XTENs of at least about 100, or about 200, or about 300, or about 400, or about 500 amino acids. In additional embodiments, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or higher, contains at least four XTENs, with each XTEN having at least 42 to about 288 amino acids. , wherein at least three XTENs are linked to the fusion protein with a distance between any two of the three XTENs of at least about 100, or about 200, or about 300, or about 400 amino acids.

In a further embodiment, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, the CFXTEN comprises at least three XTENs, with each XTEN having at least 42 to about 288 amino acids, with at least two of the three XTENs associated with the fusion protein separated by an amino acid sequence of at least 100, or about 200, or about 300 to about

- 115 041874

400 amino acids, and a third XTEN linked within the B domain (or a fragment thereof) or within the C domain (or their ends). In a further embodiment, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, the CFXTEN comprises at least four XTENs, with each XTEN having at least 42 to about 288 amino acids and, while at least three of the four XTENs associated with the fusion protein are separated by an amino acid sequence of at least 300 to about 400 amino acids, and the fourth XTEN is associated in the B domain (or fragment thereof) or within the domain C (or their ends).

In other embodiments, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, the CFXTEN comprises at least five XTENs, with four XTENs that are at least 42 to about 144 amino acids, with at least four XTENs linked to the fusion protein with a distance between any two of the four XTENs of at least about 100, 200, or about 300, or about 400 amino acids, and a fifth XTEN linked to the B domain (or fragment thereof ) or within domain C (or their ends). In a separate embodiment, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, the CFXTEN contains at least five XTENs with four XTENs that have at least 42 to about 144 amino acids. , with at least three XTENs associated with the fusion protein separated by an amino acid sequence of at least 300 to about 400 amino acids, a fourth XTEN is bound within the B domain (or fragment thereof), and a fifth XTEN is bound within the C domain (or their ends).

In a separate aspect, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2, 6 or 2.7 or 2.8 or 2.9 or 3.0 or 3.1 or 3.2 or 3.3 or 3.4 or 3.5 or more, and the composition does not contain certain sequences. In a specific embodiment of the foregoing, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, provided that the fusion protein does not contain a sequence from any of Table 1. 50 or tab. 51. In a further embodiment of the above, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, provided that the fusion protein does not contain a sequence that has the XTEN AG family of sequences. In a further embodiment of the above, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, provided that the fusion protein does not contain a sequence selected from

GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ IDNO: 33), GASPGTSSTGSP (SEQ IDNO: 34). in a further embodiment of the foregoing, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, provided that the fusion protein does not contain any of the following values, of

GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32),

GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34) and

GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS

EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59).

In a further embodiment of the foregoing, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, provided that the fusion protein does not contain the sequence, GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS

EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGTSTEPSEGSAP (SEQ W NO: 59),

PGS SPS ASTGTGPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGATGSPGS SPS ASTGTGPG

ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS

PGTSSTGSPGTPGSGTASSS (SEQ ID NO: 71), PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG

SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATTGSPGSSPSASTGTGPGSS

PSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPS

ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS (SEQ ID NO: 80).

selected from or

In a further embodiment of the foregoing, the present invention provides a CFXTEN wherein the XTEN radius ratio is at least 2.0-3.5 or greater, provided that the fusion protein does not contain an XTEN sequence consisting of

- 116 041874

GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59),

PGS SPS ASTGTGPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGATGSPGS SPS ASTGTGPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGAATGSPGTPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSS (SEQ ID NO: 71),

PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSS PSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS (SEQ ID NO: 80).

or

In a separate aspect, the present invention provides methods for creating CFXTEN with XTEN administered to maximize steric disruption of the FVIII binder, which may otherwise bind to FVIII and neutralize procoagulant activity or result in FVIII clearance or degradation. Accordingly, in a separate embodiment, the present invention provides a method that includes the steps of selecting a FVIII sequence with at least 90% sequence identity with the sequence from Table. 1, choose three or more XTEN from the table. 4, in which the ratio of radii XTEN is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or more, generate expression constructs designed to detect the indicated XTEN in, or close to, localization selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 with three or more XTENs having at least 300 to 400 amino acids, express and extract the resulting CFXTENs, and analyze the resulting fusion proteins in the assay described herein to confirm reduced binding of the CFXTEN fusion protein. Using a patentable method, CFXTEN exhibits at least 5% reduction, or at least 10% reduction, or at least 15% reduction, or at least 20% reduction, or at least 25% reduction, or reduced by at least 40%, or reduced by at least 50%, or reduced by at least 60%, or reduced by at least 70%, or reduced by at least 80% binding to the FVIII binding agent, which contains, but is not limited to, antibodies from table. 10 and exhibits procoagulant activity.

Fig. 5. Conformations of the fusion protein CFXTEN with a spacer and cleaved sequences.

In additional aspects, the present invention provides a CFXTEN configured with one or more spacer sequences inserted into, or adjacent to, an XTEN that is designed to insert into or enhance functionality or property of compositions, or as an aid in the assembly or manufacture of fusion protein compositions. Such properties include, but are not limited to, including cleavage sequence(s) to allow secretion of components, including amino acids compatible with the nucleotide of restriction sites to allow binding of XTEN-encoding nucleic acids to FVIII-encoding nucleic acids, or to facilitate the construction of expression vectors and linkers designed to reduce steric hindrance in regions of the CFXTEN fusion protein.

In one embodiment of the present invention, a spacer region can be introduced between the XTEN sequence and the FVIII component to reduce steric hindrance so that the FVIII component can adopt its desired tertiary structure and/or interact with its target substrate or processing enzyme, respectively. For spacers and methods for determining desired spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated herein by reference. In a particular embodiment of the present invention, the spacer comprises one or more peptide sequences that are 1 to 50 amino acid residues in length, or about 1 to 25 residues, or about 1 to 10 residues in length. Spacer sequences, except for cleavage sites, may contain any of the 20 naturally occurring L amino acids, and preferably have an XTEN-like property in that most of the residues will be hydrophilic amino acids that are sterically unhindered, such as, but not limited to, glycine ( G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), and aspartate (D). The spacer may be a unique residue of glycine, polyglycines or polyalanines, or preferably a mixture of combinations of glycine, serine and alanine residues. In a particular embodiment of the present invention, the spacer region, excluding the amino acids of the cleaved site, has from about 1 to 10 amino acids that consist of

- 117 041874 from amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and essentially devoid of secondary structure; for example, less than about 10%, or less than about 5%, as determined by the Chou-Fasman and/or GOR algorithm. In a separate embodiment of the present invention, the spacer region is GPEGPS (SEQ ID NO: 1612). In a further embodiment of the present invention, the spacer region is GPEGPS (SEQ ID NO: 1612) linked to a cleavage sequence from Table 1. 12. In addition, spacer sequences are created in order to avoid the introduction of T-cell epitopes, which can be achieved, in particular, by preventing or limiting the number of hydrophobic amino acids used in the spacer; the definition of epitopes is described above and in the examples.

In a particular embodiment, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction region(s) between the payload FVIII sequence and one or more XTENs inserted into the fusion protein, with the spacer sequences constituting amino acids that are compatible with nucleic acids encoding restriction sites. In a further embodiment of the present invention, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction region(s) between the FVIII payload sequence and another XTEN inserted into the fusion protein, wherein the spacer sequences contain amino acids that are compatible with nucleic acids encoding restriction sites and amino acids, and another spacer region amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). In a further embodiment of the present invention, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction region(s) between the payload FVIII sequence and another XTEN inserted into the fusion protein, wherein the spacer sequences contain amino acids that are compatible with nucleic acids encoding restriction sites, and another spacer sequence is selected from the sequences from the table. 11. The exact sequence of each spacer region is chosen to be compatible with the cloning sites in the expression vectors used for the exclusive CFXTEN construct. In a separate embodiment of the present invention, the spacer region has properties compatible with XTEN. In a separate embodiment of the present invention, the spacer region is GAGSPGAETA (SEQ ID NO: 178). In the case of XTEN sequences that are internal to the FVIII sequence, each XTEN will typically be surrounded by two spacer sequences that contain amino acids compatible with restriction sites, while an XTEN associated with N- or C- end, could only require a unique spacer region at the junction of the two, and another at the opposite end to be included in the vector. As should be apparent to any person skilled in the art, spacer sequences that contain amino acids compatible with restriction sites that are internal to FVIII can be omitted from the construct if the complete CFXTEN gene is produced synthetically.

Table 11

Spacer sequences compatible with restriction sites

spacer region Restriction enzyme GSPG (SEQ ID NO: 174) in sal ETET (SEQIDNO: 175) in sal PGSSS (SEQIDNO: 176) BbsI gap Asci GPA fsel GPSGP (SEQ GO NO: 177) Sfil AAA SacII TG Agel GT Kpnl GAGSPGAETA (SEQ ID NO: 178) Sfil ASS xhol

In additional aspects, the present invention provides CFXTEN conformations with cleaved sequences inserted into spacer sequences. In certain embodiments of the present invention, the spacer sequences in the CFXTEN fusion protein composition comprise one or more cleavage sequences that are identical or different, wherein the cleavage sequence can act in accordance with the protease, as shown in FIG. 12 in order to secrete FVIII, a FVIII component (eg, B domain), or XTEN sequence(s) from a fusion protein. In a separate embodiment of the present invention, the inclusion of a cleavage sequence in CFXTEN is obtained

- 118 041874 in order to allow secretion of a FVIII component that becomes active or more active (in relation to its ability to serve as a membrane binding site for factors IXa and X) after its secretion from XTEN. In the above embodiment of the present invention, the procoagulant activity of the FVIII component of CFXTEN is increased after cleavage by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, compared to intact CFXTEN. The cleaved sequences are close enough to the FVIII sequences, typically within 18, or within 12, or within 6, or within 2 amino acid sequences of FVIII, such that any remaining FVIII-related residues after cleavage are noticeable. do not affect the activity (eg, such as binding to a blood clotting protein) of FVIII, yet provide sufficient access to the protease to allow cleavage of the cleavage sequence to be carried out. In some cases, CFXTEN that contains cleaved sequences will also have one or more amino acids in a spacer region between FVIII and the cleavage sequence, or XTEN and the cleavage sequence to facilitate protease access; spacer amino acids contain any natural amino acid that contains glycine, serine and alanine as preferred amino acids. In a particular embodiment of the present invention, the cleaved site is a sequence that can be cleaved with a protease endogenous to a mammalian subject such that CFXTEN can be cleaved after administration to the subject. In such a case, CFXTEN may serve as a prodrug or circulating FVIII depot. In a particular construct of the above, CFXTEN could have one or two XTENs linked to the N and/or C-terminus of FVIII-BDD via cleavage sequences that can act via an activated coagulation factor and would have an additional XTEN located between the treated amino acids at positions R740 and R1689 so that XTEN can be released leaving a form of FVIII similar to natural activated FVIII. In a specific embodiment of the above construct, FVIII that is released from the fusion protein by cleavage of the cleavage sequence exhibits an increase in activity of at least about two-fold, or at least about three-fold, or at least about four-fold , or at least about five times, or at least about six times, or at least about eight times, or at least about ten times, or at least about 20 times, compared with intact fusion protein CFXTEN.

Examples of cleavage sites contemplated by the present invention include, but are not limited to, a polypeptide sequence cleavable by an endogenous mammalian protease selected from FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), elastase-2, granzyme B , MMP-12, MMP-13, MMP-17, or MMP-20, or with non-mammalian proteases such as TEV, enterokinase, PreScission™ protease (rhinovirus 3C protease) and sortase A. Sequences are known to be cleaved by previously described proteases and others known in the art. Exemplary cleavage sequences contemplated by the present invention and corresponding cut sites within the sequences are shown in Table 1. 12 as well as variants of their sequences. In the case of CFXTEN which contains an inserted cleavage sequence(s), it is generally preferred that one or more of the cleaved sequences are substrates for activated coagulation proteins. For example, thrombin (activated coagulation factor II) has an effect on the LTPRSLLV sequence (SEQ ID NO: 1618) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], which is truncated in sequence after arginine at position 4. Active FIIa is obtained by cleavage of FII with FXa in the presence of phospholipids and calcium, and further through the coagulation pathway from factor VIII. Once activated, its natural role in clotting is to break down fibrinogen, which then in turn initiates clot formation. FIIa activity is tightly controlled and occurs only if coagulation is necessary for proper hemostasis. By incorporating the sequence LTPRSLLV (SEQ ID NO: 1618) into CFXTEN between, and by binding, components of FVIII and XTEN, XTEN is removed from adjacent FVIII simultaneously with activation of either the extrinsic or intrinsic clotting pathways if clotting is physiologically required, thereby selectively releasing FVIII. In a further embodiment, the present invention provides CFXTEN with inserted cleaved FXIa sequences between FVIII and XTEN component(s) that are only effective from the initiation of the intrinsic clotting system, with the procoagulant form of FVIII being released from XTEN by FXIa to participate in the clotting cascade . Without wishing to be bound by any particular theory, it is believed that the CFXTEN of the above embodiment can isolate FVIII from other clotting factors except those within the active clotting site, thus allowing higher doses (and thus longer dosing intervals). ) with minimal security concerns.

Therefore, cleaved sequences primarily susceptible to activated

- 119 041874 nym procoagulant coagulation proteins, given in table. 12 are designed for sustained release of FVIII, which, in certain embodiments of CFXTEN, can provide a higher degree of activity of the FVIII component released from an intact form of CFXTEN, as well as an additional margin of safety in case of high doses of CFXTEN administered to a subject. In a particular embodiment, the present invention provides a CFXTEN that contains one or more cleaved sequences operably arranged to be secreted upon cleavage of FVIII from a fusion protein, wherein the one or more cleaved sequences are at least about 86%, or at least about 92%, or 100% sequence identity with the sequence selected from the table. 12. In certain embodiments of the present invention, only two or three amino acids located on either side of the cut (in total, four to six amino acids) are included in the cleavage sequence, which, in turn, is inserted into the CFXTEN of embodiments of the present invention providing eg XTEN secretion sites. In additional embodiments of the present invention, the built-in cleavage sequence from Table 12 may have one or more deletions or insertions, or one, or two, or three amino acid substitutions for any one, or two, or three amino acids in a known sequence, with the deletions, insertions, or substitutions resulting in reduced or improved sensitivity , but not without a lack of protease sensitivity, resulting in the ability to adapt the rate of secretion of FVIII from XTEN. Exemplary substitutions within the cleaved sequences that are used in the CFXTEN of the present invention are shown in Table 1. 12.

Table 12

Sequences cleaved by the protease

Proteases that act on Illustrative Sequence SEQ ID Minimal cut site SEQ ID sequences splitting NO: NO: FXIa KLTR^AET 179 KD/FL/T/R^VA/VE/GT/GV FXIa DFTR^VVG 180 KD/FL/T/R^VA/VE/GT/GV FXIIa TMTR^IVGG 181 nd Kallikrein SPFR^STGG 182 -/-/FL/RY^SR/RT/-/- FVIIa LQVR^IVGG 183 ND FIXa PLGR^IVGG 184 -/./g/r%/-/-/- FXa lEGR^TVGG 185 IA/E/GFP/R^STVVFS/-/G Flla (thrombin) LTPR^SLLV 186 -/-/PLA/R^SAG/-/-/- Elastase-2 LGPV^SGVP 187 -/-/-/VIATF-/-/-/- Granzim-V VAGD^SLEE 188 NI-1-ΙΌ^-Ι-Ι-Ι- MMP-12 GPAG^LGGA 189 G/PA/-/gW-/G/- 190 MMR-13 GPAG^LRGA 191 g/p/-/g4l/-/ga/- 192 MMR-17 APLG%RLR 193 -/PS/-/4LQ/-/LI7- MMP-20 PALP^LVAQ 194 ND TEV ENLYFQ^G 195 ENLYFQ^G/S 196 Enterokinase DDDK^IVGG 197 DDDK^IVGG 198 Protease 3S (PreScission™) LEVLFQ^GP 199 LEVLFQ^GP 200 Sortaz A LPKT^GSES 201 l/p/kead/t4g/-/eks/s 202

^ denotes a cleavage site.

ND: no data.

a list of multiple amino acids before, between, or after the slash indicates alternative amino acids that may be substituted at the position;

- denote that any amino acid can be replaced by the corresponding amino acid listed in the middle column.

6. Exemplary CFXTEN fusion protein sequences.

Non-limiting examples of fusion protein sequences that contain a unique FVIII associated with one or more XTENs are shown in Table 1. 21. Illustrative amino acid sequences from table. 21 (and the DNA sequences that encode them) contain its marks for purification purposes, which, as would be obvious to those skilled in the art, can be removed from the sequence that has an effect on the procoagulant activity of the CFXTEN fusion protein. In a separate embodiment of the present invention, CFXTEN from table. 21 further contains the amino acids at the N-terminus of their corresponding native human FVIII (namely, the sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) to

- 120 041874 help in the expression and secretion of the fusion protein CFXTEN. In a separate embodiment of the present invention, the composition of CFXTEN contains a hybrid protein that has at least about 80% sequence identity, compared with CFXTEN from table. 21, alternatively, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% sequence identity, compared to CFXTEN from table. 21, with optimal alignment. In an additional embodiment of the present invention, the composition of CFXTEN contains a hybrid protein from table. 21 in which the C-terminal sequence his-his-his-his-his-his (SEQ ID NO: 1700) is deleted. Despite this, the present invention also provides for the replacement of any sequence of FVIII from table. 1 per component FVIII CFXTEN from table. 21, and/or replacing any sequence of any of the table. 3, 4 and 13-17 on the component XTEN CFXTEN from the table. 21. Generally, the resulting CFXTEN of the above examples retains at least some of the procoagulant activity of the corresponding non-XTEN FVIII. In the above fusion proteins described above in this paragraph, the CFXTEN fusion protein may further comprise one or more cleaved sequences; for example, the sequence from the table. 12, the cleavage sequence is located between FVIII and XTEN sequences, or between adjacent XTEN-bound FVIII domains. In certain embodiments of the present invention that contains the cleavage sequence(s), the intact CFXTEN composition has less activity, but a longer half-life in its intact form, compared to the corresponding FVIII not bound to XTEN, but is designed such that when administered into the subject's body, the FVIII component is gradually released from the fusion protein by cleavage in the cleavage sequence(s) by endogenous proteases, after which the FVIII component exhibits procoagulant activity.

CFXTEN formulations of embodiments of the present invention may be assessed for activity using assays or in vivo parameters as described herein (e.g., in vitro coagulation activity assays, assays in Table 49, or pharmacodynamic effects on a preclinical model of hemophilia in human clinical trials with using methods such as those described in the examples or other methods known in the art for assessing FVIII activity) to determine the suitability of an FVIII factor configuration or sequence variant, and those CFXTEN formulations (contained after cleavage of any embedded XTEN releasing cleavage sites) that retain at least about 30% or about 40% or about 50% or about 55% or about 60% or about 70% or about 80% or about 90% or about 95% or more of the activity , compared with the natural sequence of FVIII, are considered suitable for use in the treatment of FVIII, related to conditions.

Properties of CFXTEN compositions of the present invention (a) Pharmacokinetic properties of CFXTEN.

Another object of the present invention is to provide CFXTEN fusion proteins and pharmaceutical compositions that contain CFXTEN with improved pharmacokinetic properties compared to non-XTEN FVIII. The pharmacokinetic properties of FVIII improved by binding a given XTEN to FVIII include, but are not limited to, terminal half-life, area under the curve (AUC), C _max , volume of distribution, maintenance of biologically active CFXTEN above the minimum effective concentration per unit volume of blood in over a longer period of time than FVIII not associated with XTEN. The improved properties allow for less frequent dosing and/or a longer procoagulant effect compared to a similar dose of non-XTEN FVIII. An increase in one or more of these properties may result in benefits in the treatment of factor VIII related diseases.

Exogenously administered factor VIII has been reported to have a finite half-life in humans of approximately 12-14 hours when complexed with normal von Willebrand factor protein, while in the absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham EG, et al., Br J Haematol (1982) 52(2):259-267; Bjorkman, S., et al. Clin Pharmacokinet (2001) 40:815). As a result of the improved properties provided by XTEN, CFXTEN, when used at a dose and dosing regimen determined to be appropriate for the subject and their pathological condition, can reach circulating concentrations resulting in the desired procoagulant or clinical effect over an extended period of time compared with a similar dose of the corresponding non-XTEN FVIII. As used herein, analogous dose means dose in mole equivalents/kg or international units/kg (IU/kg) in the case of a composition that is administered to a subject. It will be appreciated in the art that a similar dose of FVIII not bound to XTEN would represent a lower formulation mass, but would have substantially the same ME or mole equivalents of CFXTEN per dose.

The international unit (IU) of factor VIII is defined in the art as the coagulant activity present in 1 ml of normal human plasma. A normal, individual person without hemophilia is expected to have a factor VIII activity of about 100 IU/dl. At

- 121 041874 Hemophilia A The doses required for treatment depend on the condition. In case of minor bleeding, doses of natural or recombinant factor VIII from 20 to 40 IU/kg are usually administered as needed. In case of moderate bleeding, doses of 30 to 60 IU/kg are administered as needed, and in case of major bleeding, doses of 80 to 100 IU/kg may be needed, with repeated doses of 20 to 25 IU/kg administered every 8 -12 hours until the bleeding stops. For the prevention of bleeding in patients with severe hemophilia A, typical doses of natural or recombinant FVIII formulations are 20 to 40 IU/kg body weight over intervals of about 2 to 3 days. The standard equation for estimating the appropriate dose of an FVIII-containing composition is:

Units needed = body weight (kg) x desired increase in factor VIII (IU/dl or % of normal) x 0.5 (IU/kg per IU/dl).

In many cases, therapeutic levels of FVIII in subjects of various ages or degrees of disease have been developed and are available in the published literature or listed on the drug label in the case of approved products that contain FVIII. For example, the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis published, on the ISTH website November 29, 2000, that the most widely used indicator of hemophilia A is determined by determining circulating concentrations of FVIII procoagulant levels in blood plasma, in people with <1% (<0.01 IU/mL) factor VIII is defined as severe; 1-5% (0.01 - 0.05 IU / ml) as moderate; and >5-40% (0.05 - <0.40 IU/mL) as mild, provided that the norm is 1 IU/mL factor VIIIC (100%). Therapeutic levels can be established for new compositions, including those CFXTEN and pharmaceutical compositions containing CFXTEN disclosures, using standard methods. In the practice of the present invention, it should be understood that any dosage of CFXTEN that is effective can be used to treat cases of bleeding or maintain hemostasis. Methods for establishing therapeutic levels and dosage regimens for a given composition are known to those skilled in the art (See, for example, Goodman &Gilman's The Pharmacological Basis of Therapeutics, ^11th Edition, McGraw-Hill (2005)). For example, by using dose escalation studies in subjects with a target disease to determine efficacy or desired pharmacological effect, occurrence of side effects, and determination of circulating blood levels for a given drug or biologic, therapeutic blood levels for a given subject or population of subjects can be determined. Dose escalation studies may evaluate CFXTEN activity through studies in a subject or group of subjects with hemophilia A. Studies may monitor blood levels of the procoagulant, as well as physiological or clinical parameters known in the art, or as described herein, if one or more parameters associated with factor VIII disease or clinical parameters associated with applicable outcomes, together with observations and/or measured parameters to determine non-acting dose, adverse events, minimum effective dose, and the like, together with measurement of pharmacokinetic parameters , which establishes certain or received circulating levels in the blood. The results can then be correlated with the administered dose and therapeutic blood concentrations that match previously determined parameters or effective levels. Using these methods, the range of doses and blood concentrations can be correlated with the minimum effective dose, as well as the maximum dose and blood concentration at which the desired effect is observed and maintained or the period during which it can be provided, thereby creating therapeutic levels. in the blood and the dosage regimen for the composition. Thus, using the above methods, set the level of C _m in the blood; _n below which the CFXTEN fusion protein will not have the desired pharmacological effect, and a blood C _max level above which side effects such as thrombosis may occur (Brobrow, RS, JABFP (2005) 18(2):147-149), setting a therapeutic window for the composition.

One of skill in the art can, using the methods described herein or other methods known in the art, confirm that the administered CFXTEN remains at therapeutic blood levels to maintain hemostasis for the desired interval or requires adjustments in doses or lengths. , or XTEN sequences. Additionally, determining the appropriate dose and frequency of dosing to maintain CFXTEN within the therapeutic window establishes a therapeutically effective dosing regimen; a schedule for administering multiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in need thereof, resulting in successive C _max peaks and/or lowest C _min points that remain above therapeutically effective concentrations and result in an improvement in at least one of the measured parameter, compared to the target condition. In a separate embodiment of the present invention, CFXTEN or pharmaceutical compositions that contain CFXTEN, administered at an appropriate dose to a subject, result in fusion protein concentrations

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CFXTEN in the blood that remain above the minimum effective concentration to maintain hemostasis for at least about twice as long as the corresponding non-XTEN FVIII, and are administered at a comparable dose; alternatively, at least about three times longer; alternatively, at least about four times longer; alternatively, at least about five times longer; alternatively, at least about six times longer; alternatively, at least about seven times longer; alternatively, at least about eight times longer; alternatively at least about nine times longer, alternatively at least about ten times longer, or at least about twenty or more times longer than the corresponding non-XTEN FVIII, and administered at a comparable dose . As used herein, an appropriate dose means a dose of a drug or biologic that, when administered to a subject, can result in a desired therapeutic or pharmacological effect (eg, hemostasis) and/or blood concentration within the therapeutic window.

In the practice of the invention, CFXTENs with a longer terminal half-life are generally preferred in order to increase patient comfort, to increase the interval between doses, and to reduce the amount of drug needed to achieve a sustained effect. The improved PK parameters allow for reduced dosing of the claimed compositions as compared to non-XTEN FVIII, primarily in the case of haemophilia A subjects receiving standard prophylaxis.

As described in more detail in the examples which refer to the pharmacokinetic characteristics of XTEN-containing fusion proteins, it has been noted that increasing the overall length of XTEN, alone or in combination, confers a disproportionate increase in the terminal half-life of the fusion protein that contains XTEN. Accordingly, the present invention provides CFXTEN fusion proteins and pharmaceutical compositions that contain CFXTEN, with CFXTEN exhibiting an improved half-life when administered to a subject. In certain embodiments, the present invention provides CFXTEN monomeric fusion proteins that comprise one or more XTENs, with the number and location of XTENs chosen to confer an increased terminal half-life of CFXTEN administered to a subject compared to the corresponding FVIII not bound to XTEN and administered at a comparable dose such that the increase in terminal half-life, compared to FVIII not bound to XTEN, is at least about two times longer, or at least about three times, or at least about four times, or at least about five times, or at least about six times, or at least about seven times, or at least about eight times, or at least about nine times, or at least about ten times, or at least about 15 times, or at least 20 times, or at least 40 times or more. In additional embodiments, the present invention provides CXTEN compositions and pharmaceutical compositions that contain CFXTEN, wherein administration of the composition to a subject in need thereof results in a terminal half-life that is at least 12 hours more, or at least at least about 24 hours more, or at least about 48 hours more, or at least about 96 hours more, or at least about 144 hours more, or at least about 7 days more, or at least at least about 14 days more, or at least about 21 days more, than the same dose of non-XTEN FVIII. In a further embodiment of the present invention, administering a clotting-effective dose of the CFXTEN fusion protein to a subject in need thereof may result in savings in the time between successive doses required to maintain blood levels of about 0.1 IU/mL for at least 48 hours. , or at least 72 hours, or at least about 96 hours, or at least about 120 hours, or at least about 7 days, or at least about 14 days, or at least about 21 days between successive doses compared to non-XTEN FVIII and administered at a comparable dose.

In a separate embodiment, the present invention provides CFXTEN fusion proteins and pharmaceutical compositions that comprise CFXTEN which exhibits, when administered to a subject in need thereof, an increase in AUC of at least about 50%, or at least about 60%, or at least at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about 2000%, compared to the corresponding non-XTEN FVIII, and administered to the subject at a comparable dose. Pharmacokinetic parameters of CFXTEN can be determined using standard methods that include dosing, blood sampling at specific time intervals, and protein analysis using ELISA, HPLC, radioisotope analysis, coagulation activity assays, assays from Table. 49, or other methods known in the art or as described herein, followed by standard data calculation to obtain half-life and other PK parameters.

In a separate embodiment of the present invention, a smaller amount of ME hybrid

- 123 041874 about two times less, or about three times less, or about four times less, or about five times less, or about six times less, or about eight times less, or about 10 times less or more times less administered than the corresponding non-XTEN-bound FVIII at the dosage regimen required to maintain hemostasis, and the fusion protein achieves a similar area under the curve as in the case of the corresponding amount of non-XTEN-bound FVIII ME required for maintaining hemostasis. In a further embodiment of the present invention, the CFXTEN fusion protein or pharmaceutical compositions that contain CFXTEN require less frequent administration for standard prophylaxis in a subject with hemophilia A, with a dose of the fusion protein being administered about every four days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly to a subject, and the fusion protein reaches a similar area under the curve as the corresponding non-XTEN-bound FVIII and is administered to the subject. In other embodiments, the implementation of the present invention accumulates a smaller amount of ME of the hybrid protein administered to the subject, by about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70 %, or about 80%, or about 90% less, compared to the corresponding amount of non-XTEN-bound FVIII ME at a dosing regimen necessary to maintain a blood concentration of 0.1 IU/mL until the fusion protein reaches at least the same area under the curve as the corresponding non-XTEN FVIII. The accumulated smaller amount of ME is measured over a period of at least about one week, or about 14 days, or about 21 days, or about one month.

In a separate aspect, the present invention provides CFXTEN compositions designed to reduce binding by FVIII binding agents, thereby increasing the terminal half-life of CFXTEN administered to a subject while still retaining procoagulant activity. It is believed that the CFXTEN of the present invention has a relatively higher and/or delayed activity, achieved through reduced active clearance of molecules by adding unstructured XTEN to the clotting factor FVIII. The clearance mechanisms to remove FVIII from the circulation have not yet been elucidated. Uptake, elimination and inactivation of clotting proteins can occur in the circulatory system as well as in the extravascular space. Clotting factors are complex proteins that interact with a wide variety of other proteins, lipids, and receptors, and many of these interactions can help eliminate CF from the circulation. Protein von Willebrand factor is an example of a FVIII binding agent that is associated with FVIII. Factor VIII and von Willebrand factor (VWF) circulate in the blood as tight, non-covalently bound complexes in which VWF serves as a carrier, which most likely contributes to the protection of factor FVIII from active cleavage mechanisms, while still leading to a limitation of the final time half-life of FVIII. For example: (i) VWF stabilizes the heterodimeric structure of FVIII; (ii) VWF protects FVIII from proteolytic degradation by phospholipid-binding protease-like activated protein C and activated FX (FXa); (iii) VWF prevents FVIII from binding to negatively charged phospholipid surfaces located within activated platelets; (iv) VWF inhibits the binding of FVIII to activated FIX (FIXa), thereby preventing access of FVIII to the FX activating complex; and (v) VWF prevents cellular uptake of FVIII (Lenting, P.J., et al., J Thrombosis and Haemostasis (2007) 5(7):1353-1360). In addition, a protein-coupled LDL receptor (LRP1, also known as the α2 macroglobulin receptor or CD91) has been identified as a possible clearance receptor for FVIII, with LRP1 binding sites identified on both chains of the heterodimeric form of FVIII (Lenting PJ, et al. , J Biol Chem (1999) 274: 23734-23739; Saenko EL, et al., J Biol Chem (1999) 274:37685-37692). LRPs are involved in the clearance of a variety of ligands that include proteases, Kunitz-type inhibitors, protease-serpin complexes, lipases, and lipoproteins (Narita, et al., Blood (1998) 2:555-560). It has been shown that the light chain, rather than the heavy chain, of factor VIII binds to the open surface of the LRP1 receptor protein (Lentig et al. (J Biol Chem (1999) 274(34):23734-23739; and US Pat. No. 6,919,311) that suggests that LRP1 may play a significant role in the active clearance of proteins such as FVIII.While the VWF-FVIII interaction has a high affinity (<1 nM), the complex is nevertheless in dynamic equilibrium such that a small but significant a portion of the FVIII molecule (5-8%) circulates as free protein (Leyte A, et al., Biochem J (1989) 257: 679-683; Noe DA. Haemostasis (1996) 26: 289-303). Thus, the portion of native FVIII not protected by VWF allows active clearance mechanisms to remove unprotected FVIII from the circulation.

In a separate embodiment, the present invention provides CFXTEN that is associated with VWF but has increased protection against active clearance receptors conferred by incorporating more than two XTENs at one or more locations within the FVIII molecule (e.g., locations selected from Table 5, Table 6, Table 7, Table 8 and Table 9 or Figures 8-9), with XTEN preventing the resulting CFXTEN from interacting with these excretion receptors, with the result that the pharmacokinetic properties of CFXTEN are improved compared to the corresponding FVIII not related to XTEN. In a further embodiment, the present invention

- 124 041874 CFXTEN has a reduced binding affinity for VWF of at least 5% less, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70 % less, but nonetheless configured to have increased protection against active excretion receptors provided by the incorporation of XTEN at one or more locations within the FVIII molecule, while XTEN interferes with the interaction of factor VIII with such receptors. In previous embodiments of the present invention, CFXTEN has an increased terminal half-life of at least about 12 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 10 days, or 14 days, or 21 days, compared to non-XTEN FVIII. The present invention provides a method for generating CFXTEN with reduced clearance, wherein CFXTEN fusion proteins designed with multiple inserts are evaluated for inhibition of excretion receptor binding compared to non-XTEN FVIII using in vitro binding assays or in vivo pharmacokinetic models described herein, or other assays known in the art, and select those that show reduced binding while retaining FVIII procoagulant activity. In addition, previously described fusion proteins can be optimized to have an XTEN radius ratio increased by at least 2.0-3.5 in order to achieve pharmacokinetic properties that are further improved. Tab. 5, tab. 6, tab. 7, tab. 8 and tab. 9 and FIG. 8-9 provide non-limiting examples of XTEN insertion points within the Factor VIII sequence. Using such insertion points, the present invention provides CFXTEN compositions that have multiple XTEN conformations introduced with about 100, or about 200, or about 300, or about 400, or about 500 amino acids sharing at least three XTENs for additional enhancement. protection against active clearance mechanisms and hence increasing the terminal half-life of CFXTEN. Without wishing to be bound by any theory, XTEN formulations of high net charge CFXTEN (e.g., CFXTEN, which contains the AE XTEN family) are hypothesized, as described above, to have less non-specific interactions with various negatively charged surfaces, such as blood vessels, tissues, or various receptors, which may further contribute to a decrease in active clearance. On the other hand, XTEN formulations of CFXTEN with low (or no) net charge (e.g., CFXTEN, which contains the AG XTEN family) are expected in order to have a higher degree of interaction with surfaces that, if involved in active clearance, can potentiate activity of the associated clotting factor, taking into account the known contribution of cells (eg, platelets) and vascular surfaces to the mode of clotting and the intensity of activation of clotting factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London , F., et al Biochemistry (2000) 39(32):9850-9858). The present invention is particularly advantageous in that certain ligands that have reduced binding to the excretion receptor, either as a result of a reduced rate of association or an increased rate of dissociation, may be affected by blockade of receptor sites by administered random helix XTENs. leading to reduced binding. The choice of specific conformation of the fusion protein CFXTEN can be tested using the methods disclosed herein to confirm those conformations that reduce the degree of binding to the excretion receptor so that a reduced rate of active clearance is achieved. In a separate embodiment of the present invention, CFXTEN contains a FVIII-XTEN sequence that has one or more XTENs administered at locations selected from Table 1. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9, with the terminal half-life of CFXTEN being increased by at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six times, or at least about eight times, or at least about ten times, or at least about twenty times, compared to non-XTEN FVIII. In a further embodiment of the present invention, CFXTEN contains a FVIIIXTEN sequence that has a first and at least a second XTEN administered at a first and second location selected from Table 1. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9, with the terminal half-life of CFXTEN being increased by at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six times, or at least about eight times, or at least about ten times, or at least about twenty times, compared to non-XTEN FVIII. In another additional embodiment of the present invention, CFXTEN contains a FVIII-XTEN sequence that contains multiple XTEN sequences using three of the multiple XTEN insertion locations selected from Table 1. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9 separated by about 100, or about 200, or about 300, or about 400, or about 500 amino acids, with the terminal half-life of CFXTEN being increased by at least about two-fold, or at least about three times, or at least about four times, or at least about five times, or at least about six times, or at least about eight times, or at least about ten times, or at least about twenty times compared to

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FVIII not related to XTEN. In the previous embodiments of the present invention described above in this paragraph, XTENs built into CFXTEN conformations may be identical or may be different, and may be at least about 80%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity with the sequence from any of the table. 3, 4 and 13-17, and may optionally contain one or more split sequences from table. 12 to facilitate secretion of one or more XTENs from the CFXTEN fusion protein.

In a separate embodiment, the present invention provides a CFXTEN that enhances the pharmacokinetic properties of a fusion protein by linking one or more XTENs to the FVIII component of the fusion protein, wherein the fusion protein has an increase in average molecular weight ratio of at least about two-fold, or at least about three times, or at least about four times, or at least about five times, or at least about six times, or at least about seven times, or at least about eight times, or at least about ten times, or at least about twenty times, or at least about fifty times, and while the terminal half-life of CFXTEN, when administered to a subject, is increased by at least about two times, or at least about four times, or at least about eight times, or at least about 10 times or more, compared to the corresponding FVIII, not related with XTEN. In the above embodiment of the present invention, while at least two XTEN molecules are included in CFXTEN, the XTENs may be identical or they may have a different sequence composition, net charge, or length. XTEN may have at least about 80% or 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% sequence identity. with a sequence from any of the tables. 3, 4 and 13-17, and may optionally contain one or more split sequences from table. 12 to facilitate secretion of one or more XTENs from the CFXTEN fusion protein.

Thus, the present invention provides CFXTEN compositions in which the degree of activity, bioavailability, half-life or physicochemical characterization of the fusion protein can be cross-linked by selecting and placing the type and length of XTEN in CFXTEN compositions. Accordingly, the present invention provides compositions in which receive FVIII from table. 1 and XTEN or fragment XTEN from any of the table. 3, 4 or 13-17, for example, in a conformation selected from any of the formulas I-VIII, or XTEN introduced in the localization selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9 so that the design has the desired property.

The present invention provides methods for preparing CFXTEN compositions that can maintain the FVIII component at therapeutic levels in a subject in need of at least two times, or at least three times, or at least four times, or at least five times the time period compared to similar dosages of the corresponding non-XTEN FVIII. In a separate embodiment of the method, the subject receives standard prophylaxis to prevent bleeding events. In a further embodiment of the method, the subject is treated for bleeding. In a further embodiment of the method, the subject is treated to increase the circulating blood concentration of FVIII procoagulant above 1%, or above 1-5%, or above 5-40%, compared to normal plasma FVIII concentrations. A procoagulant, as used herein, has its general meaning in the art and generally corresponds to an activity that promotes thrombus formation, either in an in vitro assay or in vivo. The method for obtaining a composition that can maintain the FVIII component at therapeutic levels includes the steps of selecting one or more XTENs that match FVIII in terms of conjugation to provide the desired pharmacokinetic properties, taking into account a given dose and dosing regimen, creating a gene construct that encodes CFXTEN in one of the conformations, disclosed herein, transforming an appropriate host cell with an expression vector that contains a coding gene, expressing the fusion protein under appropriate culture conditions, extracting CFXTEN, administering CFXTEN to a mammal, followed by assays to test the pharmacokinetic properties, activity of the CFXTEN fusion protein (e.g., ability to maintain hemostasis, or serve as a procoagulant) and the safety of the administered composition. Those compositions that exhibit the desired properties are selected for additional use. CFXTEN made using the methods provided herein can result in increased efficacy of the administered composition by, among other properties, maintaining circulating concentrations of the FVIII procoagulant component at therapeutic levels for an extended period of time.

The present invention provides methods for analyzing CFXTEN fusion proteins of various compositions or conformations in order to provide CFXTEN with the desired degree of procoagulant and therapeutic activity and pharmacokinetic properties, as well as sufficient drug safety study results. Specific in vitro and in vivo assays or animal models are used to evaluate the activity and performance of each configured CFXTEN.

- 126 041874 and/or component FVIII built into CFXTEN, which contain, but are not limited to, analyzes in the examples, analyzes from the table. 49, as well as the following assays or other such assays known in the art for analyzing the properties and effects of FVIII. Functional assays can be performed that measure clotting activity, such as one-step coagulation activity assay and two-step coagulation activity assay (Barrowcliffe TW, Semin Thromb Hemost. (2002) 28(3):247-256), partial thromboplastin activation time assays ( aPTT) (Belaaouaj AA et al., J. Biol. Chem. (2000) 275:27123-8; Diaz-Collier JA. Haemost (1994) 71:33946), chromogenic FVIII assays (Lethagen, S., et al. , Scandinavian J Haematology (1986) 37:448-453), or animal model pharmacodynamic assays that include bleeding time or thromboelastography (TEG or ROTEM), among others. Other assays comprise determining the binding affinity of CFXTEN to a target substrate using binding or competitive binding assays such as Biacore chip-bound receptor or protein binding assays or ELISA assays as described in US Pat. document, radioreceptor assays, or other assays known in the art. Other assays to determine the binding of FVIII inhibitors to CFXTEN include the Bethesda assay or a Nijmegen modification of the Bethesda assay. Previous assays can also be used to evaluate FVIII sequence variants (assayed as unique components or as a CFXTEN fusion protein) and can be compared to natural FVIII to determine if they have the same degree of procoagulant activity as natural CF or a single fraction thereof. so that they are eligible for inclusion in CFXTEN; for example, at least about 10%, or at least about $20, or about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% activity, compared to natural FVIII.

Dose optimization is important for all drugs. A therapeutically effective dose or amount of CFXTEN will vary according to disease factors such as disease, age, sex, and weight of the individual, and the ability of the administered fusion protein to elicit the desired response in the individual. For example, a standardized unique dose of FVIII for all patients presenting with various bleeding conditions or abnormal clinical parameters (eg, neutralizing antibodies) may not always be effective. Hemophilia A patients with trauma who have undergone surgery or who have high titers of FVIII inhibitory antibodies generally require higher and more frequent dosing. Generally, the dosage level is adjusted in frequency, duration, and units according to the severity and duration of bleeding in each patient. Accordingly, CFXTEN is included in a pharmaceutically acceptable carrier, delivery device, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the fusion protein to stop bleeding, as measured by standard coagulation activity assays. Consideration of these factors is within the purview of the ordinary clinician to determine a therapeutically or pharmacologically effective amount of CFXTEN and an appropriate dosing regimen, versus an amount that would result in insufficient specific activity such that clinical improvement or bleeding control is not achieved.

The present invention provides methods for establishing a dosage regimen for CFXTEN pharmaceutical compositions of the present invention. The methods include administering successive doses of a therapeutically effective amount of a CFXTEN pharmaceutical composition using varying time periods between doses to determine if the dosing interval is sufficient to achieve and/or maintain the desired parameter, blood level, or clinical effect; such successive doses of a therapeutically effective amount within an effective range establish a therapeutically effective dosing regimen for CFXTEN in the event of a factor VIII related disease state or disease. A prophylactically effective amount corresponds to the amount of CFXTEN required for the period of time required to prevent a physiological or clinical result or event; for example, delaying the onset of a bleeding event or maintaining a blood concentration of FVIII procoagulant or equivalent to a higher threshold level (eg, 1-5% to 5-40% of normal). In methods of treatment, the amount of dose of CFXTEN administered to a subject ranges from about 5 to 300 IU/kg/dose, or from about 10 to 100 IU/kg/dose, or from about 20 to about 65 IU/kg/dose. , or about 20 to about 40 IU/kg/subject dose. The appropriate dose may also depend on other factors that may influence drug response; for example, bleeding episodes usually require higher doses at shorter intervals than prophylaxis.

In certain embodiments of the present invention, the method comprises administering a therapeutically effective amount of a pharmaceutical composition that comprises a CFXTEN fusion protein composition and at least one pharmaceutically acceptable carrier to a subject in need thereof, wherein administration results in a greater improvement in at least one parameter or physiological condition associated with FVIII or coagulop deficiency

- 127 041874 or lead to a more favorable clinical outcome, which is performed using the FVIII component of CFXTEN, compared with the effect on the parameter, disease, or clinical outcome, which is performed by administering a pharmaceutical composition that contains FVIII that is not associated with XTEN, and administered in a comparable dose. Non-limiting examples of parameters that are improved include blood concentration of FVIII procoagulant, reduced partial thromboplastin activation (aPTT) assay time, reduced one- or two-stage coagulation activity assay time, delayed onset of a bleeding event, reduced FVIII chromogenic assay time, reduced bleeding time, resolution an episode of bleeding or a decrease in the titer of Bethesda to CFXTEN, in relation to natural FVIII. In a particular embodiment of the foregoing, amelioration is accomplished by administering the CFXTEN pharmaceutical composition at a dose that achieves a circulating FVIII procoagulant concentration (or equivalent) above a threshold level (e.g., 1-5% to 5-40% of normal FVIII levels), thereby establishing therapeutically effective dose. In a further embodiment of the foregoing, amelioration is accomplished by administering multiple consecutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dosing regimen that maintains the circulating FVIII procoagulant concentration (or equivalent) above a threshold level (e.g., 1-5 to 5-40% of normal). FVIII levels) throughout the dosing period. In a further embodiment of the method, administration of at least two consecutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dosing regimen maintains a circulating FVIII procoagulant concentration (or equivalent) above about 1, 2, 3, 4, 5, 10, 15, 20, 30 or 40% normal FVIII levels for a period that is at least about three times longer; alternatively, at least about four times longer; alternatively, at least about five times longer; alternatively, at least about six times longer; alternatively, at least about seven times longer; alternatively, at least about eight times longer; alternatively at least about nine times longer or at least about ten times longer than non-XTEN FVIII and administered using a therapeutically effective dosing regimen.

In a separate embodiment of the present invention, CFXTEN or pharmaceutical compositions that contain CFXTEN, which are administered at a therapeutically effective dosage regimen, result in a gain in time of at least about three times longer; alternatively, at least about four times longer; alternatively, at least about five times longer; alternatively, at least about six times longer; alternatively, at least about seven times longer; alternatively, at least about eight times longer; alternatively, at least about nine times longer or at least about ten times longer between at least two consecutive C _max peaks and/or C _min lowest points in the case of blood levels of the fusion protein, as compared to the corresponding a biologically active protein of the fusion protein, not associated with XTEN, and administered to the subject in a similar dosing regimen. In a further embodiment of the present invention, CFXTEN administered at a therapeutically effective dosing regimen results in a similar improvement in one, or two, or three or more of the measured parameters using less frequent dosing or a lower total dosage per IU of the pharmaceutical composition fusion protein compared to the appropriate biologically active protein component(s) not bound to XTEN and administered to the subject using a therapeutically effective FVIII dosing regimen. The measured parameters comprise any of the clinical, biochemical, or physiological parameters described herein, or others known in the art for evaluating the conditions of subjects associated with factor VIII.

(b) Pharmacology and pharmaceutical properties of CFXTEN.

The present invention provides CFXTEN compositions containing XTEN-covalently bound FVIII that have enhanced pharmaceutical and pharmacological properties over non-XTEN-bound FVIII, as well as methods for enhancing the therapeutic and/or procoagulant effect of the components of the FVIII compositions. In addition, the present invention provides CFXTEN compositions with improved properties over known factor VIII fusion proteins in the art that contain albumin, immunoglobulin polypeptide partners, shorter length polypeptides, and/or repeat sequence polypeptide partners. In addition, CFXTEN fusion proteins provide significant advantages over chemical conjugates such as pegylated FVIII constructs, in particular the fact that recombinant CFXTEN fusion proteins can be made in host cell expression systems, which can reduce the time and cost of the steps. both research and product development and manufacturing, and lead to a more homogeneous, defined product with less toxicity of both the product and metabolites of CFXTEN compared to pegylated conjugates.

As a therapeutic agent, CFXTEN has several advantages over thera

- 128 041874 pia that do not contain XTEN, which contains one or more of the following non-limiting properties: increased solubility, increased thermal stability, reduced immunogenicity, increased average molecular weight, reduced renal clearance, reduced proteolysis, reduced metabolism, improved therapeutic efficacy, less frequent dosing with extended time between doses, able to maintain hemostasis in a subject with hemophilia A, ability to administer the CFXTEN formulation subcutaneously or intramuscularly, individualized absorption rate when administered subcutaneously or intramuscularly, improved lyophilization stability, improved serum/plasma stability, increased terminal half-life, increased circulating solubility, reduced binding by neutralizing antibodies, reduced active clearance, individualized substrate binding affinity, resistance to degradation, resistance to freeze-thaw, protease resistance, resistance to ubiquitination, ease of administration, compatibility with other pharmaceutical excipients or carriers, persistence in the subject, increased storage stability (eg, increased shelf life), and the like. The net effect of the improved properties is that the use of a CFXTEN formulation may result in an overall improved therapeutic effect compared to non-XTEN FVIII, lead to economic benefits associated with less frequent dosing, and/or result in better patient compliance with the treatment regimen and regimen when administered to a subject with a factor VIII related disease.

The present invention provides CFXTEN compositions and pharmaceutical compositions that contain CFXTEN, wherein administration of the composition results in an improvement in at least one of the clinical or biochemical parameters disclosed herein as being useful for evaluating a subject's diseases, diseases, or disorders. Non-limiting examples of parameters that are improved include FVIII procoagulant blood concentrations, reduced partial thromboplastin activation (aPTT) assay time, reduced one- or two-stage coagulation activity assay time, delayed onset of a bleeding event, reduced FVIII chromogenic assay time, reduced bleeding time, resolution episode of bleeding or a decrease in the titer of Bethesda CFXTEN in relation to natural FVIII. The improved pharmacokinetic properties of the subject CFXTEN allow the use of a cumulatively lower dose of ME fusion protein to maintain the parameter, compared to the corresponding non-XTEN FVIII component. In a particular embodiment of the present invention, the total dose of CFXTEN in the ME of embodiments of the present invention required to achieve and maintain an improvement in at least one parameter for about 2-7 days is at least about three times less, or at least about four times, or at least about five times, or at least about six times, or at least about eight times, or at least about 10 times less than the corresponding FVIII component , not associated with XTEN. In a further embodiment of the present invention, the total dose of a subject in ME CFXTEN required to achieve and maintain an improvement in at least one parameter over two, three, or four consecutive doses is at least about three times less, or at least about four times, or at least about five times, or at least about six times, or at least about eight times, or at least about 10 times less than the corresponding unbound FVIII component with XTEN. In addition, the present invention provides some embodiments of CFXTEN wherein the period between successive administrations that result in achieving and maintaining an improvement in at least one parameter is at least about three times, or at least about four times , or at least about five times, or at least about six times, or at least about eight times, or at least about 10 times longer than the corresponding non-XTEN FVIII component , and administered at a dose with similar ME. In addition, the present invention provides some embodiments of CFXTEN, with administration of 25 IU/kg resulting in a 30% improvement in aPTT time assay (or similar coagulation activity assay) in a subject with hemophilia A, compared to 25 IU/kg of the corresponding FVIII not associated with XTEN if analyzed about 2-7 days after administration. In another further embodiment of the present invention, the present invention provides CFXTEN, wherein administration of 25 IU/kg results in a 30% improvement in bleeding time analysis time in a subject with hemophilia A, compared to 25 IU/kg of the corresponding FVIII, not bound with XTEN if analyzed about 2-7 days after administration.

In a specific embodiment of the present invention, XTEN as a fusion partner increases the solubility of the FVIII payload. Accordingly, if it is desired to increase the pharmaceutical or physico-chemical properties of FVIII, such as degrees of solubility or stability in water, the length and/or composition of the XTEN sequence family of motifs inserted into the fusion protein can each be chosen to confer a different degree of solubility and/or or stability at the respective fusion proteins such that, in general, the pharmaceutical properties of the CFXTEN composition are improved. Fusion proteins CFXTEN can be built

- 129 041874 na and analyzed using the methods described in this document to confirm the physico-chemical properties and select the length of the XTEN sequence or adjust the location, as necessary, in order to lead to the desired properties. In a particular embodiment of the present invention, CFXTEN has a water solubility that is at least about 25% higher than non-XTEN FVIII, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1000% higher than the corresponding non-XTEN FVIII. The present invention provides methods for obtaining and recovering expressed CFXTEN from a host cell with improved solubility and ease of recovery compared to non-XTEN FVIII. In a particular embodiment of the present invention, the method comprises the steps of transforming a eukaryotic host cell with a polynucleotide encoding CFXTEN with one or more XTEN components with a combined sequence length greater than about 100, or greater than about 200, or greater than about 400, or greater than about 600, or greater than about 800 or greater than about 1000 or greater than about 2000 or greater than about 3000 amino acid residues, expressing the CFXTEN fusion protein in the host cell under suitable culture and induction conditions, and recovering the expressed fusion protein in soluble form. In a particular embodiment of the present invention, the one or more CFXTEN fusion proteins each have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity, compared with one or more XTEN selected from any of the table. 4 and 13-17, or fragments thereof, and FVIII has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95 %, or about 96%, or about 97%, or about 98%, or about 99% or 100% sequence identity, compared with FVIII selected from table. 1 and the CFXTEN components are in an N- to C-terminus conformation selected from any of the conformation embodiments disclosed herein.

VI) Use of CFXTEN compositions.

The present invention provides methods and regimens for achieving a beneficial effect on factor VIII related disease by administering compositions that contain CFXTEN. As used herein, a factor VIII disease is intended to include, but is not limited to, factor VIII deficiencies, coagulation disorders associated with factor VIII deficiency, hemophilia A, neutralization of factor VIII by anti-FVIII antibodies, or other factor VIII inhibitors, and cases of bleeding resulting from trauma or surgery or vascular injury and other such diseases, which can be improved or corrected by the introduction of factor VIII to the subject. Inventive methods benefit if the considered disadvantages and/or limitations of other therapies using factor VIII preparations that have a relatively short terminal half-life requiring frequent administration are neutralized by inhibitors or have unfavorable pharmacoeconomics.

Hemostasis is regulated by several protein factors, and such proteins, as well as their analogs, have found use in the treatment of diseases associated with factor VIII. Despite this, the use of commercially available FVIII has met with less remarkable success in the treatment of subjects suffering from similar diseases. In particular, dose optimization and dosing frequency are important for FVIII used in maintaining circulating FVIII concentrations above the threshold levels required for hemostasis, as well as treating or preventing bleeding events in subjects with hemophilia A. The fact that commercially available FVIII products have a short half-life make frequent dosing necessary in order to achieve a clinical effect, which leads to difficulties in the treatment of such patients.

As established by the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (published on the ISTH website November 29, 2000), the more widely used measure of hemophilia A severity is established by determining circulating levels of plasma FVIII procoagulant, in people with <1% (<0.01 IU/mL) factor VIII, defined as severe; 1-5% (0.01-0.05 IU/ml) as moderate severity; and >5-40% (0.05 - <0.40 IU/ml) as mild, where the norm is 1 IU/ml factor VIIIC (100%).

The present invention provides methods for treating subjects suffering from, or at risk of developing, a disease associated with factor VIII. More specifically, the present invention provides methods for treating or preventing bleeding control in a subject. The subject may be any animal, but is preferably a human. In a specific embodiment of the present invention, the method comprises administering a clotting effective amount of the CFXTEN composition to a subject in need thereof. In a further embodiment of the present invention, the method includes the step of administering to a subject with bleeding a clotting effective amount of a pharmaceutical composition that contains CFXTEN, wherein administration results in arrest or reduction of bleeding. As used herein, a clotting effective amount is an amount of a FVIII composition that, when administered to a subject, is sufficient to effect hemostasis or other applicable or desired therapeutic (including prophylactic) result. In the practice of the present invention, it should be understood that a clotting effective amount may be administered in one or more administrations. The exact clotting effective amounts of the pharmaceutical composition to be administered will be adjusted according to the judgment of the physician, although the single dose will generally depend on the severity or cause of bleeding and the amount of pre-existing FVIII in the subject. In a specific embodiment of a method for treating bleeding, a clotting effective amount of pharmaceutical compositions that contain CFXTEN is administered to a subject suffering from occasional bleeding, wherein the administration results in regression of the bleeding over a time period of at least a factor of two, or at least three times, or at least four times longer than non-XTEN FVIII, and administered to a similar subject with a similar bleeding at a comparable dose.

In a further embodiment of the present invention, administering a clotting effective amount of a CFXTEN composition to a subject with a factor VIII disease results in 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or a greater improvement in one or more biochemical, physiological, or clinical disease parameters associated with FVIII compared to FVIII not associated with XTEN, as measured between days 2 and 7 after administration. In a further embodiment of the present invention, administering a clotting effective amount of a CFXTEN composition to a subject in need thereof results in an improvement in one or more biochemical, physiological, or clinical parameters of an FVIII-associated disease over a period that is at least twice as long. , or at least four times longer, or at least five times longer, or at least six times longer, compared to the period achieved with non-XTEN FVIII and administered at a comparable dose . Non-limiting examples of parameters that are improved over longer periods include blood concentrations of FVIII procoagulant, reduced partial thromboplastin activation (aPTT) assay time, reduced one- or two-stage coagulation activity assay time, delayed onset of a bleeding event, reduced FVIII chromogenic assay time, reduced bleeding time, among other FVIII related parameters known in the art. In previous paragraph embodiments, CFXTEN is administered which contains FVIII with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity with factor VIII from table. 1 and one or more XTEN sequences with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity with XTEN from table. 4, introduced into FVIII in one or more localizations selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or, as illustrated in FIG. 8-9. In certain embodiments of the present invention, at least one CFXTEN XTEN insertion site is selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbering relative to mature natural FVIII person).

In a particular embodiment of the method of treatment, a clotting effective amount of the CFXTEN fusion protein administered to a subject suffering from hemophilia A is sufficient to increase the circulating FVIII procoagulant concentration to greater than 0.05 IU/mL and to maintain hemostasis for up to at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours, or at least about 144 hours, or at least about 168 hours or more. In a further embodiment of the present invention, administering a clotting effective amount of a pharmaceutical composition that contains CFXTEN to a subject in need thereof results in a more significant reduction in one-step coagulation activity assay time of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a subject's blood sample 2-7 days after administration, compared to the subject's time of analysis after introduction of a similar amount of the corresponding FVIII, not associated with XTEN. In a further embodiment of the present invention, administration of a therapeutically effective amount of CFXTEN or pharmaceutical compositions that contain CFXTEN to a subject in need thereof results in a greater reduction in prothrombin formation time and activity by at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a subject's blood sample 2-7 days after administration, compared to the time of formation and activity prothrombin in a subject after administration of a similar amount of the corresponding FVIII not bound to XTEN. In a further embodiment of the present invention, administration of CFXTEN or

- 131 041874 pharmaceutical compositions that contain CFXTEN to a subject in need, using a therapeutically effective amount, leads to the provision of time for the formation and activity of prothrombin within 30% of the norm in a blood sample of the subject for a period of time that is at least twice, or at least about three times, or at least about four times longer than FVIII that is not bound to XTEN, and administered to a subject using a similar dose.

In a specific embodiment of the treatment method, the CFXTEN fusion protein is formulated and administered as a pharmaceutical composition that contains CFXTEN admixed with a pharmaceutically acceptable excipient. Methods for preparing pharmaceutical compositions are well known in the art. Methods and compositions can generally be found in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. 1990 (See also Wang and Hanson, Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42-2S (1988)).

In additional aspects, the present invention provides a regimen for treating a patient with hemophilia A, said regimen comprising a composition that comprises a CFXTEN fusion protein. In a separate embodiment of a regimen for treating a hemophilia A patient, the regimen further includes the step of determining the amount of the pharmaceutical composition that contains CFXTEN to achieve hemostasis in the patient. In certain regimen embodiments, (i) a smaller amount of ME of the pharmaceutical composition that contains CFXTEN is about two times less, or about three times less, or about four times less, or about five times less, or about six times less, or about eight times less, or about 10 times less, is administered to a subject in need compared to the corresponding non-XTEN clotting factor in another similar dosing regimen, and the fusion protein achieves a similar area under the curve (based on IU/ml) and/or a similar therapeutic effect as the corresponding FVIII not associated with XTEN; (ii) the pharmaceutical composition is administered less frequently (e.g., every three days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or nearly monthly) compared to the corresponding non-XTEN FVIII , at another similar dose, and the fusion protein achieves a similar area under the curve and/or a similar therapeutic effect as the corresponding non-XTEN coagulation factor; or (iii) administering an accumulating smaller amount of ME of the pharmaceutical composition by at least about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less than the corresponding non-XTEN FVIII, with another similar dosing regimen, and the CFXTEN fusion protein achieves a similar therapeutic effect as the corresponding non-XTEN FVIII. The accumulated less ME is measured over a period of at least about one week, or about 14 days, or about 21 days, or about one month. In previous embodiments of the present invention, the therapeutic effect can be determined using any of the measured parameters described herein, which include, but are not limited to, FVIII procoagulant blood concentration, reduced partial thromboplastin activation (aPTT) assay time, reduced one-step or two-stage coagulation activity assay, delayed onset of a bleeding event, reduced FVIII chromogenic assay time, reduced bleeding time, resolution of a bleeding episode, or decreased Bethesda CFXTEN titer relative to natural FVIII, fibrinogen levels, or other assays known in the art to evaluate FVIII coagulopathy. In a further embodiment, the present invention provides CFXTEN for use in a regimen for treating hemophilia in a subject, which comprises administering the CFXTEN composition in two or more consecutive doses to the subject in an effective amount, wherein administration results in at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% higher improvement in at least one, two, or three disease-related parameters compared to non-related FVIII XTEN, and administered using a similar dose.

In a separate aspect, the present invention relates to a method for the prevention or treatment of bleeding in a patient, optionally a patient with hemophilia A, which has a pre-existing anti-FVIII inhibitor(s). Inhibitory antibodies against FVIII commonly develop in hemophiliacs, where the average incidence of inhibitor development is 15-30%, primarily in hemophiliacs who are highly susceptible to FVIII concentrates (Algiman et al. Natural antibodies to factor VIII (anti-hemophilic factor) in healthy individuals PNAS USA (1992) 89: 3795-3799). Despite this, inhibitory antibodies have also been observed in patients with autoimmune disorders, malignancies (such as lymphoproliferative diseases, lymphomas, and solid tumors), during pregnancy, and in the postpartum state. Inhibition can also occur if antibodies prevent FVIII from binding to FIX and FX. At the same time or in addition, anti-FVIII antibodies may interfere with the binding of von Willebrand factor and/or phospholipids to FVIII, which affects the clotting and/or half-life of FVIII. The presence of inhibitory

- 132 041874 antibodies are often first detected with symptoms such as mild bruising and uncontrolled bleeding and are usually consistent with acquired hemophilia. Ahtu-FVIII antibodies can be detected by a variety of methods that include quantification of anti-FVIII activity in a coagulation activity assay, ELISA for FVIII inhibitors, and purification using chromatography and immunoadsorption (Algiman et al., 1992). Accordingly, in the treatment or prevention of any disease associated with or determined by the presence of inhibitory antibodies to FVIII, patentable methods are used. In a specific embodiment, the present invention provides a method of treating a patient who has a pre-existing anti-FVIII inhibitor, the method of which comprises the step of administering to the patient a clotting effective amount of a CFXTEN fusion protein to be administered in order to achieve hemostasis, which is clotting effective the amount of fusion protein administered is reduced compared to the amount of non-XTEN-bound FVIII (or natural FVIII) that must be administered in order to achieve hemostasis. In the method, the reduced amount of CFXTEN is about two times, or three times, or four times, or five times less in IU/kg, compared to the corresponding non-XTEN FVIII. In a further embodiment of the method, the amount of CFXTEN administered as a dose to achieve hemostasis is at least 20 to 40 IU/kg less, or 30 to 60 IU/kg less, or 40 to 80 IU/kg less, or 60 to 100 IU/kg less, or 100 to 140 IU/kg less, or 120 to 180 IU/kg less, or 140 to 200 IU/kg less, compared to the corresponding FVIII not associated with XTEN, or with natural FVIII, necessary in order to achieve hemostasis. In a further embodiment, the present invention provides a method for treating a bleeding event in a subject with hemophilia A that has a titer of at least 10, or 20, or 30, or 40, or 50, or 75, or 100, or 150, or 200 or more units of Bethesd against non-XTEN FVIII, provided that the dose of the CFXTEN fusion protein required to stop a bleeding episode is at least two times, or three times, or four times, or five times, or six times, or seven times, or eight times, or nine times, or 10 times less than the amount of non-XTEN-related FVIII (or natural FVIII) that had to be administered in order to achieve state of hemostasis in a similar subject. One of skill in the art will appreciate that the amount of procoagulant administered to maintain hemostasis will depend on the severity of the FVIII deficiency and/or the frequency or duration of bleeding.

Part of the purpose of the present invention relates to the use of CFXTEN with reduced binding FVIII inhibitors that bind the A2 and/or C2 domains of factor VIII, as a drug. Such a drug is preferably used to maintain hemostasis in a patient suffering from hemophilia, while such a patient has circulating FVIII inhibitors directed against the A2 domain and/or the C2 domain of factor VIII. In a separate embodiment, the present invention provides a method of treatment, the method of which comprises the step of administering to a patient with an A2 domain binding inhibitor a clotting effective amount of a CFXTEN fusion protein, wherein CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or less binding to an inhibitor that binds the A2 domain of FVIII as compared to non-XTEN-bound FVIII or native FVIII, and that the introduction leads to hemostasis. In a further embodiment, the present invention provides a method of treatment, the method of which comprises the step of administering to a patient with a C2 domain-binding inhibitor an effective amount of a CFXTEN fusion protein for clotting, wherein CFXTEN exhibits at least 10%, or 20%, or 30%, or 40 %, or 50%, or 60%, or 70%, or 80% or less binding to an inhibitor that binds the C2 domain of FVIII compared to FVIII not bound to XTEN or native FVIII, and given that administration leads to hemostasis. The reduced binding of a subject's CFXTEN can be assayed directly by an ELISA that detects FVIII inhibitors, or measured indirectly by demonstrating reduced FVIII inhibitory activity of CFXTEN, compared to natural FVIII in the presence of an inhibitor, as measured by a factor VIII chromogenic assay or a one-step assay, as described herein, or other suitable folding methods known in the art. In addition, a CFXTEN subject can be measured for reduced (or no) inhibition in the presence of known inhibitors using the modified Bethesda assay. In accordance with a specific aspect of the present invention, CFXTEN used in the methods has reduced reactivity against one or more of the antibodies from table. 10 as well as naturally occurring antibodies that are present in patients with hemophilia. For testing purposes, similar and different inhibitory antibodies may be obtained from humans (e.g., from the sera of patients who have inhibitory antibodies) or may be obtained from mice, guinea pigs, horses, goats, primates, and other mammals by immunization with FVIII, or fragments, in particular with a fragment containing all or part of the A2 or C2 domain, whether in polyclonal or monoclonal form.

The present invention further contemplates that CFXTEN, which are used in accordance with the methods provided herein, can be administered in combination with other treatments and compositions (eg, other clotting proteins) used to treat diseases associated with factor VIII, or a disease in which the clotting factor is an adjuvant therapy; for example, cases of bleeding due to trauma or surgery.

In additional aspects, the present invention provides methods for producing a medicament for a disease associated with factor VIII, which contains a combination of the sequence of factor VIII selected from table. 1, with one or more XTEN selected from the table. 4, entered into one or more insertion sites selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 for what results in a drug that retains at least some of the activity of native FVIII. The present invention provides a method for preparing a pharmaceutical composition which includes the step of combining the drug of the above embodiment with at least one pharmaceutically acceptable carrier. In a specific embodiment of the method for preparing a drug for a factor VIII related disease, factor VIII has a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity, compared with the sequence selected from the table. 1 and one or more XTENs have a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity, compared with a sequence selected from any of the table. 3, 4 and 13-17, or a fragment thereof, given that one or more XTEN is administered in one or more locations selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9. In a specific embodiment of the above, at least one XTEN insertion site is selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905, and 1910 (relative to natural mature numbering). human FVIII). In a further embodiment of the method, CFXTEN comprises a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity, compared with a sequence selected from any of the table. 21.

In additional aspects, the present invention provides a method for preparing a CFXTEN composition in order to achieve the desired pharmacokinetic, pharmacological or pharmaceutical properties. In general, the steps involved in building and preparing inventive fusion protein compositions, as illustrated in FIG. 11-13 comprise: (1) a selection of FVIIIs (eg, natural proteins, sequences from Table 1, analogues or derivatives with activity) for the treatment of a particular disease; (2) selecting one or more XTENs (e.g., sequences with at least 80% identity with the sequences shown in Table 4) that will confer the desired pharmacokinetic and physicochemical characteristics of the resulting CFXTENs (e.g., administering a CFXTEN composition to a subject results in that the fusion protein is maintained above 0.05-0.4 IU/ml for a longer period compared to non-XTEN FVIII); (3) establishing the desired conformation from the N- to the C-terminus of CFXTEN in order to achieve the desired potency or PK parameters (e.g., selecting one or more insertion sites from Table 5, Table 6, Table 7, Table 8 and Table 9); (4) determining the structure of the expression vector that encodes the configured CFXTEN; (5) transformation of a suitable host with an expression vector; and (6) expressing and recovering the resulting isolated CFXTEN fusion protein. In a separate embodiment of the method for producing CFXTEN, the XTEN for insertion is evaluated using Equation IV, in order to maximize the ratio of XTEN radii in the case of a fusion protein construct, with XTEN resulting in values greater than 2.0, or 2.1, or 2.2 or 2.3 or 2.4 or 2.5 or 2.6 or 2.7 or 2.8 or 2.9. or 3.0 are preferred. For those CFXTENs in which an increase in half-life or an increase in the period of time spent above the minimum effective clotting concentration is desirable, the XTEN selected for inclusion will typically have at least about 144, or about 288, or about 432, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues, provided that the unique XTEN is built into CFXTEN. In a further embodiment of the present invention, the CFXTEN comprises a first XTEN of the above length, and at least a second XTEN of about 36, or about 42, or about 72, or about 144, or about 288, or about 576, or about 864, or about 875 , or about 912, or about 923, or about 1000 or more amino acid residues. The location of XTEN within the fusion protein may contain one, two, three, four, five or more locations selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9. In a specific embodiment of the present invention, the construction method comprises inserting XTEN into FVIII at at least one site selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbering relative to mature native human FVIII).

- 134 041874

In additional aspects, the present invention provides methods for preparing a CFXTEN composition to improve ease of manufacture that result in increased stability, increased water solubility, and/or formulation convenience, compared to natural FVIII. In a separate embodiment, the present invention comprises a method for increasing the water solubility of FVIII, which includes the step of linking FVIII with at least about 80%, or about 90%, or about 95% identity with the sequence from table. 1 to one or more XTEN in one, two, three, four, five or more locations selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9, while XTEN is a sequence with at least about 80%, or about 90%, or about 95% sequence identity, compared with a sequence from any of the tables. 3, 4 and 13-17, so that a higher concentration in the soluble form of the produced CFXTEN, under physiological conditions, can be achieved compared to FVIII in the unfused state. In a specific embodiment, the CFXTEN contains FVIII associated with two, three, four, or five XTENs that have at least about 24, or about 36, or about 48, or about 60, or about 72, or about 84, or about 96 , or about 144, or about 288 amino acid residues, introduced within the sites selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or FIG. 8-9, in which the solubility of the fusion protein under physiological conditions is at least three times higher than the corresponding FVIII not associated with XTEN, or, in addition, at least four times, or five times, or six times, or seven times, or eight times, or nine times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 50 times, or at least least 60 times or more FVIII not associated with XTEN. Factors contributing to the property of XTEN to confer increased water solubility of CF when incorporated into a fusion protein include high solubility of the XTEN fusion partner and low degree of self-aggregation between XTEN molecules in solution, as well as broadening the hydrophilicity of the outer loops of FVIII into which XTEN is introduced. In certain embodiments of the present invention, the method results in a CFXTEN fusion protein wherein the water solubility is at least about 20% greater, or at least about 30% greater, or at least about 50% greater, or at least about 75% more, or at least about 90% more, or at least about 100% more, or at least about 150% more, or at least about 200% more, or at least about 400% more, or at least about 600% more, or at least about 800% more, or at least about 1000% more, or at least about 2000% more under physiological conditions compared to non-fused FVIII. In a separate embodiment of the present invention, the CFXTEN fusion protein XTEN is a sequence with at least about 80%, or about 90%, or about 95% sequence identity, as compared to a sequence from any of Table. 3, 4 and 13-17. In a further embodiment, the present invention comprises a method for increasing the shelf life of FVIII, which comprises the step of linking FVIII to one or more XTENs at one or more sites selected from Table 1. 5, tab. 6, tab. 7, tab. 8 and tab. 9, while the shelf life of the resulting CFXTEN is extended compared to the FVIII in the unfilled state. As used herein, shelf life refers to the period of time for which the procoagulant activity of FVIII or CFXTEN that is in solution, lyophilized, or in any other storage formulation remains stable without undue loss of activity or activity remains within release specifications. established for the release of a pharmaceutical composition. FVIII that degrades or aggregates generally has reduced functional activity or reduced bioavailability compared to that which remains in solution. Factors that drive the ability of the method to increase the shelf life of FVIII when incorporated into a fusion protein include increased water solubility, reduced self-aggregation in solution, and increased heat stability of the XTEN fusion partner. In particular, the low tendency of XTEN to aggregate facilitates pharmaceutical formulation methods that contain higher concentrations of CF drug, and the heat stability of XTEN contributes to the property of the CFXTEN fusion proteins to remain soluble and functional for an extended period of time. The method results in extended or extended shelf life CFXTEN fusion proteins that exhibit higher activity relative to the FVIII standard subjected to the same storage and handling conditions. The standard may be a non-fused bulk FVIII or a commercially available pharmaceutical composition of FVIII. In a particular embodiment of the present invention, the method includes the step of formulating isolated CFXTEN with one or more pharmaceutically acceptable excipients that enhance the ability of XTEN to maintain its unstructured conformation and CFXTEN to remain soluble in the composition for a time that is longer than the corresponding non-fused FVIII. . In a separate embodiment of the present invention, the method includes binding FVIII selected from table. 1 with one or more XTEN selected from any of the table. 3, 4 and 13-17, entered in one or more sites selected from the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 and admixture with at least one pharmaceutically acceptable excipient to form a pharmaceutical

- 135 041874 compositions that retain more than about 100% procoagulant activity, or more than about 105, 110, 120, 130, 150 or 200% procoagulant activity of the FVIII standard, which is subjected to the same storage and handling condition, when compared at a time point of at least at least 90 days, or at least 6 months, or at least 12 months. Shelf life can also be evaluated in terms of functional activity remaining after storage, normalized to functional activity at the start of storage. In certain embodiments of the present invention, the CFXTEN pharmaceutical compositions of the present invention retain more than about 50% procoagulant activity, or about 60%, 70%, 80%, or more than 90% of the FVIII standard procoagulant activity when exposed under the same conditions for the same period up to 2 weeks, or 4 weeks, or 6 weeks or longer under various temperature conditions. In a particular embodiment of the present invention, the CFXTEN pharmaceutical composition retains at least about 50%, or about 60%, or at least about 70%, or at least about 80%, and more preferably at least about 90% or more than its initial activity in solution when heated to 80°C for 10 min. In a further embodiment of the present invention, the CFXTEN pharmaceutical composition retains at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or alternatively at least about 90% or more of its initial activity in solution when heated or maintained at 37°C for about 7 days. In a further embodiment of the present invention, the pharmaceutical composition of CFXTEN retains at least about 80% or more of its functional activity after exposure to a temperature of from about 30° C. to about 70° C. for a period of time from about 1 to about 18 hours. In previous embodiments of the present of the invention described in this paragraph above, the retained potency of CFXTEN pharmaceutical compositions at a given point in time is at least about two times, or at least about three times, or at least about four times, or at least about five times, or at least about six times higher than the corresponding pharmaceutical composition, which contains FVIII, not associated with XTEN.

Nucleotide sequences of the present invention

The present invention provides isolated polynucleotides that encode CFXTEN fusion fusion proteins and sequences complementary to polynucleotide molecules encoding CFXTEN fusion fusion proteins that contain homologous variants thereof. In additional aspects, the present invention encompasses methods for producing polynucleotides encoding CFXTEN fusion proteins and sequences complementary to polynucleotide molecules encoding a CFXTEN fusion protein that contains homologous variants thereof. In general, and as illustrated in FIG. 11-13, methods for obtaining a polynucleotide sequence encoding a CFXTEN fusion protein and expressing the resulting gene product include generating nucleic acids encoding FVIII and XTEN, ligating in-frame components, incorporating the coding gene into an expression vector corresponding to a host cell, transforming the corresponding host cell with expression vector and culturing the host cell under conditions that cause or allow the fusion protein to be expressed in the transformed host cell, thereby producing a biologically active CFXTEN polypeptide that is recovered as an isolated fusion protein using standard protein purification methods known in the art. . Standard recombinant molecular biology techniques are used to generate the polynucleotides and expression vectors of the present invention.

In accordance with the present invention, nucleotide sequences that encode CFXTEN (or its complement) are used to generate recombinant DNA molecules that direct the expression of CFXTEN fusion proteins in appropriate host cells. For the purposes of the present invention, a nucleotide encoding a signal sequence corresponding to natural human FVIII (encodes MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) is added to any of the coding constructs described herein to aid in the expression and secretion of the CFXTEN fusion protein. In a particular embodiment of the present invention, the additional nucleotide is

ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613) _{Or its} complementary sequence.

Several cloning strategies are suitable for carrying out the present invention, many of which are used to generate a construct that contains a gene encoding a CFXTEN composition fusion protein of the present invention, or its complement. In certain embodiments of the present invention, a cloning strategy is used to create a gene that encodes a monomeric CFXTEN containing at least a first FVIII and at least a first XTEN polypeptide, or a complement thereof. In a separate embodiment of the above, the gene contains a sequence encoding FVIII, or a sequence variant. In further embodiments of the present invention, a cloning strategy using

- 136 041874 to create a gene that encodes a monomeric CFXTEN containing nucleic acids that encodes at least the first FVIII molecule or its complement, and the first and at least the second XTEN, or their complement, which is used to transform the host cell to expression of the fusion protein of the CFXTEN compositions. In the previous embodiments of the present invention described above in this paragraph, the genes may further comprise nucleic acids encoding spacer sequences that also encode the cleavage sequence(s).

In designing the desired XTEN sequences, it has been found that the non-repeating nature of the XTEN inventive compositions is achieved despite the use of a molecular approach to building blocks in the design of XTEN coding sequences. This was achieved by using a library of polynucleotides encoding the peptide motif sequences described above, which are then ligated and/or multimerized to generate genes encoding XTEN sequences (see FIGS. 11 and 12 and Examples). Thus, if the XTEN of an expressed fusion protein can consist of several units of only four different sequence motifs, because the motifs themselves are composed of non-repeating amino acid sequences, in general, the XTEN sequence appears to be non-repeating. Accordingly, in a particular embodiment of the present invention, the XTEN-encoding polynucleotides comprise a plurality of polynucleotides that encode non-repetitive sequences, or motifs operably linked into a frame, in which the resulting expressed XTEN amino acid sequences are non-repetitive.

In a separate approach, a construct is first generated that contains the DNA sequence corresponding to the CFXTEN fusion protein. DNA encoding FVIII compositions obtained synthetically, from a commercial source, or from a cDNA library obtained using standard methods from tissue or isolated cells, is believed to retain the FVIII mRNA and to express it at a detectable level. If necessary, a coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, et al., supra, to detect precursors and handle mRNA intermediates that may not be reverse transcribed into cDNA. In this case, a polymerase chain reaction (PCR) technique can be used to amplify the target DNA or RNA coding sequence to obtain sufficient material to generate CFXTEN constructs that contain the FVIII gene. Analyzes can be performed to confirm that the hybridized full length genes are the desired FVIII gene(s). Using these standard methods, DNA can be readily obtained from a cDNA library obtained from such sources. The FVIII encoding gene(s) can also be generated using standard synthetic methods known in the art (e.g., automatic nucleic acid synthesis using, for example, one of the methods described in Engels et al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA sequences obtained from public databases, patents or literary sources. Such procedures are well known in the art and are well described in the scientific and patent literature. For example, sequences can be obtained from Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or Model Protein GenBank Accession Numbers (e.g., Locus ID, NP_XXXXX and XP_XXXXX) available from the National Center for Biotechnology website. Information (NCBI), available online at ncbi.nlm.nih.gov, which corresponds to entries in the CAS Registry or the GenBank database that contain the amino acid sequence of a protein of interest, or fragment, or protein variant. In a separate embodiment of the present invention, FVIII encodes a gene encoding a protein sequence from table. 1, or a fragment or variant thereof.

A gene or polynucleotide encoding a portion of the subject's FVIIIa CFXTEN protein, in the case of an expressed fusion protein that contains a unique FVIII, is then cloned into a construct that is a plasmid or other vector under the control of appropriate transcriptional and translational sequences in the case of a high level of protein expression in a biological system . At a later stage, the second gene or polynucleotide encoding XTEN is genetically fused to the nucleotides encoding the N- and/or C-terminus of the FVIII gene by cloning it in a construct adjacent to and in frame with the FVIII encoding gene(s). This second step occurs via a ligation or multimerization step. In the previous embodiments of the present invention described above in this paragraph, it should be understood that the generated gene constructs can alternatively be supplemented with appropriate genes that encode appropriate fusion proteins.

The gene encoding XTEN can be created in one or more steps, either entirely synthetically or by synthesis in combination with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and PCR with overlapping primers, which contain the methods more fully described in the examples. The methods disclosed herein can be used, for example, to ligate short polynucleotide sequences encoding

- 137 041874 XTEN genes into longer XTEN genes of the required length and sequence. In a specific embodiment of the present invention, the method ligates two or more oligonucleotides with an optimized codon encoding an XTEN motif or sequence segment of about 9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to 42 amino acids, or about 42 up to about 144 amino acids, or about 144 to about 288 amino acids, or from 288 to about 864 amino acids or more, or any combination of the above motif ranges or segment lengths.

In addition, the disclosed method is used to multimerize XTEN coding sequences into longer sequences of the required length; for example, a gene encoding 36 amino acids XTEN can be dimerized into a gene encoding 72 amino acids, then 144, then 288, and so on. Even with multimerization, XTEN polypeptides can be designed such that the XTEN-coding gene has a sparse or almost non-repetitive codon pattern chosen for the shortest unit motif used, which can reduce recombination and increase the stability of the encoded gene in the transformed host.

Genes encoding XTEN with non-repetitive sequences are assembled from oligonucleotides using standard gene synthesis techniques. The construction of the gene can be performed using algorithms that optimize using the codon and amino acid composition. In a separate method of the present invention, a library of relatively short XTEN-encoding polynucleotide constructs is created and then assembled as described above. The resulting genes are then assembled with genes encoding FVIII or FVIII regions as illustrated in FIG. 11 and 12, and the resulting genes are used to transform the host cell and obtain and restore CFXTEN to evaluate its properties, as described in this document.

In additional aspects, the present invention provides isolated nucleotides that comprise a polynucleotide sequence encoding embodiments of the CFXTEN fusion protein described herein. In a particular embodiment of the present invention, the isolated nucleotide comprises a polynucleotide sequence selected from (a) a sequence that has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity, compared to a sequence of comparable length selected from Table. 21 at optimal alignment, or (b) the complement of polynucleotide (a). In a further embodiment of the present invention, the isolated nucleotide comprises the sequence ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO; 1613) linked to the 5' end of the nucleotide (a) or a sequence complement linked to the 3' end (b).

polynucleotide libraries.

In additional aspects, the present invention provides libraries of polynucleotides that encode XTEN sequences used to assemble genes that encode XTEN of the required length and sequence. In certain embodiments of the present invention, the XTEN coding library constructs contain polynucleotides that encode polypeptide segments of a specific length. As a first step, a library of oligonucleotides can be assembled that encode motifs of 9-14 amino acid residues. In a preferred embodiment of the present invention, libraries of oligonucleotides are assembled that encode 12 amino acid motifs.

Segments of the XTEN coding sequence may be dimerized or multimerized into longer coding sequences, as illustrated schematically in FIG. 13. Dimerization or multimerization can be accomplished by ligation, primer overlap PCR, assembly PCR, or similar cloning techniques known in the art. This process can be repeated several times until the resulting XTEN coding sequences reach sequence organization and length, providing XTEN coding genes. As will be appreciated, a library of polynucleotides that code for, for example, 12 amino acid motifs can be dimerized and/or ligated into a library of polynucleotides that code for 36 amino acids. Libraries encode motifs of various lengths; for example, 9-14 amino acid motifs lead to libraries encoding 27 to 42 amino acids as contemplated by the present invention. In turn, a library of polynucleotides that encode 27 to 42 amino acids, and preferably 36 amino acids (as described in the examples) can be sequentially dimerized into a library that contains sequentially longer lengths of polynucleotides that encode XTEN sequences of the required length for inclusion in the gene. encoding the CFXTEN fusion protein as disclosed herein.

A more efficient way to optimize the DNA sequence encoding XTEN is based on combinatorial libraries. The gene encoding XTEN can be constructed and synthesized in a segment such that multiple codon variants are obtained for each segment. These segments can be arbitrarily combined into libraries of genes, such that each member of the library encodes the same amino acid sequences, but the members of the library contain a large number of codon variants. Such libraries can be screened for genes that result in high expression and/or low abundance of processing products. The method for assembling a hybrid gene is illustrated in FIG. 18. The genes in FIG. 18 are assembled from 6 main fragments and each fragment is available in 4 different codon variants. This leads to the theoretical diversity of 4096.

In certain embodiments of the present invention, libraries are assembled from polynucleotides that encode amino acids restricted to a specific XTEN family sequence; for example, the sequence AD, AE, AF, AG, AM or AQ from the table. 4. In additional embodiments of the present invention, the libraries contain sequences that encode two or more sequence family motifs from Table. 3. names and sequences of representative, non-limiting polynucleotide sequences from libraries that encode 36-mers are presented in table. 13-17, and the methods used to create them are described in more detail in the respective examples. In additional embodiments of the present invention, libraries that encode XTEN are constructed from segments of polynucleotide codons linked in a randomized sequence that encodes amino acids, with at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 97%, or at least about 98 %, or at least about 99% of the codons are selected from the group consisting of codons for the amino acids glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) . The libraries can in turn be used for serial dimerization or ligation, in order to reach a library of polynucleotide sequences that encode for XTEN sequences, for example from 42, 48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well as intermediate lengths in which the encoded XTEN may have one or more of the properties disclosed herein when expressed as a component of the CFXTEN fusion protein. In some cases, library polynucleotide sequences may also contain additional bases used as sequencing islands, described more fully below.

Fig. 14 is a schematic block diagram of representative, non-limiting assembly steps for an XTEN polynucleotide construct and a CFXTEN polynucleotide construct in embodiments of the present invention. Individual oligonucleotides 501 are annealed into sequence motifs 502, such as a 12 amino acid motif (12-mer), which are ligated to additional sequence motifs from the library to create a pool that spans the desired length of XTEN 504, and also ligated at a lower concentration of oligo-containing BbsI and restriction sites KpnI 503. The resulting pool of ligation products is gel-purified and the band is cut to the desired XTEN length, resulting in an isolated XTEN gene with a stopper sequence of 505. The XTEN gene is cloned into a spacer vector. In this case, the vector encodes an additional CBD 506 sequence and a GFP 508 gene. Cleavage is then performed with BbsI/HindIII to remove 507 and 508 and place a stop codon. The resulting product is then cloned into a BsaI/HindIII cleavable vector that contains the gene encoding FVIII, resulting in the 500 gene encoding the FVIII-XTEN fusion protein.

You can clone a library of XTEN-coding genes into one or more expression vectors known in the art. To facilitate the identification of well-expressed members of the library, the library can be constructed as a reporter protein fusion. Non-limiting examples of suitable reporter genes are green fluorescent protein, luciferase, alkaline phosphatase, and beta-galactosidase. By screening, XTEN short sequences can be identified that can be expressed at high concentration in a preferred host organism. Subsequently, a library of random XTEN dimers can be generated and the high expression test repeated. Subsequently, the resulting constructs can be tested for a number of properties such as expression level, protease stability, or serum binding.

In one aspect, the present invention provides polynucleotide sequences encoding components of a fusion protein, with the creation of the sequence undergoing codon optimization. Of particular interest is codon optimization to increase the expression of polypeptide compositions and to improve the genetic stability of the coding gene in source organisms. For example, codon optimization is of particular importance for XTEN sequences that are rich in glycine or that have highly repetitive amino acid sequences. Codon optimization is performed using computer programs (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53), some of which minimize the ribosomal gap (Coda Genomics Inc.). In one embodiment of the present invention, it is possible to perform codon optimization by constructing a codon library, provided that all members of the library encode the same amino acid sequence, but where the codon usage varies. Such libraries can be screened for high expression

- 139 041874 and genetically stable members, which are particularly suitable for large-scale production of XTEN-containing products. There are a number of properties to consider when creating XTEN sequences. You can minimize the repetition in coding DNA sequences. In addition, the use of codons that are rarely used by the source organism (eg, the AGG and AGA arginine codons and the leucine codon in E. coli) can be avoided or minimized. In the case of E. coli, two glycine codons, GGA and GGG, are rarely used in highly expressed proteins. Thus, codon optimization of a gene encoding XTEN sequences may be highly desirable. DNA sequences that have a high level of glycine, resulting in a high GC content, which can lead to instability or low levels of expression. Thus, if possible, it is preferable to choose codons such that the GC content of the XTEN coding sequence is appropriate for the production organism to be used to make the XTEN.

In a particular embodiment of the present invention, polynucleotide libraries are constructed using the disclosed methods, where all members of the library encode the same amino acid sequence, but where the codon usage for the corresponding amino acids in the sequence is changed or optimized for the intended host cell. Such libraries can be screened for high expression and genetically stable members, which are particularly suitable for large scale production of XTEN-containing products. In a separate embodiment of the present invention, the libraries are optimized for expression in a eukaryotic host cell.

If desired, the clones in the library can be ordered in order to eliminate isolates that contain unwanted sequences. The original XTEN short sequence library allows for some change in the amino acid sequence. For example, individual codons can be randomized such that the number of hydrophilic amino acids can be observed at a particular position. During iterative multimerization, it is possible to screen the obtained library members for other characteristics such as solubility or protease resistance in addition to the high level expression test.

Once an XTEN-coding gene of the required length and properties has been selected, it is genetically fused at the desired location on the nucleotides encoding the FVIII(s) gene(s) by cloning it into a construct adjacent to and in frame with the FVIII-encoding gene, or alternatively between nucleic acids encoding adjacent FVIII domains, or alternatively within a sequence encoding a given FVIII domain, or alternatively in-frame with nucleic acids encoding a spacer/cleavage sequence associated with a terminal XTEN. The present invention provides various permutations of the above, depending on the CFXTEN to be encoded. For example, a gene encoding a CFXTEN fusion protein that contains a FVIII and two XTENs such as those depicted in formula VI as shown above, the gene will have FVIII encoding polynucleotides encoding two XTENs that may be identical or different in composition and sequence length. . In a separate non-limiting embodiment of the above, FVIII polynucleotides can encode factor VIII and polynucleotides encoding the C-terminus, an XTEN can encode a 288 amino acid XTEN, and polynucleotides encoding an internal XTEN adjacent to the C-terminus of the A2 domain can encode a 144 amino acid XTEN. The step of cloning FVIII genes into an XTEN construct can occur via a ligation or multimerization step, as shown in FIG. 14. The constructs encoding the CFXTEN fusion proteins can be constructed in various conformations of the XTEN, CF components and spacer sequences, such as the conformations of formulas I-VIII. In a particular embodiment of the present invention, the construct comprises polynucleotide sequences that are complementary to or that encode the monomeric component polypeptide in the following order (5' to 3') of FVIII internal to the B domain of XTEN and C-terminal of XTEN. In a further embodiment of the present invention, the construct comprises polynucleotide sequences complementary to those, or those encoding the monomeric polypeptide of the components in the following order (5' to 3') FVIIII, a C-terminal spacer region, and XTEN. Spacer polynucleotides may optionally contain sequences encoding cleaved sequences. As will be apparent to those skilled in the art, various permutations of FVIII domains and administered XTENs are possible.

Homology, sequence similarity, or sequence identity of a nucleotide or amino acid sequences can also be determined conventionally using known software or computer programs such as BestFit or Gap pair comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711 ). BestFit uses the local homology algorithm from Smith and Waterman (Advances in Applied Mathematics. 1981. 2: 482-489) to find the best segment of identity or similarity between two sequences. Gap provides total sequence alignments with all other similar sequence using the method of Needleman and Wunsch, (Journal of Molecular Biology. 1970. 48:443-453). When used, primary structure benchmarking programs

- 140 041874 sequences, such as BestFit, to determine the degree of sequence homology, similarity or identity, can be used by default, or to optimize the results of identity, similarity or homologues, an appropriate scoring matrix can be selected.

Nucleotide sequences that are complementary are those that are capable of base pairing according to standard Watson-Crick complementarity rules. As used herein, the term complementary sequences means nucleotide sequences that are substantially complementary, that can be assessed using the same nucleotide comparison set forth above, or determined to hybridize with polynucleotides that encode CFXTEN sequences under stringent conditions, such as described in this document.

The resulting polynucleotides encoding the CFXTEN fusion proteins can then be individually cloned into an expression vector. The nucleotide sequence is introduced into the vector using various procedures. In general, DNA is introduced into the appropriate restriction endonuclease site(s) using methods known in the art. Vector components typically contain, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcriptional stop codon. Construction of suitable vectors that contain one or more of these components uses standard ligation techniques that are known to those skilled in the art. Such methods are well known in the art, well described in the scientific and patent literature.

Various vectors are public. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage, which can be easily subjected to recombinant DNA techniques, and the choice of vector often depends on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, ie. a vector that exists as an extrachromosomal substance whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome of the host cell and replicates along with the chromosome(s) into which it has been integrated. The representative plasmids illustrated in FIG. 17 are shown with coding regions for various FVIII conformations and XTEN components.

The present invention provides for the use of plasmid vectors that contain replication and control sequences that are compatible with, and recognized by, host cells and are operably linked to the CFXTEN gene for controlled expression of the CFXTEN fusion protein. The vector usually carries a site of replication as well as sequences encoding proteins that are capable of conferring phenotypic selection in transformed cells. Such vector sequences are well known for various bacteria, yeasts and viruses. Applicable expression vectors that can be used contain, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. The expression vector corresponds to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of a DNA encoding a fusion protein in a suitable host. The requirements are that the vectors are reproducible and viable in the chosen host cell. If necessary, vectors with low or high copy number can be used.

Other suitable vectors include, but are not limited to, SV40 and pcDNA derivatives, and known bacterial plasmids such as col EI, pCRl, pBR322, pMal-C2, pET, pGEX, as described by Smith, et al., Gene 57:31- 40 (1988), pMB9 and their derivatives, plasmids such as RP4, phage DNA such as numerous phage I derivatives such as NM989, as well as other phage DNA such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as the 2-micron plasmid or derivatives of the 2m plasmid, as well as centromeric and integrative bifunctional yeast vectors; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids that have been modified to use phage DNA or expression control sequences; and the like. Yeast expression systems that can also be used in the present invention include, but are not limited to, pYES2 unfused vector (Invitrogen), pYESHisA, B, C fusion (Invitrogen), pRS vectors, and the like.

The vector control sequences contain a promoter to effect transcription, an additional operator sequence to direct such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control the termination of transcription and translation. A promoter can be any DNA sequence that exemplifies transcriptional activity in the selected host cell and can be derived from protein-coding genes either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of DNA encoding a variant FVIII polypeptide in mammalian cells are the SV40 promoter (Subramani et al., Mol.

- 141 041874

cell. Biol. 1 (1981), 854-864), MT-1 promoter (metalothionein gene) (Palmiter et al., Science 222 (1983), 809-814), CMV promoter (Boshart et al., Cell 41:521-530, 1985) or the major adenovirus 2 late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). The vector can also carry sequences such as UCOE (Universal Chromatin Opening Elements). Examples of suitable promoters for use in filamentous fungal host cells are, for example, the ADH3 promoter or the tpiA promoter. Examples of other promoters used are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger, A. niger or A. awamoriglucoamylase (gluA) acid-fast α-amylase, lipase Rhizomucor miehei, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. TAKA-amylase and gluA promoters are preferred.

Promoters suitable for use in expression vectors with prokaryotic hosts include β-lactamase and lactose promotion systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, tryptophan (trp) promotion system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36776] and hybrid promoters such as the fusion promoter [deBoer et al., Proc. Natl. Acad. sci. USA, 80:21-25 (1983)], all are operably linked to DNA encoding CFXTEN polypeptides. Promoters for use in bacterial systems may also contain a Shine-Dalgarno (S.D.) sequence operably linked to DNA encoding CFXTEN polypeptides.

The present invention contemplates the use of other expression systems which include, for example, a baculovirus expression system with both non-fused transfer vectors such as, but not limited to, pVL941 Summers, et al., Virology 84:390-402 (1978)), pVL1393 ( Invitrogen), pVL1392 (Summers, et al., Virology 84:390-402 (1978) and Invitrogen) and pBlueBacIII (Invitrogen), and fusion transfer vectors such as, but not limited to, pAc700 (Summers, et al. al., Virology 84:390-402 (1978)), pAc701 and pAc70-2 (such as pAc700, with different reading frames), pAc360 Invitrogen) and pBlueBacHisA, B, C (Invitrogen).

Examples of suitable promoters for directing the transcription of DNA encoding a FVIII polypeptide variant in mammalian cells are the CMV promoter (Boshart et al., Cell 41:521-530, 1985), the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol 2:1304-1319, 1982). The vector may also carry sequences such as UCOE (Universal Chromatin Opening Elements).

The DNA sequences encoding CFXTEN may further, if desired, be operably linked to an appropriate terminator such as the hGH terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or TPI1 terminators (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., EMBO J. 4, 1985, pp. 2093-2099). Expression vectors may also contain a set of RNA splice sites downstream of the promoter and downstream of the insertion site of the CFXTEN sequence, which contains adenovirus-derived splicing sites. In addition, contained in the expression vectors is a polyadenylation signal located in the forward direction from the insertion site. Particularly preferred polyadenylation signals comprise an early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), a polyadenylation signal from the Elb 5 region of adenovirus, the hGH terminator (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981). Expression vectors may also contain a non-coding viral leader, such as the three-component adenovirus 2 leader, located between the promoter and RNA splicing sites; and enhancer sequences such as the SV40 enhancer.

To direct the CFXTEN of the present invention into the secretory pathway of host cells, a secretory signal sequence (also known as a leader sequence, preprosequence, or presequence) may be contained in a recombinant vector. The secretory signal sequence is operably linked to DNA sequences encoding CFXTEN, typically located 5' to the DNA sequence encoding the CFXTEN fusion protein. The secretory signal sequence may generally be associated with the naturally occurring FVIII protein or may form a gene encoding another secreted protein. Non-limiting examples include OmpA, PhoA and DsbA when expressed in E. coli, ppLalpha, DEX4, invertase signal sequence, acid phosphatase signal sequence, CPY or INU1 in case of yeast expression, and IL2L, SV40, IgG kappa and IgG lambda in case of mammalian expression. . The signal sequences are typically proteolytically removed from the protein during the translocation and secretion process, generating a specific N-terminus. The methods are disclosed in Arnau, et al., Protein Expression and Purification 48: 1-13 (2006).

Procedures used to ligate a DNA sequence encoding CFXTEN, a promoter and optionally a terminator and/or a secretory signal sequence, respectively, and insert them into suitable vectors that contain the information necessary for the replica

- 142 041874 are well known to those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001). Thus, a DNA fusion molecule encoding the monomeric CFXTEN fusion protein is obtained within the construct. Optionally, this fused DNA molecule can be transported or cloned into another construct that is a more appropriate expression vector. At this point, a host cell capable of expressing the fused DNA molecule can be transformed with the fused DNA molecule.

Non-limiting examples of mammalian cell lines for use in the present invention are COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al. al., J. Gen. Virol. 36:59-72, 1977), BHK-570 (ATCC CRL 10314), CHO-K1 (ATCC CCL 61), CHO-S (Invitrogen 11619-012), and 293- F (Invitrogen R790-7) and parental and derived cell lines known in the art useful for FVIII expression. The tk-ts 13 BHK cell line is also available from ATCC with accession number CRL 1632. In addition, a number of other cell lines can be used in the present invention, which contains Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (hepatoma rats; ATCC CRL 1548), TCMK (ATCC CCL 139), human lungs (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61), and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad Sci USA 77:4216-4220, 1980).

Examples of suitable yeast cells include Saccharomyces spp. or Schizosaccharomyces spp., in specific strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and obtaining heterologous polypeptides are described in, for example, US Pat. No. 4,599,311, US Pat. No. 4,931,373, US Pat. No. 4,870,008, US 5,037,743 and US Pat. Transformed cells are selected for phenotype as determined by a selectable marker, usually resistance or the ability of a drug to grow in the absence of a particular nutrient, such as leucine. A preferred vector for use in yeast is the POT1 vector disclosed in US Pat. No. 4,931,373. DNA sequences encoding CFXTEN can be predicted by a signal sequence and optionally a leader sequence, eg as described above. Further examples of suitable yeast cells are strains of Kluyveromyces such as K. lactis, Hansenula, e.g. H. polymorpha or Pichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp 3459-3465; US Pat. No. 4,882,279). Examples of other fungal cells are filamentous fungal cells, eg Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in specific strains of A. oryzae, A. nidulans or A. niger. Use of Aspergillus spp. for protein expression is described in eg EP 272 277, EP 238 023, EP 184 438. The transformation of F. oxysporum can be, for example, performed as described in Malardier et al., 1989, Gene 78: 147-156. The transformation of Trichoderma spp. can be carried out, for example, as described in EP 244 234.

Other suitable cells that may be used in the present invention include, but are not limited to, strains of prokaryotic host cells such as Escherichia coli (e.g. DH5-a strain), Bacillus subtilis, Salmonella typhimurium, or strains of the genus Pseudomonas, Streptomyces and Staphylococcus. Non-limiting examples of suitable prokaryotes include those of the genus: Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus; Listeria; oceanobacillus; paracoccus; Pseudomonas; Staphylococcus; Streptococcus; Streptomyces; Thermo-plasma; and Vibrio.

Methods for transfecting mammalian cells and expressing DNA sequences introduced into cells are described in, for example, Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumannet al., EMBO J. 1 (1982), 841-845.

Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb , Virology 52d:456-467, 1973), transfection of many commercially available substances such as FuGENEG Roche Diagnostics, Mannheim, Germany) or lipofectamine (Invitrogen) or by electroporation (Neumann et al., EMBO J. 1:841-845 , 1982). To identify and select cells that express exogenous DNA, a gene that confers a selectable phenotype (selectable marker) is typically introduced into cells along with the gene or cDNA of interest. Preferred selectable markers contain genes that confer resistance to drugs such as neomycin, hygromycin, puromycin, zeocin and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplifiable selectable marker is the dihydrofolate reductase (DHFR) sequence. Additional examples of selectable markers are well known to those skilled in the art and include reporters such as improved green fluorescent.

- 143 041874 protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT). Selectable markers are provided by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., incorporated herein by reference). One skilled in the art will readily be able to select suitable selectable markers. Any known selectable marker can be used as long as it is capable of being expressed simultaneously with the nucleic acid encoding the gene product.

The selectable markers can be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they can be introduced on the same plasmid. If, with the same plasmid, the selectable marker and the gene of interest can be under the control of different promoters or the same promoter, the latter mechanism receives a bicistronic message. Designs of this type are known in the art (eg, Levinson and Simonsen, US Pat. No. 4,713,339). It may also be useful in adding additional DNA, known as carrier DNA, to a mixture that is injected into cells.

After the cells take up the DNA, they are grown in an appropriate growth medium, typically 1-2 days, to start expressing the gene of interest. As used herein, the term appropriate growth medium means a medium that contains nutrients and other components necessary for cell growth and expression of CFXTEN, of interest generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts. , phospholipids, proteins and growth factors. To obtain gamma carboxylated proteins, the medium contains vitamin K, preferably at a concentration of about 0.1 µg/ml to about 5 µg/ml. Drug selection is then used to select if cells grow that express the selectable marker in a stable form. In the case of cells that have been transfected with an amplifiable selectable marker, drug concentration can be increased in order to select for increased copy number of cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of the FVIII polypeptide variant of interest.

The transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions allowing expression of the CFXTEN polypeptide, after which the resulting peptide can be recovered from culture as an isolated fusion protein. The medium used for culturing the cells may be any conventional medium suitable for growing host cells, such as minimal or complex medium containing appropriate additives. Suitable media are available from commercial suppliers or can be obtained from published recipes (eg, in the American Type Culture Collection catalogues). Culture conditions such as temperature, pH, and the like are those previously used for the host cell selected for expression and will be apparent to those skilled in the art.

Gene expression is measured directly in the sample, for example, using conventional Southern blot, Northern blot to quantify mRNA transcription [Thomas, Proc. Natl. Acad. sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis) or in situ hybridization using an appropriately labeled probe, based on the sequence provided herein. In addition, antibodies can be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid DNA-RNA duplexes or DNA protein duplexes. Antibodies, in turn, can be labeled and analyzed if the duplex is bound to the surface such that when the duplex is formed on the surface, the presence of the antibody bound to the duplex can be detected.

Gene expression, alternatively, can be measured using immunological fluorescent methods such as immunohistochemical staining of cell or tissue sections and analysis of cell culture or body fluids, or detection of selectable markers to quantify gene product expression directly. Antibodies useful for immunohistochemical staining and/or analysis of fluid samples can be either monoclonal or polyclonal, and can be obtained from any mammal. For convenience, antibodies can be made to a natural FVIII polypeptide sequence, or to a synthetic peptide based on the DNA sequences provided herein, or to an exogenous sequence fused to FVIII and encoding a specific antibody epitope. Examples of selectable markers are well known to those skilled in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal), or chloramphenicol acetyltransferase (CAT). Expressed CFXTEN polypeptide product(s) can be purified by methods known in the art or by methods disclosed herein. Methods such as size exclusion chromatography, affinity purification (for example, using an anti-FVIII antibody column), salt fractionation, and

- 144 041874 no-exchange chromatography, gel permeation chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography and gel electrophoresis; each subject to the recovery and purification of the hybrid protein is obtained using the appropriate host cells. Additional purification may be performed using conventional chemical purification methods such as high performance liquid chromatography. Some expressed CFXTEN may require refolding during isolation and purification. Purification methods are described in Robert K. Scopes, Protein Purification: Principles and Practice, Charles R. Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra. Separations by multi-step purification are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and below, et al., J. Chromatogr. A. 679:67-83 (1994). For therapeutic purposes, these CFXTEN fusion proteins of the present invention are desirable to be substantially pure. Thus, in a preferred embodiment of the present invention, the CFXTEN of the present invention is purified to at least about 90 to 95% homogeneity, preferably to at least about 98% homogeneity. Purity can be assessed using, for example, gel electrophoresis, HPLC, and amino-terminal protein sequencing.

VIII) Pharmaceutical compositions.

The present invention provides pharmaceutical compositions that contain CFXTEN. In a specific embodiment of the present invention, the pharmaceutical composition comprises the CFXTEN fusion protein disclosed herein admixed with at least one pharmaceutically acceptable carrier. The CFXTEN polypeptides of the present invention may be formulated according to known methods to obtain pharmaceutically useful compositions, wherein the polypeptide is combined in admixture with a pharmaceutically acceptable carrier base such as aqueous solutions, buffers, diluents, and/or pharmaceutically acceptable suspensions, emulsions, stabilizers, or excipients. substances. Examples of anhydrous solvents include propylethylene glycol, polyethylene glycol and vegetable oils. Pharmaceutical formulations are prepared for storage by mixing the active ingredient CFXTEN which is of the desired purity with additional physiologically acceptable carriers, excipients (eg sodium chloride, calcium salt, sucrose or polysorbate) or stabilizers (eg sucrose, trehalose, raffinose, arginine, calcium salt, glycine or histidine) as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), as lyophilized compositions or aqueous solutions.

The pharmaceutical composition may be provided as a lyophilized powder for reconstitution prior to administration. In a further embodiment of the present invention, the pharmaceutical composition may be provided in liquid form in a vial, the content of which may be administered directly to a patient. In addition, the composition is provided as a liquid in a pre-filled syringe for administering the composition. In a further embodiment of the present invention, the composition is provided as a liquid in a pre-filled vial that can be built into a pump.

Pharmaceutical compositions may be administered by any suitable route or route, including subcutaneously, subcutaneously via infusion pump, intramuscularly and intravenously. It should be borne in mind that the preferred route will vary depending on the disease and age of the patient, and the severity of the disease being treated.

In a separate embodiment of the present invention, the pharmaceutical composition of CFXTEN in liquid form or after dilution (if provided as a lyophilized powder) contains a clotting factor VIII with an activity of at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/mL, or at least 300 IU/mL, or at least 400 IU/mL, or at least 500 IU/mL activity, or at least 600 IU/mL activity, the composition of which is capable of increasing Factor VIII activity to at least 1.5% of normal blood plasma levels for at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours after administration of the Factor VIII pharmaceutical composition to a subject in need of standard prophylaxis. In a further embodiment of the present invention, the pharmaceutical composition of CFXTEN in liquid form or after dilution (if provided as a lyophilized powder) contains clotting factor VIII with an activity of at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least 300 IU/ml, or at least 400 IU/ml, or at least 500 IU/ml, or an activity of at least 600 IU/ml, the composition of which is able to increase the activity of factor VIII to at least 2.5% of normal blood plasma levels for at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours after administration to a subject in need of standard prophylaxis. It is specifically contemplated that the pharmaceutical compositions of the foregoing may be formulated to contain one or more excipients, buffers, or other ingredients known in the art to be compatible with intravenous, subcutaneous, or intramuscular administration. Thus, in the embodiments of the present invention described above in this paragraph, the pharmaceutical composition is administered subcutaneously, intramuscularly or intravenously.

The compositions of the present invention can be formulated using various excipients. Suitable excipients include microcrystalline cellulose (e.g. Avicel PH102, Avicel PH101), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonium ethyl methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M, Premium CR Methocel K100M, Methocel E5, Opadry ®), magnesium stearate, talc, triethyl citrate, aqueous dispersion of ethyl cellulose (Surelease®) and protamine sulfate. The delayed release agent may also contain a carrier which may contain, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delay agents. Pharmaceutically acceptable salts may also be used in these delayed release agents, for example mineral salts such as hydrochlorides, hydrobromides, phosphates or sulfates, as well as organic acid salts such as acetates, propionates, malonates or benzoates. The composition may also contain liquids such as water, saline, glycerol and ethanol, as well as substances such as wetting agents, emulsifying agents or pH buffering agents. Liposomes can also be used as a carrier.

In a further embodiment of the present invention, the compositions of the present invention are encapsulated in liposomes which have been shown to be useful in delivering effective active agents in a controlled manner over extended periods of time. Liposomes are closed bilayer membranes containing an entrapped volume of water. Liposomes can also be unilamellar vesicles having a single membrane bilayer, or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) lipid tails are oriented towards the center of the bilayer, while the hydrophilic (polar) heads are oriented towards the aqueous phase. In a separate embodiment of the present invention, the liposome can be applied with a conformationally labile water-soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, especially the liver and spleen. Suitable hydrophilic polymers in the case of surrounding liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinyl methyl ether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, and hydrophilic peptide sequences as described in US Pat. No. 6,316,024; 6126966; 6056973; 6043094, the contents of which are provided by reference in their entirety. Additional liposomal methods are described in US Pat. Nos. 6,759,057; 6406713; 6352716; 6316024; 6294191; 6126966; 6056973; 6043094; 5965156; 5916588; 5874104; 5215680; and 4,684,479, the contents of which are incorporated herein by reference. They describe liposomes and lipid-coated microvesicles and methods for their manufacture. Thus, one skilled in the art, in view of both the disclosures of the present invention and those disclosed in these other patents, can produce sustained release liposomes for the polypeptides of the present invention.

In the case of liquid formulations, it is a desirable property that the formulation be supplied in a form that can pass through 25, 28, 30, 31, 32 gauge needles for intravenous, intramuscular, intraarticular, or subcutaneous administration.

Syringe infusion pumps can also be used as delayed release agents. Such devices are described in US Pat. Nos. 4,976,696; 4933185; 5017378; 6309370; 6254573; 4435173; 4398908; 6572585; 5298022; 5176502; 5492534; 5318540; and 4988337, the contents of which are incorporated herein by reference. A person skilled in the art, in view of both disclosed in the present invention and disclosed in other patents, can receive a syringe infusion pump for delayed release of the compositions of the present invention.

IX) Pharmaceutical kits.

In additional aspects, the present invention provides a kit to facilitate the use of CFXTEN polypeptides. The kit contains the pharmaceutical composition provided herein, a container, and a label or package insert on, or associated with, the container. Suitable containers include, for example, vials, test tubes, syringes, etc., formed from various materials such as glass or plastic. The container contains the pharmaceutical composition in the form of a composition that is effective in treating a disease associated with FVIII and may have a sterile inlet (for example, the container may have an intravenous solution bottle or a vial that has a stopper pierceable by a hypodermic needle). The leaflet in the package insert may give the approved indications for the drug, instructions for reconstituting

- 146 041874 administration and / or administration of the drug in the case of use in the case of a specified symptom, appropriate dosage and safety information, and information identifying the lot number and expiration date of the medicinal product. In a further embodiment of the foregoing, the kit may comprise a second container which may carry an appropriate diluent for the pharmaceutical composition, the use of which will be provided to the user at the appropriate concentration to be delivered to the subject.

Examples

Example 1. Construction of segments of the XTEN_AD36 motif.

The following example describes the construction of a gene collection with an optimized codon encoding 36 amino acid motif sequences. As a first step, the spacer vector pCW0359 is constructed based on the pET vector and which contains the T7 promoter. pCW0359 encodes a cellulose binding domain (CBD) and a TEV protease recognition site followed by a spacer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites are introduced such that, upon cleavage, they generate compatible DNA sticky ends. The spacer sequence follows the truncated variant of the GFP gene and a histidine tag. The spacer sequence contains stop codons and thus E. coli cells carry out non-fluorescent colonies of the spacer form of the pCW0359 plasmid. The spacer vector pCW0359 was digested with BsaI and KpnI to remove the spacer segment, and the resulting vector fragment was isolated by agarose gel purification. The sequences are designated XTEN_AD36, reflecting the AD family of motifs. Their segments have the amino acid sequence [X] ₃ where X is a 12 amino acid peptide with the sequences: GESPGGSSGSES (SEQ ID NO: 19), GSEGSSGPGESS (SEQ ID NO: 20), GSSESGSSEGGP (SEQ ID NO: 21) or GSGGEPSESGSS (SEQ ID NO: 21). SEQ ID NO: 22). The insert is produced by renaturating the following pairs of phosphorylated synthetic oligonucleotide pairs:

ADI for: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC (SEQ ID

NO: 1619)

ADlrev: ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC (SEQ ID

NO: 1620)

AD2for: AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC (SEQ ID

NO: 1621)

AD2rev: ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT (SEQ ID

NO: 1622)

AD3for: AGGTTCYTCYGAAACGGGTTCTTCYGARGGYGGTCC (SEQ ID

NO: 1623)

AD3rev: ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA (SEQ ID

NO: 1624)

AD4for: AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC (SEQ ID

NO: 1625)

The phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the unphosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627) are also annealed. Renatured oligonucleotide pairs are ligated, resulting in a mixture of products of varying lengths that represent varying numbers of repetitions from the 12 amino acids ligated to the BbsI/KpnI segment. Products corresponding to a length of 36 amino acids are isolated from the mixture by preparative agarose gel electrophoresis and ligated into the pCW0359 spacer vector cleaved with BsaI/KpnI. Most of the clones in the resulting library, designated LCW0401, show green fluorescence upon induction, illustrating the fact that the XTEN_AD36 sequence was ligated in-frame to the GFP gene and that a large proportion of the XTEN_AD36 sequences had good expression levels. 96 isolates from the LCW0401 library were tested for high fluorescence by punching them onto an agar plate that contained IPTG. Similar isolates were evaluated by PCR and 48 isolates were identified which contained 36 amino acid segments as well as strong fluorescence. These isolates are sequenced and 39 clones are identified that contain the correct XTEN_AD36 segments. The names of the files of nucleotide and amino acid structures and sequences of these segments are given in Table. 13.

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Table 13

DNA and amino acid sequences of 36-mer AD motifs (SEQ ID NOS 203-278, respectively, in order of appearance)

File name LCW0401_00 1GFPN_A01.abl LCW0401_00 2GFPN_B01.abl LCW0401_00 3GFPN_C01.abl LCW0401_00 4GFPN_D01.abl Amino acid sequence GSGGEPSESGSSGES PGGSSGSESGESPGG SSGSES GSEGSSGPGESSGES PGGSSGSESGSSESG SSEGGP GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSES GSGGEPSESGSSGSS ESGSSEGGPGSGGEP SESGSS Nucleotide sequence GGTTCTGGTGGCGAACCGTCCGAGTCTGGT AGCTCAGGTGAATCTCCGGGTGGCTCTAGC GGTTCCGAGTCAGGTGAATCTCCTGGTGGT TCCAGCGGTTCCGAGTCA GGTAGCGAAGGTTCTTCTGGTCCTGGCGAG TCTTCAGGTGAATCTCCTGGTGGTTCCAGC GGTTCTGAATCAGGTTCCTCCGAAAGCGGT TCTTCCGAGGGCGGTCCA GGTTCCTCTGAAAGCGGTTCTTCCGAAGGT GGTCCAGGTTCCTCTGAAAGCGGTTTCTTCT GAGGGTGGTCCAGGTGAATCTCCGGGTGG CTCCAGCGGTTCCGAGTCA GGTTCCGGTGGCGAACCGTCTGAATCTGGT AGCTCAGGTTCTTCTGAAAGCGGTTCTTCC GAGGGTGGTCCAGGTTCTGGTGGTGAACC TTCCGAGTCTGGTAGCTCA LCW0401_00 7GFP- N_F01.abl GSSESGSSEGGPGSE GSSGPGESSGSEGSS GPGESS GGTTCTTCCGAAAGCGGTTCTTCTGAGGGT GGTCCAGGTAGCGAAGGTTCTTCCGGTCCA GGTGAGTCTTCAGGTAGCGAAGGTTCTTCT GGTCCTGGTGAATCTTCA LCW0401_00 8GFP- N_G01.abl GSSESGSSEGGPGES PGGSSGSESGSEGSS GPGESS GGTTCCTCTGAAAGCGGTTCTTCCGAGGGT GGTCCAGGTGAATCTCCAGGTGGTTCCAGC GGTTCTGAGTCAGGTAGCGAAGGTTCTTCT GGTCCAGGTGAATCCTCA LCW0401_01 2GFP- N_H01.abl GSGGEPSESGSSGSG GEPSESGSSGSEGSS GPGESS GGTTCTGGTGGTGAACCGTCTGAGTCTGGT AGCTCAGGTTCCGGTGGCGAACCATCCGA ATCTGGTAGCTCAGGTAGCGAAGGTTCTTC CGGTCCAGGTGAGTCTTCA LCW0401_01 5GFP- N_A02.abl GSSESGSSEGGPGSE GSSGPGESSGESPGG SSGSES GGTTCTTCCGAAAGCGGTTCTTCCGAAGGC GGTCCAGGTAGCGAAGGTTCTTCTGGTCCA GGCGAATCTTCAGGTGAATCTCCTGGTGGC TCCAGCGGTTCTGAGTCA LCW0401_01 6GFP- N_B02.abl GSSESGSSEGGPGSS ESGSSEGGPGSSESG SSEGGP GGTTCCTCCGAAAGCGGTTCTTCTGAGGGC GGTCCAGGTTCCTCCGAAAGCGGTTCTTCC GAGGGCGGTCCAGGTTCTTCTGAAAGCGG TTCTTCCGAGGGCGGTCCA LCW0401_02 OGFP- N_E02.abl GSGGEPSESGSSGSE GSSGPGESSGSSESG SSEGGP GGTTCCGGTGGCGAACCGTCCGAATCTGGT AGCTCAGGTAGCGAAGGTTCTTCTGGTCCA GGCGAATCTTCAGGTTCCTCTGAAAGCGGT TCTTCTGAGGGCGGTCCA LCW0401_02 2GFP- N_F02.abl GSGGEPSESGSSGSS ESGSSEGGPGSGGEP SESGSS GGTTCTGGTGGTGAACCGTCCGAATCTGGT AGCTCAGGTTCTTCCGAAAGCGGTTCTTCT GAAGGTGGTCCAGGTTCCGGTGGCGAACC TTCTGAATCTGGTAGCTCA LCW0401_02 4GFP- N_G02.abl GSGGEPSESGSSGSS ESGSSEGGPGESPGG SSGSES GGTTCTGGTGGCGAACCGTCCGAATCTGGT AGCTCAGGTTCCTCCGAAAGCGGTTTCTTCT GAAGGTGGTCCAGGTGAATCTCCAGGTGG TTCTAGCGGTTCTGAATCA LCW0401_02 6GFP- N_H02.abl GSGGEPSESGSSGES PGGSSGSESGSEGSS GPGESS GGTTCTGGTGGCGAACCGTCTGAGTCTGGT AGCTCAGGTGAATCTCCTGGTGGCTCCAGC GGTTCTGAATCAGGTAGCGAAGGTTTCTTCT GGTCCTGGTGAATCTTCA LCW0401_02 7GFP- N_A03.abl GSGGEPSESGSSGES PGGSSGSESGSGGEP SESGSS GGTTCCGGTGGCGAACCTTCCGAATCTGGT AGCTCAGGTGAATCTCCGGGTGGTTCTAGC GGTTCTGAGTCAGGTTCTGGTGGTGAACCT TCCGAGTCTGGTAGCTCA LCW0401_02 8GFP- N_B03.abl GSSESGSSEGGPGSS ESGSSEGGPGSSESG SSEGGP GGTTCCTCTGAAAGCGGTTCTTCTGAGGGC GGTCCAGGTTCTTCCGAAAGCGGTTCTTCC GAGGGCGGTCCAGGTTCTTCCGAAAGCGG TTCTTCTGAAGGCGGTCCA LCW0401_03 0 GFP- GESPGGSSGSESGSE GSSGPGESSGSEGSS GGTGAATCTCCGGGTGGCTCCAGCGGTTCT GAGTCAGGTAGCGAAGGTTCTTCCGGTCC

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N_C03.abl GPGESS GGGTGAGTCCTCAGGTAGCGAAGGTTCTTC CGGTCCTGGTGAGTCTTCA LCW0401_03 1GFP- N_D03.abl GSGGEPSESGSSGSG GEPSESGSSGSSESG SSEGGP GGTTCTGGTGGCGAACCTTCCGAATCTGGT AGCTCAGGTTCCGGTGGTGAACCTTCTGAA TCTGGTAGCTCAGGTTCTTCTGAAAGCGGT TCTTCCGAGGGCGGTCCA LCW0401_03 3GFP- N_E03.abl GSGGEPSESGSSGSG GEPSESGSSGSGGEP SESGSS GGTTCCGGTGGTGAACCTTCTGAATCTGGT AGCTCAGGTTCCGGTGGCGAACCATCCGA GTCTGGTAGCTCAGGTTCCGGTGGTGAACC ATCCGAGTCTGGTAGCTCA LCW0401_03 7GFP- N_F03.abl GSGGEPSESGSSGSS ESGSSEGGPGSEGSS GPGESS GGTTCCGGTGGCGAACCTTCTGAATCTGGT AGCTCAGGTTCCTCCGAAAGCGGTTTCTTCT GAGGGCGGTCCAGGTAGCGAAGGTTCTTC TGGTCCGGGCGAGTCTTCA LCW0401_03 8GFP- N_G03.abl GSGGEPSESGSSGSE GSSGPGESSGSGGEP SESGSS GGTTCCGGTGGTGAACCGTCCGAGTCTGGT AGCTCAGGTAGCGAAGGTTCTTCTGGTCCG GGTGAGTCTTCAGGTTCTGGTGGCGAACCG TCCGAATCTGGTAGCTCA LCW0401_03 9GFP- N_H03.abl GSGGEPSESGSSGES PGGSSGSESGSGGEP SESGSS GGTTCTGGTGGCGAACCGTCCGAATCTGGT AGCTCAGGTGAATCTCCTGGTGGTTCCAGC GGTTCCGAGTCAGGTTCTGGTGGCGAACCT TCCGAATCTGGTAGCTCA LCW0401_04 OGFP- N_A04.abl GSSESGSSEGGPGSG GEPSESSGSSGSSESG SSEGGP GGTTCTTCCGAAAGCGGTTCTTCCGAGGGC GGTCCAGGTTCCGGTGGTGAACCATCTGA ATCTGGTAGCTCAGGTTCTTCTGAAAGCGG TTTCTTCTGAAGGTGGTCCA LCW0401_04 2GFP- N_C04.abl GSEGSSGPGESSGES PGGSSGSESGSEGSS GPGESS GGTAGCGAAGGTTCTTCCGGTCCTGGTGAG TCTTCAGGTGAATCTCCAGGTGGCTCTAGC GGTTCCGAGTCAGGTAGCGAAGGTTTCTTCT GGTCCTGGCGAGTCCTCA LCW0401_04 6GFP- N_D04.abl GSSESGSSEGGPGSS ESGSSEGGPGSSESG SSEGGP GGTTCCTCTGAAAGCGGTTCTTCCGAAGGC GGTCCAGGTTCTTCCGAAAGCGGTTTCTTCT GAGGGCGGTCCAGGTTCCTCCGAAAGCGG TTCTTCTGAGGGTGGTCCA LCW0401_04 7GFP- N_E04.abl GSGGEPSESGSSGES PGGSSGSESGESPGG SSGSES GGTTCTGGTGGCGAACCTTCCGAGTCTGGT AGCTCAGGTGAATCTCCGGGTGGTTCTAGC GGTTCCGAGTCAGGTGAATCTCCGGGTGGT TCCAGCGGTTCTGAGTCA LCW0401_05 1GFP- N_F04.abl GSGGEPSESGSSGSE GSSGPGESSGESPGG SSGSES GGTTCTGGTGGCGAACCATCTGAGTCTGGT AGCTCAGGTAGCGAAGGTTCTTCCGGTCCA GGCGAGTCTTCAGGTGAATCTCCTGGTGGC TCCAGCGGTTCTGAGTCA LCW0401_05 3GFP- N_H04.abl GESPGGSSGSESGES PGGSSGSESGESPGG SSGSES GGTGAATCTCCTGGTGGTTCCAGCGGTTCC GAGTCAGGTGAATCTCCAGGTGGCTCTAG CGGTTCCGAGTCAGGTGAATCTCCTGGTGG TTCTAGCGGTTCTGAATCA LCW0401_05 4GFP- N_A05.abl GSEGSSGPGESSGSE GSSGPGESSGSGGEP SESGSS GGTAGCGAAGGTTCTTCCGGTCCAGGTGA ATCTTCAGGTAGCGAAGGTTCTTCTGGTCC TGGTGAATCCTCAGGTTCCGGTGGCGAACC ATCTGAATCTGGTAGCTCA

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LCW0401_05 9GFP- N_D05.abl GSGGEPSESGSSGSE GSSGPGESSGESPGG SSGSES GGTTCTGGTGGCGAACCATCCGAATCTGGT AGCTCAGGTAGCGAAGGTTCTTCTGGTCCT GGCGAATCTTCAGGTGAATCTCCAGGTGG CTCTAGCGGTTCCGAATCA LCW0401_06 OGFP- N_E05.abl GSGGEPSESGSSGSS ESGSSEGGPGSGGEP SESGSS GGTTCCGGTGGTGAACCGTCCGAATCTGGT AGCTCAGGTTCCTCTGAAAGCGGTTCTTCC GAGGGTGGTCCAGGTTCCGGTGGTGAACC TTCTGAGTCTGGTAGCTCA LCW0401_06 1GFP- N_F05.abl GS SESGS SEGGPGSG GEPSESGSSGSEGSS GPGESS GGTTCCTCTGAAAGCGGTTCTTCTGAGGGC GGTCCAGGTTCTGGTGGCGAACCATCTGA ATCTGGTAGCTCAGGTAGCGAAGGTTCTTC CGGTCCGGGTGAATCTTCA LCW0401_06 3GFP- N_H05.abl GSGGEPSESGSSGSE GSSGPGESSGSEGSS GPGESS GGTTCTGGTGGTGAACCGTCCGAATCTGGT AGCTCAGGTAGCGAAGGTTCTTCTGGTCCT GGCGAGTCTTCAGGTAGCGAAGGTTCTTCT GGTCCTGGTGAATCTTCA LCW0401_06 6GFP- N_B06.abl GSGGEPSESGSSGSS ESGSSEGGPGSGGEP SESGSS GGTTCTGGTGGCGAACCATCCGAGTCTGGT AGCTCAGGTTCTTCCGAAAGCGGTTCTTCC GAAGGCGGTCCAGGTTCTGGTGGTGAACC GTCCGAATCTGGTAGCTCA LCW0401_06 7GFP- N_C06.abl GSGGEPSESGSSGES PGGSSGSESGESPGG SSGSES GGTTCCGGTGGCGAACCTTCCGAATCTGGT AGCTCAGGTGAATCTCCGGGTGGTTCTAGC GGTTCCGAATCAGGTGAATCTCCAGGTGGT TCTAGCGGTTCCGAATCA LCW0401_06 9GFP- N_D06.abl GSGGEPSESGSSGSG GEPSESGSSGESPGG SSGSES GGTTCCGGTGGTGAACCATCTGAGTCTGGT AGCTCAGGTTCCGGTGGCGAACCGTCCGA GCTTGGTAGCTCAGGTGAATCTCCGGGTGG TTCCAGCGGTTCCGAATCA LCW0401_07 OGFP- N_E06.abl GSEGSSGPGESSGSS ESGSSEGGPGSEGSS GPGESS GGTAGCGAAGGTTCTTCTGGTCCGGGCGA ATCCTCAGGTTCCTCCGAAAGCGGTTCTTC CGAAGGTGGTCCAGGTAGCGAAGGTTCTT CCGGTCCTGGTGAATCTTCA LCW0401_07 8GFP- N_F06.abl GS SESGS SEGGPGES PGGSSGSESGESPGG SSGSES GGTTCCTCTGAAAGCGGTTTCTTCTGAAGGC GGTCCAGGTGAATCTCCGGGTGGCTCCAG CGGTTCTGAATCAGGTGAATCTCCTGGTGG CTCCAGCGGTTCCGAGTCA LCW0401_07 9GFP- N_G06.abl GSEGSSGPGESSGSE GSSGPGESSGSGGEP SESGSS GGTAGCGAAGGTTCTTCTGGTCCAGGCGA GTCTTCAGGTAGCGAAGGTTCTTCCGGTCC TGGCGAGTCTTCAGGTTCCGGTGGCGAACC GTCCGAATCTGGTAGCTCA

Example 2. Building XTEN_AE36 segments.

A library of codons encoding 36 amino acid XTEN sequences was constructed. The XTEN sequence is designated XTEN_AE36. Their segments have the amino acid sequence [X] ₃ where X is a 12 amino acid peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 23), GSEPATSGSETP (SEQ ID NO: 24), GTSESATPESGP (SEQ ID NO: 25) or GTSTEPSEGSAP ( SEQ ID NO: 26). The insert is produced by renaturating the following pairs of phosphorylated synthetic oligonucleotide pairs:

AElfor: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA (SEQ W NO:

1628)

AElrev: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT (SEQ GO NO:

1629)

AE2for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC (SEQ ID NO:

1630)

AE2rev: ACTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT (SEQ ID NO:

1631)

AE3for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC (SEQ ID NO:

1632)

AE3rev: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT (SEQ ID NO:

1633)

AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC (SEQ ID NO:

1634)

AE4rev: ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT (SEQ ID NO:

1635)

The phosphorylated oligonucleotide 3KpnIstopperFor is also renatured: AGGTTCGTCTTCAC

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TCGAGGGTAC (SEQ ID NO: 1626) and non-phosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). Renatured oligonucleotide pairs are ligated, resulting in a mixture of products with different lengths, which represent different numbers of repetitions from the 12 amino acids ligated to the BbsI/KpnI segment. Products corresponding to a length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the pCW0359 spacer vector cleaved with BsaI/KpnI. Most of the clones in the resulting library, designated LCW0402, showed green fluorescence after induction, illustrating the fact that the XTEN_AE36 sequence was ligated in frame with the GFP gene and most of the XTEN_AE36 sequences showed good expression. 96 isolates from the LCW0402 library were tested for high fluorescence by punching them onto an agar plate that contained IPTG. Similar isolates were evaluated by PCR and 48 isolates were identified which contained 36 amino acid segments as well as strong fluorescence. These isolates are sequenced and 37 clones are identified that contain the correct XTEN_AE36 segments. The names of the files of nucleotide and amino acid structures and sequences of these segments are given in Table. 14.

Table 14

DNA and amino acid sequences of 36-mer AE motifs (SEQ ID NOS 279-352, respectively, in order of appearance)

File name Amino acid sequence Nucleotide sequence LCW0402_00 2GFP- N_A07.abl GSPAGSPTSTEEG TSESATPESGPGT STEPSEGSAP GGTAGCCCGGCAGGCTCTCCGACCTCTACT GAGGAAGGTACTTCTGAAAGCGCAACCCCG GAGTCCGGCCCAGGTACCTCTACCGAACCG TCTGAGGGCAGCGCACCA LCW0402_00 3GFP- N_B07.abl GTSTEPSEGSAPG TSTEPSEGSAPGT STEPSEGSAP GGTACTTCTACCGAACCGTCCGAAGGCAGC GCTCCAGGTACCTCTACTGAACCTTCCGAG GGCAGCGCTCCAGGTACCTCTACCGAACCT TCTGAAGGTAGCGCACCA LCW0402_00 4GFP- N_C07.abl GTSTEPSEGSAPG TSESATPESGPGT SESATPESGP GGTACCTCTACCGAACCGTCTGAAGGTAGC GCACCAGGTACCTCTGAAAGCGCAACTCCT GAGTCCGGTCCAGGTACTTCTGAAAGCGCA ACCCCGGAGTCTGGCCCA LCW0402_00 5GFP- N_D07.abl GTSTEPSEGSAPG TSESATPESGPGT SESATPESGP GGTACTTCTACTGAACCGTCTGAAGGTAGC GCACCAGGTACTTCTGAAAGCGCAACCCCG GAATCCGGCCCAGGTACCTCTGAAAGCGCA ACCCCGGAGTCCGGCCCA LCW0402_00 6GFP- N_E07.abl GSEPATSGSETPG TSESATPESGPGS PAGSPTSTEE GGTAGCGAACCGGCAACCTCCGGCTCTGAA ACCCCAGGTACCTCTGAAAGCGCTACTCCT GAATCCGGCCCAGGTAGCCCGGCAGGTTCT CCGACTTCCACTGAGGAA LCW0402_00 8GFP- N_F07.abl GTSESATPESGPG SEPATSGSETPGT STEPSEGSAP GGTACTTCTGAAAGCGCAACCCCTGAATCC GGTCCAGGTAGCGAACCGGCTACTTCTGGC TCTGAGACTCCAGGTACTTCTACCGAACCG TCCGAAGGTAGCGCACCA LCW0402_00 9GFP- N_G07.abl GSPAGSPTSTEEG SPAGSPTSTEEGSEPATSGSETP GGTAGCCCGGCTGGCTCTCCAACCTCCACT GAGGAAGGTAGCCCGGCTGGCTCTCCAACC TCCACTGAAGAAGGTAGCGAACCGGCTACC TCCGGCTCTGAAACTCCA LCW0402_01 1GFP- N_A08.abl GSPAGSPTSTEEG TSESATPESGPGT STEPSEGSAP GGTAGCCCGGCTGGCTCTCCTACCTCTACTG AGGAAGGTACTTCTGAAAGCGCTACTCCTG AGTCTGGTCCAGGTACCTCTACTGAACCGT CCGAAGGTAGCGCTCCA LCW0402_01 2GFP- N_B08.abl GSPAGSPTSTEEG SPAGSPTSTEEGT STEPSEGSAP GGTAGCCCCTGCTGGCTCTCCGACTTCTACTG AGGAAGGTAGCCCGGCTGGTTCTCCGACTT CTACTGAGGAAGGTACTTCTACCGAACCTT CCGAAGGTAGCGCTCCA LCW0402_01 3GFP- N_C08.abl GTSESATPESGPG TSTEPSEGSAPGT STEPSEGSAP GGTACTTCTGAAAGCGCTACTCCGGAGTCC GGTCCAGGTACCTCTACCGAACCGTCCGAA GGCAGCGCTCCAGGTACTTCTACTGAACCT TCTGAGGGTAGCGCTCCA

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LCW0402_01 4GFP- N_D08.abl GTSTEPSEGSAPG SPAGSPTSTEEGT STEPSEGSAP GGTACCTCTACCGAACCTTCCGAAGGTAGC GCTCCAGGTAGCCCGGCAGGTTCTCCTACT TCCACTGAGGAAGGTACTTCTACCGAACCT TCTGAGGGTAGCGCACCA LCW0402_01 5GFP- N_E08.abl GSEPATSGSETPG SPAGSPTSTEEGT SESATPESGP GGTAGCGAACCGGCTACTTCCGGCTCTGAG ACTCCAGGTAGCCCTGCTGGCTCTCCGACC TCTACCGAAGAAGGTACCTCTGAAAGCGCT ACCCCTGAGTCTGGCCCA LCW0402_01 6GFP- N_F08.abl GTSTEPSEGSAPG TSESATPESGPGT SESATPESGP GGTACTTCTACCGAACCTTCCGAGGGCAGC GCACCAGGTACTTCTGAAAGCGCTACCCCT GAGTCCGGCCCAGGTACTTCTGAAAGCGCT ACTCCTGAATCCGGTCCA LCW0402_02 OGFP- N_G08.abl GTSTEPSEGSAPG SEPATSGSETPGS PAGSPTSTEE GGTACTTCTACTGAACCGTCTGAAGGCAGC GCACCAGGTAGCGAACCGGCTACTTCCGGT TCTGAAACCCCAGGTAGCCCAGCAGGTTCT CCAACTTCTACTGAAGAA LCW0402_02 3GFP- N_A09.abl GSPAGSPTSTEEG TSESATPESGPGS EPATSGSETP GGTAGCCCTGCTGGCTCTCCAACCTCCACC GAAGAAGGTACCTCTGAAAGCGCAACCCCT GAATCCGGCCCAGGTAGCGAACCGGCAACC TCCGGTTCTGAAACCCCA LCW0402_02 4GFP- N_B09.abl GTSESATPESGPG SPAGSPTSTEEGS PAGSPTSTEE GGTACTTCTGAAAGCGCTACTCCTGAGTCC GGCCCAGGTAGCCCGGCTGGCTCTCCGACT TCCACCGAGGAAGGTAGCCCGGCTGGCTCT CCAACTTCTACTGAAGAA LCW0402_02 5GFP- N_C09.abl GTSTEPSEGSAPG TSESATPESGPGT STEPSEGSAP GGTACCTCTACTGAACCTTCTGAGGGCAGC GCTCCAGGTACTTCTGAAAGCGCTACCCCG GAGTCCGGTCCAGGTACTTCTACTGAACCG TCCGAAGGTAGCGCACCA LCW0402_02 6GFP- N_D09.abl GSPAGSPTSTEEG TSTEPSEGSAPGS EPATSGSETP GGTAGCCCGGCAGGCTCTCCGACTTCCACC GAGGAAGGTACCTCTACTGAACCTTCTGAG GGTAGCCGCTCCAGGTAGCGAACCGGCAACC TCTGGCTCTGAAACCCCA LCW0402_02 7GFP- N_E09.abl GSPAGSPTSTEEG TSTEPSEGSAPGT STEPSEGSAP GGTAGCCCAGCAGGCTCTCCGACTTCCACT GAGGAAGGTACTTCTACTGAACCTTCCGAA GGCAGCGCACCAGGTACCTCTACTGAACCT TCTGAGGGCAGCGCTCCA LCW0402_03 2GFP- N_H09.abl GSEPATSGSETPG TSESATPESGPGS PAGSPTSTEE GGTAGCGAACCTGCTACCTCCGGTTCTGAA ACCCCAGGTACCTCTGAAAGCGCAACTCCG GAGTCTGGTCCAGGTAGCCCTGCAGGTTCT CCTACCTCCACTGAGGAA LCW0402_03 4GFP- N_A10.abl GTSESATPESGPG TSTEPSEGSAPGT STEPSEGSAP GGTACCTCTGAAAGCGCTACTCCGGAGTCT GGCCCAGGTACCTCTACTGAACCGTCTGAG GGTAGCCGCTCCAGGTACTTCTACTGAACCG TCCGAAGGTAGCGCACCA LCW0402_03 6GFP- N_C10.abl GSPAGSPTSTEEG TSTEPSEGSAPGT STEPSEGSAP GGTAGCCCGGCTGGTTCTCCGACTTCCACC GAGGAAGGTACCTCTACTGAACCTTCTGAG GGTAGCCGCTCCAGGTACCTCTACTGAACCT TCCGAAGGCAGCGCTCCA LCW0402 03 GTSTEPSEGSAPG GGTACTTCTACCGAACCGTCCGAGGGCAGC

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9GFP- N_E10.abl TSTEPSEGSAPGT STEPSEGSAP GCTCCAGGTACTTCTACTGAACCTTCTGAA GGCAGCGCTCCAGGTACTTCTACTGAACCT TCCGAAGGTAGCGCACCA LCW0402_04 OGFP- N_F10.abl GSEPATSGSETPG TSESATPESGPGT STEPSEGSAP GGTAGCGAACCTGCAACCTCTGGCTCTGAA ACCCCAGGTACCTCTGAAAGCGCTACTCCT GAATTCTGGCCCAGGTACTTCTACTGAACCG TCCGAGGGCAGCGCACCA LCW0402_04 1GFP- N_G10.abl GTSTEPSEGSAPG SPAGSPTSTEEGT STEPSEGSAP GGTACTTCTACCGAACCGTCCGAGGGTAGC GCACCAGGTAGCCCAGCAGGTTCTCCTACC TCCACCGAGGAAGGTACTTCTACCGAACCG TCCGAGGGTAGCGCACCA LCW0402_05 OGFP- N_All.abl GSEPATSGSETPG TSESATPESGPGS EPATSGSETP GGTAGCGAACCGGCAACCTCCGGCTCTGAA ACTCCAGGTACTTCTGAAAGCGCTACTCCG GAATCCGGCCCAGGTAGCGAACCGGCTACT TCCGGCTCTGAAACCCCA LCW0402_05 1GFP- N_Bll.abl GSEPATSGSETPG TSESATPESGPGS EPATSGSETP GGTAGCGAACCGGCAACTTCCGGCTCTGAA ACCCCAGGTACTTCTGAAAGCGCTACTCCT GAGTCTGGCCCAGGTAGCGAACCTGCTACC TCTGGCTCTGAAACCCCA LCW0402_05 9GFP- N_Ell.abl GSEPATSGSETPG SEPATSGSETPGT STEPSEGSAP GGTAGCGAACCGGCAACCTCTGGCTCTGAA ACTCCAGGTAGCGAACCTGCAACCTCCGGC TCTGAAACCCCAGGTACTTCTACTGAACCTT CTGAGGGCAGCGCACCA LCW0402_06 OGFP- N_Fll.abl GTSESATPESGPG SEPATSGSETPGS EPATSGSETP GGTACTTCTGAAAGCGCTACCCCGGAATCT GGCCCAGGTAGCGAACCGGCTACTTCTGGT TCTGAAACCCCAGGTAGCGAACCGGCTACC TCCGGTTCTGAAACTCCA LCW0402_06 1GFP- N_Gll.abl GTSTEPSEGSAPG TSTEPSEGSAPGT SESATPESGP GGTACCTCTACTGAACCTTCCGAAGGCAGC GCTCCAGGTACCTCTACCGAACCGTCCGAG GGCAGCGCACCAGGTACTTCTGAAAGCGCA ACCCCTGAATCCGGTCCA LCW0402_06 5GFPN_A12.abl GSEPATSGSETPG TSESATPESGPGT SESATPESGP GGTAGCGAACCGGCAACCTCTGGCTCTGAA ACCCCAGGTACCTCTGAAAGCGCTACTCCG GAATTCTGGTCCAGGTACTTCTGAAAGCGCT ACTCCGGAATCCGGTCCA LCW0402_06 6GFP- N_B12.abl GSEPATSGSETPG SEPATSGSETPGT STEPSEGSAP GGTAGCGAACCTGCTACCTCCGGCTCTGAA ACTCCAGGTAGCGAACCGGCTACTTCCGGT TCTGAAACTCCAGGTACCTCTACCGAACCT TCCGAAGGCAGCGCACCA LCW0402_06 7GFP- N_C12.abl GSEPATSGSETPG TSTEPSEGSAPGS EPATSGSETP GGTAGCGAACCTGCTACTTCTGGTTCTGAA ACTCCAGGTACTTCTACCGAACCGTCCGAG GGTAGCCGCTCCAGGTAGCGAACCTGCTACT TCTGGTTCTGAAACTCCA LCW0402_06 9GFP- N_D12.abl GTSTEPSEGSAPG TSTEPSEGSAPGS EPATSGSETP GGTACCTCTACCGAACCGTCCGAGGGTAGC GCACCAGGTACCTCTACTGAACCGTCTGAG GGTAGCCGCTCCAGGTAGCGAACCGGCAACC TCCGGTTCTGAAACTCCA LCW0402_07 3GFP- GTSTEPSEGSAPG SEPATSGSETPGS GGTACTTCTACTGAACCTTCCGAAGGTAGC GCTCCAGGTAGCGAACCTGCTACTTCTGGT N_F12.abl PAGSPTSTEE TCTGAAACCCCAGGTAGCCCGGCTGGCTCT CCGACCTCCACCGAGGAA LCW0402_07 4GFP- N_G12.abl GSEPATSGSETPG SPAGSPTSTEEGT SESATPESGP GGTAGCGAACCGGCTACTTCCGGCTCTGAG ACTCCAGGTAGCCCAGCTGGTTCTCCAACC TCTACTGAGGAAGGTACTTCTGAAAGCGCT ACCCCTGAATCTGGTCCA LCW0402_07 5GFP- N_H12.abl GTSESATPESGPG SEPATSGSETPGT SESATPESGP GGTACCCTTGAAAGCGCAACTCCTGAGTCT GGCCCAGGTAGCGAACCTGCTACCTCCGGC TCTGAGACTCCAGGTACCTCTGAAAGCGCA ACCCCGGAATCTGGTCCA

Example 3. Building XTEN_AF36 segments.

A library of codons was constructed, encoding sequences of 36 amino acids in length, the sequences are designated XTEN_AF36. Their segments have the amino acid sequence [X] ₃ where X is a 12 amino acid peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 27), GTSTPESGSASP (SEQ ID NO: 28), GTSPSGESSTAP (SEQ ID NO: 29) or GSTSSTAESPGP ( SEQ ID NO: 30). The insert is produced by renaturating the following pairs of phosphorylated synthetic oligonucleotide pairs:

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AFI for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC (SEQ ID NO: 1636)

AFlrev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA (SEQ ID NO: 1637)

AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC (SEQ ID NO: 1638)

AF2rev: ACCTGGAGAWGCRGARCGCTTTCMGGRGTAGARGT (SEQ ID NO: 1639)

AF3for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC (SEQ W NO: 1640)

AF3rev: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT (SEQ ID NO: 1641)

AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC (SEQ ID NO: 1642)

AF4rev: ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA (SEQ ID NO: 1643)

The phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the unphosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627) are also annealed. The renatured oligonucleotide pairs are ligated, resulting in a mixture of products with different lengths, representing a different number of repetitions from the 12 amino acids ligated to the BbsI/KpnI segment. Using preparative agarose gel electrophoresis, products corresponding to a length of 36 amino acids are isolated from the mixture and ligated into the pCW0359 spacer vector cleaved with BsaI/KpnI. Most of the clones in the resulting library, designated LCW0403, showed green fluorescence after induction, illustrating the fact that the XTEN_AF36 sequence was ligated in frame with the GFP gene and most of the XTEN_AF36 sequences showed good expression. 96 isolates from the LCW0403 library were tested for high fluorescence by punching them onto an agar plate that contained IPTG. such isolates are evaluated by PCR and 48 isolates are identified which contain 36 amino acid segments as well as strong fluorescence. These isolates are sequenced and 44 clones are identified that contain the correct XTEN_AF36 segments. The names of the files of nucleotide and amino acid structures and sequences of these segments are given in Table. 15.

Table 15

DNA and amino acid sequences of AF 36-mer motifs SEQ ID NOS 353-440, respectively, in order of appearance)

File name Amino acid sequence Nucleotide sequence LCW0403_00 4GFPN_A01.abl GTTPESGSASPG TSPSGESTAPGT SPSGESTAPG GGTACTTCTACTCCGGAAAAGCGGTTCCGCA TCTCCAGGTACTTCTCCTAGCGGTGAATCTT CTACTGCTCCAGGTACCTCTCCTAGCGGCG AATTTCTACTGCTCCA LCW0403_00 5GFPN_B01.abl GTPPSGESTAPG STSSTAESPGPGT SPSGESTAPG GGTACTTCTCCGAGCGGTGAATCTTCTACC GCACCAGGTTCTACTAGCTCTACCGCTGAA TCTCCGGGCCCAGGTACTTCTCCGAGCGGT GAATCTTCTACTGCTCCA LCW0403_00 6GFPN_C01.abl GSTSSTAESPGPG TSPSGESTAPGT STPESGSASP GGTTCCACCAGCTCTACTGCTGAATCTCCTG GTCCAGGTACCTCTCCTAGCGGTGAATCTTC TACTGCTCCAGGTACTTCTACTCCTGAAAGC GGCTCTGCTTCTCCA LCW0403_00 7GFPN_D01.abl GSTSSTAESPGPG STSSTAESPGPGT SPSGESTAP GGTTTCTACCAGCTCTACTGCAGAATCTCCTG GCCCAGGTTCCACCAGCTCTACCGCAGAAT CTCCGGGTCCAGGTACTTCCCCTAGCGGTG AATCTTCTACCGCACCA LCW0403 00 GSTSSTAESPGPG GGTTCTACTAGCTCTACTGCTGAATCTCCTG

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8GFP- N_E0Eabl TSPSGESTAPGT STPESGSASP GCCCAGGTACTTCTCCTAGCGGTGAATCTTC TACCGCTCCAGGTACCTCTACTCCGGAAAG CGGTTCTGCATCTCCA LCW0403_01 OGFP- N_F0Eabl GSTSSTAESPGPG TSTPESGSASPGS TSESPSGTAP GGTTCTACCAGCTCTACCGCAGAATCTCCT GGTCCAGGTACCTCTACTCCGGAAAGCGGC TCTGCATCTCCAGGTTCTACTAGCGAATCTC CTTTCTGGCACTGCACCA LCW0403_01 1GFPN_G0Eabl GSTSSTAESPGPG TSTPESGSASPGT STPESGSASP GGTTCTACTAGCTCTACTGCAGAATCTCCTG GCCCAGGTACCTCTACTCCGGAAAGCGGCT CTGCATCTCCAGGTACTTCTACCCCTGAAA GCGGTTCTGCATCTCCA LCW0403_01 2GFPN_H0Eabl GSTSESPSSGTAPG TSPSGSGESTAPGS TSESPSSGTAP GGTTCTACCAGCGAATCTCCTTCTGGCACC GCTCCAGGTACCTCTCCTAGCGGCGAATCT TCTACCGCTCCAGGTTCTACTAGCGAATCTC CTTCTGGCACTGCACCA LCW0403_01 3GFP- N_A02.abl GSTSSTAESPGPG STSSTAESPGPGT SPSGESTAP GGTTCCACCAGCTCTACTGCAGAATCTCCG GGCCCAGGTTCTACTAGCTCTACTGCAGAA TCTCCGGGTCCAGGTACTTCTCCTAGCGGC GAATCTTCTACCGCTCCA LCW0403_01 4GFP- N_B02.abl GSTSSTAESPGPG TSTPESGSASPGS TSESPSGTAP GGTTCCACTAGCTCTACTGCAGAATCTCCTG GCCCAGGTACCTCTACCCCTGAAAGCGGCT CTGCATCTCCAGGTTCTACCAGCGAATCCC CGTCTGGCACCGCACCA LCW0403_01 5GFP- N_C02.abl GSTSSTAESPGPG STSSTAESPGPGT SPSGESTAP GGTTCTACTAGCTCTACTGCTGAATCTCCGG GTCCAGGTTCTACCAGCTCTACTGCTGAATC TCCTGGTCCAGGTACCTCCCCGAGCGGTGA ATCTTCTACTGCACCA LCW0403_01 7GFP- N_D02.abl GSTSSTAESPGPG STSESPSGTAPGS TSSTAESPGP GGTTCTACCAGCTCTACCGCTGAATCTCCTG GCCCAGGTTCTACCAGCGAATCCCCGTCTG GCACCGCACCAGGTTCTACTAGCTCTACCG CTGAATCTCCGGGTCCA LCW0403_01 8GFP- N_E02.abl GSTSSTAESPGPG STSSTAESPGPGS TSSTAESPGPG GGTTCTACCAGCTCTACCGCAGAATCTCCT GGCCCAGGTTCCACTAGCTCTACCGCTGAA TCTCCTGGTCCAGGTTCTACTAGCTCTACCG CTGAATCTCCTGGTCCA LCW0403_01 9GFP- N_F02.abl GSTSESSPSGTAPG STSSTAESPGPGS TSSTAESPGP GGTTCTACTAGCGAATCCCCTTCTGGTACTG CTCCAGGTTCCACTAGCTCTACCGCTGAATC TCCTGGCCCAGGTTCCACTAGCTCTACTGCA GAATCTCCTGGTCCA LCW0403_02 3GFP- N_H02.abl GSTSESPSSGTAPG STSESPSGTAPGS TSESPSSGTAP GGTTCTACTAGCGAATCTCCTTCTGGTACCG CTCCAGGTTCTACCAGCGAATCCCCGTCTG GTACTGCTCCAGGTTCTACCAGCGAATCTC CTTCTGGTACTGCACCA LCW0403_02 4GFP- N_A03.abl GSTSSTAESPGPG STSSTAESPGPGS TSSTAESPGPG GGTTCCACCAGCTCTACTGCTGAATCTCCTG GCCCAGGTTTCTACCAGCTCTACTGCTGAAT CTCCGGGCCCAGGTTCCACCAGCTCTACCG CTGAATCTCCGGGTCCA LCW0403_02 5GFP- GSTSSTAESPGPG STSSTAESPGPGT GGTTCCACTAGCTCTACCGCAGAATCTCCT GGTCCAGGTTCTACTAGCTCTACTGCTGAAT

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N_B03.abl SPSGESTAP CTCCGGGTCCAGGTACCTCCCCTAGCGGCG AATCTTCTACCGCTCCA LCW0403_02 8GFP- N_D03.abl GSSPSASTGTGPG SSTPSGATGSPGS STPSGATGSP GGTTCTAGCCCTTCTGCTTCCACCGGTACCG GCCCAGGTAGCTCTACTCCGTCTGGTGCAA CTGGCCTCTCCAGGTAGCTCTACTCCGTCTGG TGCAACCGGCTCCCCA LCW0403_02 9GFP- N_E03.abl GTPPSGESTAPG TSTPESGSASPGS TSSTAESPGP GGTACTTCCCCTAGCGGTGAATCTTCTACTG CTCCAGGTACCTCTACTCCGGAAAGCGGCT CCGCATCTCCAGGTTCTACTAGCTCTACTGC TGAATCTCCTGGTCCA LCW0403_03 OGFP- N_F03.abl GSTSSTAESPGPG STSSTAESPGPGT STPESGSASP GGTTCTACTAGCTCTACCGCTGAATCTCCGG GTCCAGGTTTCTACCAGCTCTACTGCAGAAT CTCCTGGCCCAGGTACTTCTACTCCGGAAA GCGGTTCCGCTTCTCCA LCW0403_03 1GFP- N_G03.abl GTSPSGESTAPG STTSSTAESPGPGT STPESGSASP GGTACTTCTCCTAGCGGTGAATCTTCTACCG CTCCAGGTTCTACCAGCTCTACTGCTGAATC TCCTGGCCCAGGTACTTCTACCCCGGAAAG CGGCTCCGCTTCTCCA LCW0403_03 3GFP- N_H03.abl GSTSESSPSGTAPG STSSTAESPGPGS TSSTAESPGP GGTTCTACTAGCGAATCCCCTTCTGGTACTG CACCAGGTTTCTACCAGCTCTACTGCTGAAT CTCCGGGCCCAGGTTCCACCAGCTCTACCG CAGAATCTCCTGGTCCA LCW0403_03 5GFP- N_A04.abl GSTSSTAESPGPG STSESPSGTAPGS TSSTAESPGP GGTTCCACCAGCTCTACCGCTGAATCTCCG GGCCCAGGTTCTACCAGCGAATCCCCTTCT GGCACTGCACCAGGTTCTACTAGCTCTACC GCAGAATCTCCGGGCCCA LCW0403_03 6GFP- N_B04.abl GSTSSTAESPGPG TSPSGESTAPGT STPESGSASP GGTTTCTACCAGCTCTACTGCTGAATCTCCGG GTCCAGGTACTTCCCCGAGCGGTGAATCTT CTACTGCACCAGGTACTTCTACTCCGGAAA GCGGTTCCGCTTCTCCA LCW0403_03 9GFP- N_C04.abl GSTSESPSSGTAPG STSESPSGTAPGT SPSGESTAP GGTTCTACCAGCGAATCTCCTTCTGGCACC GCTCCAGGTTCTACTAGCGAATCCCCGTCT GGTACCGCACCAGGTACTTCTCCTAGCGGC GAATCTTCTACCGCACCA LCW0403_04 1GFP- N_D04.abl STSESPSGTAPG STSESPSGTAPGT STPESGSASP GGTTCTACCAGCGAATCCCCTTCTGGTACTG CTCCAGGTTCTACCAGCGAATCCCCTTCTGG CACCGCACCAGGTACTTCTACCCCTGAAAG CGGCTCCGCTTCTCCA LCW0403_04 4GFP- N_E04.abl GTSTPESGSASPG STSSTAESPGPGS TSSTAESPGP GGTACCTCTACTCCTGAAAGCGGTTCTGCA TCTCCAGGTTCCACTAGCTCTACCGCAGAA TCTCCGGGCCCAGGTTCTACTAGCTCTACTG CTGAATCTCCTGGCCCA LCW0403_04 6GFP- N_F04.abl GSTSESPSSGTAPG STSESPSGTAPGT SPSGESTAP GGTTCTACCAGCGAATCCCCTTCTGGCACT GCACCAGGTTCTACTAGCGAATCCCCTTCT GGTACCGCACCAGGTACTTCCGAGCGGC GAATCTTCTACTGCTCCA LCW0403_04 7GFP- N_G04.abl GSTSSTAESPGPG STSSTAESPGPGS TSESPSSGTAP GGTTCTACTAGCTCTACCGCTGAATCTCCTG GCCCAGGTTCCACTAGCTCTACCGCAGAAT CTCCGGGCCCAGGTTCTACTAGCGAATCCC

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CTTCTGGTACCGCTCCA LCW0403_04 9GFP- N_H04.abl GSTSSTAESPGPG STSSTAESPGPGT STPESGSASP GGTTCCACCAGCTCTACTGCAGAATCTCCT GGCCCAGGTTCTACTAGCTCTACCGCAGAA TCTCCTGGTCCAGGTACCTCTACTCCTGAAA GCGGTTCCGCATCTCCA LCW0403_05 1GFP- N_A05.abl GSTSSTAESPGPG STSSTAESPGPGS TSESPSSGTAP GGTTCTACTAGCTCTACTGCTGAATCTCCGG GCCCAGGTTCTACTAGCTCTACCGCTGAAT CTCCGGGTCCAGGTTCTACTAGCGAATCTC CTTCTGGTACCGCTCCA LCW0403_05 3GFP- N_B05.abl GTSPSGESTAPG STSESPSGTAPGSTSSTAESPGP GGTACCTCCCCGAGCGGTGAATCTTCTACT GCACCAGGTTCTACTAGCGAATCCCCTTCT GGTACTGCTCCAGGTTCCACCAGCTCTACT GCAGAATCTCCGGGTCCA LCW0403_05 4GFP- N_C05.abl GSTSESPSSGTAPG TSPSGESTAPGS TSSTAESPGP GGTTCTACTAGCGAATCCCCGTCTGGTACT GCTCCAGGTACTTCCCCTAGCGGTGAATCTT CTACTGCTCCAGGTTCTACCAGCTCTACCGC AGAATCTCCGGGTCCA LCW0403_05 7GFP- N_D05.abl GSTSSTAESPGPG STSESPSGTAPGT SPSGESTAP GGTTCTACCAGCTCTACCGCTGAATCTCCTG GCCCAGGTTCTACTAGCGAATCTCCGTCTG GCACCGCACCAGGTACTTCCCCTAGCGGTG AATCTTCTACTGCACCA LCW0403_05 8GFP- N_E05.abl STSESPSGTAPG STSESPSGTAPGT STPESGSASP GGTTCTACTAGCGAATCTCCTTCTGGCACTG CACCAGGTTCTACCAGCGAATCTCCGTCTG GCACTGCACCAGGTACCTCTACCCCCTGAAA GCGGTTCCGCTTCTCCA LCW0403_06 OGFP- N_F05.abl GTTPESGSASPG STSESPSGTAPGSTSSTAESPGP GGTACCTCTACTCCGGAAAAGCGGTTCCGCA TCTCCAGGTTCTACCAGCGAATCCCCGTCTG GCACCGCACCAGGTTCTACTAGCTCTACTG CTGAATCTCCGGGCCCA LCW0403_06 3GFP- N_G05.abl GSTSSTAESPGPG TSPSGESTAPGT SPSGESTAPG GGTTCTACTAGCTCTACTGCAGAATCTCCG GGCCCAGGTACCTCTCCTAGCGGTGAATCT TCTACCGCTCCAGGTACTTCTCCGAGCGGT GAATCTTCTACCGCTCCA LCW0403_06 4GFP- N_H05.abl GTSPSGESSTAPGTSPSGESTAPGTSPSGESSTAPG GGTACCTCCCCTAGCGGCGAATCTTCTACT GCTCCAGGTACCTCTCCTAGCGGCGAATCT TCTACCGCTCCAGGTACCTCCCCTAGCGGT GAATCTTCTACCGCACCA LCW0403_06 5GFP- N_A06.abl GSTSSTAESPGPG TSTPESGSASPGS TSESPSGTAP GGTTCCACTAGCTCTACTGCTGAATCTCCTG GCCCAGGTACTTCTACTCCGGAAAGCGGTT CCGCTTCTCCAGGTTCTACTAGCGAATCTCC GCTTGGCACCGCACCA LCW0403_06 6GFP- N_B06.abl GSTSESPSSGTAPG TSPSGESTAPGT SPSGESSTAPG GGTTCTACTAGCGAATCTCCGTCTGGCACT GCTCCAGGTACTTCTCCTAGCGGTGAATCTT CTACCGCTCCAGGTACTTCCCCTAGCGGCG AATCTTCTACCGCTCCA LCW0403_06 7GFP- N_C06.abl GSTSESPSGTAPG TSTPESGSASPGS TSSTAESPGP GGTTCTACTAGCGAATCTCCTTCTGGTACCG CTCCAGGTACTTCTACCCCTGAAAGCGGCT CCGCTTCTCCAGGTTCCACTAGCTCTACGC TGAATCTCCGGGTCCA LCW0403_06 8GFP- N_D06.abl GSTSSTAESPGPG STSSTAESPGPGS TSESPSSGTAP GGTTCCACTAGCTCTACTGCTGAATCTCCTG GCCCAGGTTCTACCAGCTCTACCGCTGAAT CTCCTGGCCCAGGTTCTACCAGCGAATCTC CGTCTGGCACCGCACCA LCW0403_06 9GFP- N_E06.abl GSTSESPSGTAPG TSTPESGSASPGT STPESGSASP GGTTCTACTAGCGAATCCCCGTCTGGTACC GCACCAGGTACTTCTACCCCGGAAAGCGGC TCTGCTTCTCCAGGTACTTCTACCCCGGAAA GCGGCTCCGCATCTCCA LCW0403_07 OGFP- N_F06.abl GSTSESPSGTAPG TSTPESGSASPGT STPESGSASP GGTTCTACTAGCGAATCCCCGTCTGGTACT GCTCCAGGTACTTCTACTCCTGAAAGCGGT TCCGCTTCTCCAGGTACCTCTACTCCGGAAA GCGGTTCTGCATCTCCA

Example 4. Building XTEN_AG36 segments.

Build a library of codons encoding sequences of 36 amino acids. The sequences are designated XTEN_AG36. Their segments have the amino acid sequence [X] ₃ where X is a 12 amino acid peptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33) or GASPGTSSTGSP ( SEQ ID NO: 34). The insert is produced by renaturating the following pairs of phosphorylated synthetic oligonucleotide pairs:

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AGlfor: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC (SEQ ID NO: 1644)

AGlrev: ACTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT (SEQ W NO: 1645)

AG2for: AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC (SEQ ID NO: 1646)

AG2rev: ACTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT (SEQ ID NO: 1647)

AG3for: AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC (SEQ ID NO: 1648)

AG3rev: ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA (SEQ ID NO: 1649)

AG4for: AGGTGCWTCYCCKGGYACYAGCTTCTACYGGTTCTCC (SEQ ID NO: 1650)

AG4rev: ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC (SEQ W NO: 1651)

The phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the unphosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627) are also annealed. The renatured oligonucleotide pairs are ligated, resulting in a mixture of products with different lengths, representing a different number of repetitions from the 12 amino acids ligated to the BbsI/KpnI segment. Using preparative agarose gel electrophoresis, products corresponding to a length of 36 amino acids are isolated from the mixture and ligated into the pCW0359 spacer vector cleaved with BsaI/KpnI. Most of the clones in the resulting library, designated LCW0404, showed green fluorescence after induction, illustrating the fact that the XTEN_AG36 sequence was ligated in frame to the GFP gene and most of the XTEN_AG36 sequences showed good expression. 96 isolates from the LCW0404 library are tested for high fluorescence by punching them onto an agar plate that contains IPTG. Similar isolates were evaluated by PCR and 48 isolates were identified which contained 36 amino acid segments as well as strong fluorescence. These isolates are sequenced and 44 clones are identified that contain the correct XTEN_AG36 segments. The names of the files of nucleotide and amino acid structures and sequences of these segments are given in Table. 16.

Table 16

DNA and amino acid sequences of 36-mer AG motifs (SEQ ID NOS 441-528, respectively, in order of appearance)_____________________

File name Amino acid sequence Nucleotide sequence LCW0404_00 1GFP- N_A07.abl GASPGTSSTGSPGT PGSGTASSSPPGSSTP SGATGSP GGTGCATCCCCGGGCACTAGCTCTACCGGT TCTCCAGGTACTCCTGGTAGCGGTACTGCTT CTTCTTCTCCAGGTAGCTCTACTCCTTCTGG TGCTACTGGTTCTCCA LCW0404_00 3GFP- N_B07.abl GSSTPSGATGSPGS SPSASTGTGPGSSTP SGATGSP GGTAGCTCTACCCCTTCTGGTGCTACCGGCT CTCCAGGTTCTAGCCCGTCTGCTTCTACCGG TACCGGTCCAGGTAGCTCTACCCCTTCTGGT GCTACTGGTTCTCCA LCW0404_00 6GFP- N_C07.abl GASPGTSSTGSPGS SPSASTGTGPGSSTP SGATGSP GGTGCATCTCCGGGTACTAGCTCTACCGGT TCTCCAGGTTCTAGCCCTTCTGCTTCCACTG GTACCGGCCCAGGTAGCTCTACCCCGTCTG GTGCTACTGGTTCCCCA LCW0404_00 7GFP- N_D07.abl GTPGSGTASSSPGS STPSGATGSPGASP GTSSTGSP GGTACTCCGGGCAGCGGTACTGCTTCTTCCT CTCCAGGTAGCTCTACCCCTTCTGGTGCAAC TGGTTCCCCAGGTGCATCCCCTGGTACTAG CTCTACCGGTTCTCCA LCW0404_00 9 GFP- GTPGSGTASSSPGA SPGTSSTGSPGSRPS GGTACCCCTGGCAGCGGTACTGCTTCTTCTT CTCCAGGTGCTTCCCCTGGTACCAGCTCTAC

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N_E07.abl ASTGTGP CGGTTCTCCAGGTTCTAGACCTTCTGCATCC ACCGGTACTGGTCCA LCW0404_01 1GFP- N_F07.abl GASPGTSSTGSPGS STPSGATGSPGASP GTSSTGSP GGTGCATCTCCTGGTACCAGCTCTACCGGTT CTCCAGGTAGCTCTACTCCTTCTGGTGCTAC TGGCTCTCCAGGTGCTTCCCCGGGTACCAG CTCTACCGGTTCTCCA LCW0404_01 2GFP- N_G07.abl GTPGSGTASSSPGS STPSGATTGSPGSSTP SGATGSP GGTACCCCGGGCAGCGGTACCGCATCTTCC TCTCCAGGTAGCTCTACCCCGTCTGGTGCTA CCGGTTCCCCAGGTAGCTCTACCCCGTCTG GTGCAACCGGCTCCCCA LCW0404_01 4GFP- N_H07.abl GASPGTSSTGSPGA SPGTSSTGSPGASP GTSSTGSP GGTGCATCTCCGGGCACTAGCTCTACTGGT TCTCCAGGTGCATCCCCTGGCACTAGCTCTA CTGGTTCTCCAGGTGCTTCTCCTGGTACCAG CTCTACTGGTTCTCCA LCW0404_01 5GFP- N_A08.abl GS STPSGATGSPGS SPSASTGTGPGASP GTSSTGSP GGTAGCTCTACTCCGTCTGGTGCAACCGGC TCCCCAGGTTCTAGCCCGTCTGCTTCCACTG GTACTGGCCCAGGTGCTTCCCCGGGCACCA GCTCTACTGGTTCTCCA LCW0404_01 6GFP- N_B08.abl GS STPSGATGSPGS STPSGATTGSPGTPG SGTASSSP GGTAGCTCTACTCCTTCTGGTGCTACCGGTT CCCCAGGTAGCTCTACTCCTTCTGGTGCTAC TGGTTCCCCAGGTACTCCGGGCAGCGGTAC TGCTTCTTCCTCTCCA LCW0404_01 7GFP- N_C08.abl GS STPSGATGSPGS STPSGATGSPGASP GTSSTGSP GGTAGCTCTACTCCGTCTGGTGCAACCGGT TCCCCAGGTAGCTCTACTCCTTCTGGTGCTA CTGGCTCCCCAGGTGCATCCCCTGGCACCA GCTCTACCGGTTCTCCA LCW0404_01 8GFP- N_D08.abl GTPGSGTASSSPGS SPSASTGTGPGSSTP SGATGSP GGTACTCCTGGTAGCGGTACCGCATCTTCCT CTCCAGGTTCTAGCCCTTCTGCATCTACCGG TACCGGTCCAGGTAGCTCTACTCCTTCTGGT GCTACTGGCTCTCCA LCW0404_02 3GFP- N_F08.abl GASPGTSSTGSPGS SPSASTGTGPGTPG SGTASSSP GGTGCTTCCCCGGGCACTAGCTCTACCGGT TCTCCAGGTTCTAGCCCTTCTGCATCTACTG GTACTGGCCCAGGTACTCCGGGCAGCGGTA CTGCTTCTTCCTCTCCA LCW0404_02 5GFP- N_G08.abl GS STPSGATGSPGS STPSGATGSPGASP GTSSTGSP GGTAGCTCTACTCCGTCTGGTGCTACCGGCT CTCCAGGTAGCTCTACCCCTTCTGGTGCAAC CGGCTCCCCCAGGTGCTTCTCCGGGTACCAG CTCTACTGGTTCTCCA LCW0404_02 9GFP- N_A09.abl GTPGSGTASSSPGS STPSGATTGSPGSSPS ASTGTGP GGTACCCCTGGCAGCGGTACCGCTTCTTCCT CTCCAGGTAGCTCTACCCCGTCTGGTGCTAC TGGCTCTCCAGGTTCTAGCCCGTCTGCATCT ACCGGTACCGGCCCA LCW0404_03 OGFP- N_B09.abl GSTPSGATGSPGT PGSGTASSSPGTPG SGTASSSP GGTAGCTCTACTCCTTCTGGTGCAACCGGCT CCCCAGGTACCCCGGGCAGCGGTACCGCAT CTTCCTCTCCAGGTACTCCGGGTAGCGGTA CTGCTTCTTCTTCTCCA LCW0404_03 1GFP- N_C09.abl GTPGSGTASSSPGS STPSGATGSPGASP GTSSTGSP GGTACCCCGGGTAGCGGTACTGCTTCTTCCT CTCCAGGTAGCTCTACCCCTTCTGGTGCAAC CGGCCTCTCCAGGTGCTTCTCCGGGCACCAG CTCTACCGGTTCTCCA

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LCW0404_03 4GFP- N_D09.abl GS STPSGATGSPGS STPSGATGSPGASP GTSSTGSP GGTAGCTCTACCCCGTCTGGTGCTACCGGC TCTCCAGGTAGCTCTACCCCGTCTGGTGCA ACCGGCCTCCCCAGGTGCATCCCCGGGTACT AGCTCTACCGGTTCTCCA LCW0404_03 5GFP- N_E09.abl GASPGTSSTGSPGT PGSGTASSSPPGSSTP SGATGSP GGTGCTTCTCCGGGCACCAGCTCTACTGGTT CTCCAGGTACCCCGGGCAGCGGTACCGCAT CTTCTTCTCCAGGTAGCTCTACTCCTTCTGG TGCAACTGGTTCTCCA LCW0404_03 6GFP- N_F09.abl GSPSASTGTGPGS STPSGATTGSPGTPG SGTASSSP GGTTCTAGCCCGTCTGCTTCCACCGGTACTG GCCCAGGTAGCTCTACCCCGTCTGGTGCAA CTGGTTCCCCAGGTACCCCTGGTAGCGGTA CCGCTTCTTCTTCTCCA LCW0404_03 7GFP- N_G09.abl GASPGTS STGSPGS SPSASTGTGPGSSTP SGATGSP GGTGCTTCTCCGGGCACCAGCTCTACTGGTT CTCCAGGTTCTAGCCCTTCTGCATCCACCGG TACCGGTCCAGGTAGCTCTACCCCTTCTGGT GCAACCGGCTCTCCA LCW0404_04 OGFP- N_H09.abl GASPGTS STGSPGS STPSGATTGSPGSSTP SGATGSP GGTGCATCCCCGGGCACCAGCTCTACCGGT TCTCCAGGTAGCTCTACCCCGTCTGGTGCTA CCGGCTCTCCAGGTAGCTCTACCCCGTCTG GTGCTACTGGCTCTCCA LCW0404_04 1GFP- N_A10.abl GTPGSGTASSSPGS STPSGATTGSPGTPG SGTASSSP GGTACCCCTGGTAGCGGTACTGCTTCTTCCT CTCCAGGTAGCTCTACTCCGTCTGGTGCTAC CGGTTCTCCAGGTACCCCGGGTAGCGGTAC CGCATCTTCTTCTCCA LCW0404_04 3GFP- N_C10.abl GSPSASTGTGPGS STPSGATTGSPGSSTP SGATGSP GGTTCTAGCCCTTCTGCTTCCACCGGTACTG GCCCAGGTAGCTCTACCCCTTCTGGTGCTAC CGGCTCCCCCAGGTAGCTCTACTCCTTCTGGT GCAACTGGCTCTCCA LCW0404_04 5GFP- N_D10.abl GASPGTS STGSPGS SPSASTGTGPGSSPS ASTGTGP GGTGCTTCTCCTGGCACCAGCTCTACTGGTT CTCCAGGTTCTAGCCCTTCTGCTTCTACCGG TACTGGTCCAGGTTCTAGCCCTTCTGCATCC ACTGGTACTGGTCCA LCW0404_04 7GFP- N_F10.abl GTPGSGTASSSPGA SPGTSSTGSPGASP GTSSTGSP GGTACTCCTGGCAGCGGTACCGCTTCTTCTT CTCCAGGTGCTTCTCCTGGTACTAGCTCTAC TGGTTCTCCAGGTGCTTCTCCGGGCACTAGC TCTACTGGTTCTCCA LCW0404_04 8GFP- N_G10.abl GSSTPSGATGSPGA SPGTSSTGSPGSSTP SGATGSP GGTAGCTCTACCCCGTCTGGTGCTACCGGTT CCCCAGGTGCTTCTCCTGGTACTAGCTCTAC CGGTTCTCCAGGTAGCTCTACCCCGTCTGGT GCTACTGGCTCTCCA LCW0404_04 9GFP- N_H10.abl GSTPSGATGSPGT PGSGTASSSPPGSSTP SGATGSP GGTAGCTCTACCCCGTCTGGTGCTACTGGTT CTCCAGGTACTCCGGGCAGCGGTACTGCTT CTTCCTCTCCAGGTAGCTCTACCCCTTCTGG TGCTACTGGCTCTCCA LCW0404_05 OGFP- N_AlEabl GASPGTS STGSPGS SPSASTGTGPGSSTP SGATGSP GGTGCATCTCCTGGTACCAGCTCTACTGGTT CTCCAGGTTCTAGCCCTTCTGCTTCTACCGG TACCGGTCCAGGTAGCTCTACTCCTTCTGGT GCTACCGGTTCTCCA LCW0404_05 1 GFP- GS STPSGATGSPGS STPSGATGGSPGSSTP GGTAGCTCTACCCCGTCTGGTGCTACTGGCT CTCCAGGTAGCTCTACTCCTTCTGGTGCTAC

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N_Bll.abl SGATGSP TGGTTCCCCAGGTAGCTCTACCCCGTCTGGT GCAACTGGCTCTCCA LCW0404_05 2GFPN_Cll.abl GASPGTSSTGSPGT PGSGTASSSPGASP GTSSTGSP GGTGCATCCCCGGGTACCAGCTCTACCGGT TCTCCAGGTACTCCTGGCAGCGGTACTGCA TCTTCCTCTCCAGGTGCTTCTCCGGGCACCA GCTCTACTGGTTCTCCA LCW0404_05 3GFPN_Dll.abl GSSTPSGATGSPGS SPSASTGTGPGASP GTSSTGSP GGTAGCTCTACTCCTTCTGGTGCAACTGGTT CTCCAGGTTCTAGCCCGTCTGCATCCACTGG TACCGGTCCAGGTGCTTCCCCTGGCACCAG CTCTACCGGTTCTCCA LCW0404_05 7GFPN_Ell.abl GASPGTSSTGSPGS STPSGATGSPGSSPS ASTGTGP GGTGCATCTCCTGGTACTAGCTCTACTGGTT CTCCAGGTAGCTCTACTCCGTCTGGTGCAA CCGGCTCTCCAGGTTCTAGCCCTTCTGCATC TACCGGTACTGGTCCA LCW0404_06 OGFP- N_Fll.abl GTPGSGTASSSPGS STPSGATGSPGASP GTSSTGSP GGTACTCCTGGCAGCGGTACCGCATCTTCC TCTCCAGGTAGCTCTACTCCGTCTGGTGCAA CTGGTTCCCCAGGTGCTTCTCCGGGTACCA GCTCTACCGGTTCTCCA LCW0404_06 2GFPN_Gll.abl GSTPSGATGSPGT PGSGTASSSPPGSSTP SGATGSP GGTAGCCTTACCCCGTCTGGTGCAACCGGC TCCCCAGGTACTCCTGGTAGCGGTACCGCT TCTTTCTTCTCCAGGTAGCTCTACTCCGTCTG GTGCTACCGGCTCCCCA LCW0404_06 6GFPNHll.abl GSSPSASTGTGPGS SPSASTGTGPGASP GTSSTGSP GGTTCTAGCCCTTCTGCATCCACCGGTACCG GCCCAGGTTCTAGCCCGTCTGCTTCTACCGG TACTGGTCCAGGTGCTTCTCCGGGTACTAG CTCTACTGGTTCTCCA LCW0404_06 7GFP- N_A12.abl GTPGSGTASSSPGS STPSGATGSPGSNP SASTGTGP GGTACCCCGGGTAGCGGTACCGCTTCTTCTT CTCCAGGTAGCTCTACTCCGTCTGGTGCTAC CGGCCTCTCCAGGTTCTAACCCTTCTGCATCC ACCGGTACCGGCCCA LCW0404_06 8GFP- N_B12.abl GSSPSASTGTGPGS STPSGATGSPGASP GTSSTGSP GGTTCTAGCCCTTCTGCATCTACTGGTACTG GCCCAGGTAGCTCTACTCCTTCTGGTGCTAC CGGCCTCTCCAGGTGCTTCTCCGGGTACTAG CTCTACCGGTTCTCCA LCW0404_06 9GFP- N_C12.abl GSSTPSGATGSPGA SPGTSSTGSPGTPGS GTASSSP GGTAGCTCTACCCCTTCTGGTGCAACCGGC TCTCCAGGTGCATCCCCGGGTACCAGCTCT ACCGGTTCTCCAGGTACTCCGGGTAGCGGT ACCGCTTCTTCCTCTCCA LCW0404_07 OGFP- N_D12.abl GSSTPSGATGSPGS STPSGATTGSPGSSTP SGATGSP GGTAGCTCTACTCCGTCTGGTGCAACCGGT TCCCCAGGTAGCTCTACCCCTTCTGGTGCAA CCGGCTCCCCAGGTAGCTCTACCCCTTCTGG TGCAACTGGCTCTCCA LCW0404_07 3GFP- N_E12.abl GASPGTSSTGSPGT PGSGTASSSPPGSSTP SGATGSP GGTGCTTCTCCTGGCACTAGCTCTACCGGTT CTCCAGGTACCCCTGGTAGCGGTACCGCAT CTTCCTCTCCAGGTAGCTCTACTCCTTCTGG TGCTACTGGTTCCCCA LCW0404_07 5GFP- N_F12.abl GSSTPSGATGSPGS SPSASTGTGPGSSPS ASTGTGP GGTAGCTCTACCCCGTCTGGTGCTACTGGCT CCCCAGGTTCTAGCCCTTCTGCATCCACCGG TACCGGTCCAGGTTCTAGCCCGTCTGCATCT ACTGGTACTGGTCCA LCW0404_08 OGFP- N_G12.abl GASPGTSSTGSPGS SPSASTGTGPGSSPS ASTGTGP GGTGCTTCCCCGGGCACCAGCTCTACTGGT TCTCCAGGTTCTAGCCCGTCTGCTTCTACTG GTACTGGTCCAGGTTCTAGCCCTTCTGCTTC CACTGGTACTGGTCCA LCW0404_08 1GFP- N_H12.abl GASPGTSSTGSPGS SPSASTGTGPGTPG SGTASSSP GGTGCTTCCCCGGGTACCAGCTCTACCGGT TCTCCAGGTTCTAGCCCTTCTGCTTCTACCG GTACCGGTCCAGGTACCCCTGGCAGCGGTA CCGCATCTTCTCTCCA

Example 5. Building XTEN_AE864.

XTEN_AE864 is built by serial dimerization of XTEN_AE36 with AE72, 144, 288, 576 and 864. A collection of XTEN_AE72 segments is built from 37 different XTEN_AE36 segments. E. soy cultures that carry all 37 different 36-amino acid segments are mixed and the plasmid is isolated. This plasmid pool is digested with Bsal/Ncol in order to obtain a small fragment as an insert. A similar plasmid pool was digested with Bbsl/Ncol in order to obtain a large fragment as a vector. The insert and vector fragments are ligated to double in length and the ligation mixture is transformed into BL21Gold(DE3) cells to produce XTEN_AE72 colonies.

This XTEN_AE72 segment library is referred to as LCW0406. All clones from LCW0406 were combined and dimerized again using the same method as described above to obtain the LCW0410 XTEN_AE144 library. All clones from LCW0410 are combined and dimerized again using the same method as described above to obtain the LCW0414 of XTEN_AE288 library. Two

- 161 041874 Isolates LCW0414.001 and LCW0414.002 are randomly selected from the library and sequenced to verify identities. All clones from LCW0414 are combined and dimerized again using the same method as described above to obtain the LCW0418 of XTEN_AE576 library. 96 isolates from the LCW0418 library were tested for high GFP fluorescence. 8 isolates with the desired insert sizes by PCR and strong fluorescence were sequenced and 2 isolates (LCW0418.018 and LCW0418.052) were selected for further use based on sequencing and expression data.

A specific clone pCW0432 XTEN_AE864 was built by combining LCW0418.018 XTEN_AE576 and LCW0414.002 XTEN_AE288 using the same dimerization method as described above.

Example 6. Building XTEN_AM144.

The XTEN_AM144 segment collection is built starting from 37 different XTEN_AE36 segments, 44 XTEN_AF36 segments, and 44 XTEN_AG36 segments.

E. coli cultures that carry all 125 different 36-amino acid segments are mixed and the plasmid is isolated. This plasmid pool is digested with BsaI/NcoI in order to obtain a small fragment as an insert. The same plasmid pool is digested with BbsI/NcoI in order to obtain a large fragment as a vector. The insert and vector fragments are ligated resulting in a length doubling, and the ligation mixture is transformed into BL21Gold(DE3) cells to produce XTEN_AM72 colonies.

This XTEN_AM72 segment library is referred to as LCW0461. All clones from LCW0461 are combined and dimerized again using the same method as described above to obtain the LCW0462 library. 1512 Isolates from the LCW0462 library were examined for protein expression. Individual colonies are transferred to 96-well plates and cultured overnight as starter cultures. These starter cultures are diluted in fresh self-inducing medium and cultured for 20-30 hours. Expression is measured using a plate reader using fluorescence with excitation at 395 nm and emission at 510 nm. 192 isolates show a high level of expression and are subjected to DNA sequencing. Most of the clones in the LCW0462 library show good expression and similar physicochemical properties, suggesting that most combinations of XTEN_AM36 segments give usable XTEN sequences. Thirty isolates from LCW0462 are selected as the preferred collection of XTEN_AM144 segments in the case of constructing multifunctional proteins that contain multiple XTEN segments. The names of the files of nucleotide and amino acid structures and sequences of these segments are given in Table. 17.

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Table 17

DNA and amino acid sequences of AM144 segments (SEQ ID NOS 529-594, respectively, in order of appearance)

Clone Truncated Sequence Protein sequence LCW462 g GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCC GTPGSGTASSSP 1 AGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCC AGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCC CAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG GAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGG TCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCG CTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCG GCCCAGGTTCTAGCCCGTCTGCTTCTACCGGTACTG GTCCAGGTGCTTCTCCGGGTACTAGCTCTACTGGTT CTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGC GCACCAGGTACCTCTACTGAACCGTCTGAGGGTAG CGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTG АААСТССА GSSTPSGATGSP GSSTPSGATGSP GSPAGSPTSTEE GTSESATPESGP GTSTEPSEGSAP GSSPSASTGTGP GSSPSASTGTGP GASPGTSSTGSP GTSTEPSEGSAP GTSTEPSEGSAP GSEPATSGSETP LCW462 g GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACC GSTSESPSGTAP 5 AGGTTCTACTAGCGAATCCCCTTCTGGTACCGCAC CAGGTACTTCTCCGAGCGGCGAATCTTCTACTGCTC CAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCT CCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGC ACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG GTCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTT CTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCT CTCCAGGTGCTTCCCCGGGTACCAGCTCTACCGGTT CTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTG CACCAGGTTCTACCAGCGAATCTCCGTCTGGCACT GCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGC ТТСТССА GSTSESPSGTAP GTSPSGESSTAP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GASPGTSSTGSP GSSTPSGATGSP GASPGTSSTGSP GSTSESPSGTAP GSTSESPSGTAP GTSTPESGSASP LCW462 g GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACC GTSTEPSEGSAP 9 AGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCC CAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGT CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGC TCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCG GTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGC GCACCAGGTACTTCTACTGAACCTTCCGAAGGTAG CGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTG AAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCC ACCGAGGAAGGTGCTTCTCCTGGCACCAGCTCTAC TGGTTCTCCAGGTTCTAGCCCTTCTGCTTCTACCGG TACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGG TACTGGTCCA GTSESATPESGP GTSESATPESGP GTSTEPSEGSAP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GSEPATSGSETP GSPAGSPTSTEE GASPGTSSTGSP GSSPSASTGTGP GSSPSASTGTGP LCW462 g GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCC GSEPATSGSETP 10 AGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTC CAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGT CCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCT CCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGC ACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCG CACCAGGTGCATCTCCGGGTACTAGCTCTACCGGT TCTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACC GGCCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG TTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCG GTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTG GCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACC GGTTCTCCA GTSESATPESGP GTSESATPESGP GSTSESPSGTAP GSTSESPSGTAP GTSPSGESSTAP GASPGTSSTGSP GSSPSASTGTGP GSSTPSGATGSP GSSTPSGATGSP GSSTPSGATGSP GASPGTSSTGSP LCW462 g GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA GASPGTSSTGSP

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15 GGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCA GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCA GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCC AGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCC CAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT CCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGG TCCAGGTACCTCTACCGAACCGTCCGAAGGCAGCG CTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGC GCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAG CGCACCAGGTACCTCTACTGAACCGTCTGAGGGTA GCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCT GAAACTCCA GSSPSASTGTGP GSSTPSGATGSP GTSESATPESGP GSEPATSGSETP GSEPATSGSETP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GSEPATSGSETP LCW462_r 16 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCC AGGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGG AAGGTACTTCTACCGAACCTTCTGAGGGTAGCGCA CCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGA CTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCT GGTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACT GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTC TGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA GCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCT GAAACTCCAGGTACTTCTACCGAACCGTCCGAGGG TAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTT CTGAAACTCCA GTSTEPSEGSAP GSPAGSPTSTEE GTSTEPSEGSAP GTSESATPESGP GSEPATSGSETP GTSESATPESGP GSPAGSPTSTEE GTSESATPESGP GTSTEPSEGSAP GSEPATSGSETP GTSTEPSEGSAP GSEPATSGSETP LCW462_r 20 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCC AGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTC CAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCA CCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGC TCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCG CTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGC GCACCAGGTACTTCTACCGAACCTTCCGAGGGCAG CGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGT CCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA TCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGG TAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTT CTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACC TCCACCGAGGAA GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GTSESATPESGP GTSTEPSEGSAP GSEPATSGSETP GSPAGSPTSTEE LCW462_r 23 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCC AGGTACTTCTACTGAACCTTCTGAAGGCAGCGCTC CAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCA CCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCT CCAGGTTCTACCAGCGAATCCCCTTCTGGCACCGC ACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT CTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAA ACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATC TGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCA GCGCACCAGGTACTTCTACTGAACCGTCTGAAGGT AGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGA GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GSTSESPSGTAP GSTSESPSGTAP GTSTPESGSASP GSEPATSGSETP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP

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ATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGG AGTCCGGCCCA GTSESATPESGP LCW462 g GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCA GSSTPSGATGSP 24 GGTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCA GSSPSASTGTGP GGTAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA GSSTPSGATGSP GGTAGCCCCTGCTGGCTCTCCGACTTCTACTGAGGA GSPAGSPTSTEE AGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGG GSPAGSPTSTEE AAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCT GTSTEPSEGSAP CCAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCT GASPGTSSTGSP CCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGC GSSPSASTGTGP CCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCT GTPGSGTASSSP CCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGC GSTSSTAESPGP CCAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCT GTSPSGESTAP CCAGGTACCTCTACTCCGGAAAAGCGGTTCTGCATC TCCA GTSTPESGSASP LCW462 g GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCC GTSTEPSEGSAP 27 AGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTC GTSESATPESGP CAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA GTSTEPSEGSAP CCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGC GTSTEPSEGSAP ACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCG GTSESATPESGP GCCCAGGTACCCTTGAAAGCGCAACCCCGGAGTCC GTSESATPESGP GGCCCAGGTACTCCTGGCAGCGGTACCGCTTCTTC GTPGSGTASSSP TTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGG GASPGTSSTGSP TTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGG GASPGTSSTGSP TTCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTAC GSPAGSPTSTEE TGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA GSPAGSPTSTEE CTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGT AGCGCTCCA GTSTEPSEGSAP LCW462 g GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGA GSPAGSPTSTEE 28 AGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC GTSTEPSEGSAP CAGGTACCCTACTGAACCTTCTGAGGGCAGCGCT GTSTEPSEGSAP CCAGGTACCTCTACCGAACCGTCTGAAGGTAGCGC GTSTEPSEGSAP ACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCCG GTSESATPESGP GTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCT GTSESATPESGP GGCCCAGGTACCCCGGGTAGCGGTACTGCTTCTTC GTPGSGTASSSP CTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGG GSSTPSGATGSP CTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCG GASPGTSSTGSP GTTCTCAGGTACCTCTACTGAACCTTCTGAGGGC GTSTEPSEGSAP AGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGA GTSESATPESGP GTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAG GTAGCGCACCA GTSTEPSEGSAP LCW462 g GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCC GSEPATSGSETP 38 AGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCC GTSESATPESGP CAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACC GSEPATSGSETP CCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTC GSSTPSGATGSP CCCAGGTACTCCTGGTAGCGGTACCCGCTTCTTCTTC GTPGSGTASSSP TCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTC GSSTPSGATGSP CCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTC GASPGTSSTGSP TCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTC GSSTPSGATGSP

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TCCAGGTGCTTCCCCGGGTACCAGCTCTACCGGTTC TCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAA CTCCAGGTACTTCTACCGAACCGTCCGAGGGTAGC GCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA AACTCCA GASPGTSSTGSP GSEPATSGSETP GTSTEPSEGSAP GSEPATSGSETP LCW462_r 39 GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCC AGGTACCTCTACCGAACCGTCCGAGGGCAGCGCAC CAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGT CCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGA GGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTG AGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGC GCTCCAGGTAGCCCGGCTGGTTCTCCGACTTCCAC CGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTA GCGCTCCAGGTACCTCTACTGAACCTTCCGAAGGC AGCGCTCCAGGTGCTTCCCCGGGCACCAGCTCTAC TGGTTCTCCAGGTTCTAGCCCGTCTGCTTCTACTGG TACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGG TACTGGTCCA GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GSPAGSPTSTEE GSPAGSPTSTEE GTSTEPSEGSAP GSPAGSPTSTEE GTSTEPSEGSAP GTSTEPSEGSAP GASPGTSSTGSP GSSPSASTGTGP GSSPSASTGTGP LCW462_r 41 GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCA GGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCA GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGA AGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCC CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC CCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCT CCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTC TCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTGG TCCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGC TCCAGGTTCTACCAGCGAATCCCCTTCTGGCACCG CACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCT TCTCCA GSSTPSGATGSP GASPGTSSTGSP GSSTPSGATGSP GSPAGSPTSTEE GTSESATPESGP GSEPATSGSETP GASPGTSSTGSP GSSTPSGATGSP GSSPSASTGTGP GSTSESPSGTAP GSTSESPSGTAP GTSTPESGSASP LCW462_r 42 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCA GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACC AGGTACTTCTCCTAGCGGCGAATCTTCTACCGCAC CAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGC CCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGC TCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG CACCAGGTACCTCTACTGAACCTTCTGAGGGCAGC GCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTC CGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTA GCGCACCAGGTAGCTCTACCCCGTCTGGTGCTACC GGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTACC GGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACT GGCTCTCCA GSTSESPSGTAP GSTSESPSGTAP GTSPSGESSTAP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GTSTEPSEGSAP GSSTPSGATGSP GASPGTSSTGSP GSSTPSGATGSP LCW462_r 43 GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCC AGGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCC AGGTACTTCCGAGCGGTGAATCTTCTACCGCTCC AGGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCC AGGTTTCTACCAGCTCTACTGCAGAATCTCCTGGCC GSTSSTAESPGP GSTSSTAESPGP GSTSSTAESPGP GSTSSTAESPGP GSTSSTAESPGP GSTSSTAESPGP

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CAGGTACTTCTACTCCGGAAAAGCGGTTCCGCTTCTC CAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTC CAGGTTTCTACCAGCTCTACTGCTGAATCTCCTGGCC CAGGTACTTCTACCCCGGAAAGCGGCTCCGCTTCT CCAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGC AC CCAGGTTCTACTAGCGAATCACTCGTGTCTGGCACGCACTCGTCTGTCCACCGCACAGGTCCTGCC GTSTPESGSASP GTSPSGESSTAP GSTSSTAESPGP GTSTPESGSASP GSTSSTAESPGP GSTSESPSSGTAP GTSPSGESSTAP LCW462_r 45 GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCC AGGTTCTACCAGCGAATCCCCGTCTGGCACCGCAC CAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGCC CAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCT CCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGC ACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG GTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCT GGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAG CGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTA GCGCACCAGGTACTTCTGAAAGCGCTACTCCGGAG TCCGGTCCAGGTACCTCTACCGAACCGTCCGAAGG CAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG GTAGCGCTCCC GTSTPESGSASP GSTSESPSGTAP GSTSSTAESPGP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP LCW462_r 47 GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACC AGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTC CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACT CCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGC ACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCG GCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCC GGCCCAGGTGCATCTCCGGGTACTAGCTCTACCGG TTCTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTAC CGGCCCAGGTAGCTCTACCCCGTCTGGTGCTACTG GTTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACC GGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACT GGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTAC CGGTTCTCCA GTSTEPSEGSAP GTSTEPSEGSAP GSEPATSGSETP GTSTEPSEGSAP GTSESATPESGP GTSESATPESGP GASPGTSSTGSP GSSPSASTGTGP GSSTPSGATGSP GSSTPSGATGSP GSSTPSGATGSP GASPGTSSTGSP LCW462_r 54 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCC AGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCC CAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCA CCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAAC CCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTG GCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCAGGTAGCTCTACTCCGTCTGGTGCTACCGG CTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGG CTCCCCAGGTGCTTCTCCGGGTACCAGCTCTACTGG TTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGG TTCCCCAGGTGCTTCTCCTGGTACTAGCTCTACCGG TTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG CTCTCCA GSEPATSGSETP GSEPATSGSETP GTSTEPSEGSAP GSEPATSGSETP GTSESATPESGP GTSTEPSEGSAP GSSTPSGATGSP GSSTPSGATGSP GASPGTSSTGSP GSSTPSGATGSP GASPGTSSTGSP GSSTPSGATGSP LCW462r GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCC GTSTEPSEGSAP

AGGTACTTCTACTGAACCTTCTGAAGGCAGCGCTC GTSTEPSEGSAP

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CAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCA CCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGG TCCAGGTACCTCTACCGAACCGTCCGAAGGCAGCG CTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGC GCTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACT GCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACC GCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACC GCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACT GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTC TGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA GCGCTCCA GTSTEPSEGSAP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GSTSESPSGTAP GTSPSGESSTAP GTSPSGESSTAP GSPAGSPTSTEE GTSESATPESGP GTSTEPSEGSAP LCW462_r 57 GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCC AGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCC CAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAG GAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGA GGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCG GCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGC GCACCAGGTACCTCTACTGAACCTTCCGAAGGCAG CGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCA GCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAA TCCGGTCCAGGTAGCTCTACTCCGTCTGGTGCAAC CGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGG TACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTA CTGGTTCTCCA GTSTEPSEGSAP GSEPATSGSETP GSPAGSPTSTEE GSPAGSPTSTEE GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GSSTPSGATGSP GSSPSASTGTGP GASPGTSSTGSP LCW462_r 61 GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCC AGGTAGCCCTGCTGGCTCTCCGACCTCTACCGAAG AAGGTACCTCTGAAAGCGCTACCCCTGAGTCTGGC CCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGC TCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCG CACCAGGTACTTCTGAAAGCGCAACCCCTGAATCC GGTCCAGGTACCTCTACTCCGGAAAGCGGTTCCGC ATCTCCAGGTTCTACCAGCGAATCCCCGTCTGGCA CCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTC CGGGCCCAGGTACTTCTGAAAGCGCTACTCCGGAG TCCGGTCCAGGTACCTCTACCGAACCGTCCGAAGG CAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG GTAGCGCTCCA GSEPATSGSETP GSPAGSPTSTEE GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GTSTPESGSASP GSTSESPSGTAP GSTSSTAESPGP GTSESATPESGP GTSTEPSEGSAP GTSTEPSEGSAP LCW462_r64 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCC AGGTACTTCTACTGAACCTTCTGAAGGCAGCGCTC CAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCA CCAGGTACCTCTACCGAACCGTCTGAAGGTAGCGC ACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCCG GTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCT GGCCCAGGTACTCCTGGCAGCGGTACCGCATCTTC CTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACTGG TTCCCCAGGTGCTTCTCCGGGTACCAGCTCTACCGG TTCTCCAGGTTCCACCAGCTCTACTGCTGAATCTCC TGGTCCAGGTACCTCTCCTAGCGGTGAATCTTCTAC TGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGC GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GTSESATPESGP GTPGSGTASSSP GSSTPSGATGSP GASPGTSSTGSP GSTSSTAESPGP GTSPSGESSTAP GTSTPESGSASP

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TTSTSSA LCW462 g GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGA GSPAGSPTSTEE 67 AGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCC GTSESATPESGP CAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCA GTSTEPSEGSAP CCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG GTSESATPESGP TCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGA GSEPATSGSETP CTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC GTSTEPSEGSAP GCACCAGGTAGCCCGGCTGGTTTCCCGACTTCCAC GSPAGSPTSTEE CGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTA GTSTEPSEGSAP GCGCTCCAGGTACCTCTACTGAACCTTCCGAAGGC GTSTEPSEGSAP AGCGCTCCAGGTACTTCTACCGAACCGTCCGAGGG GTSTEPSEGSAP CAGCGCTCCAGGTACTTCTACTGAACCTTCTGAAG GTSTEPSEGSAP GCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAA GGTAGCGCACCA GTSTEPSEGSAP LCW462 g GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACC GTSPSGESTAP 69 AGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCC GSTSSTAESPGP CAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTC GTSPSGESTAP CAGGTACCCTTGAAAGCGCTACTCCGGAGTCTGGC GTSESATPESGP CCAGGTACCCTACTGAACCGTCTGAGGGTAGCGC GTSTEPSEGSAP TCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG GTSTEPSEGSAP CACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTG GSSPSASTGTGP GCCCAGGTAGCTCTACTCCTTCTGGTGCTACCGGCT GSSTPSGATGSP CTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTT GASPGTS STGSP CTCCAGGTACTTCTACTCCGGAAACGGGTTCCGCA GTSTPESGSASP TCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACT GTSPSGESTAP GCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACT GCTCCA GTSPSGESTAP LCW462 g GGTACCCTTGAAAGCGCTACTCCGGAGTCTGGCCC GTSESATPESGP 70 AGGTACCCTACTGAACCGTCTGAGGGTAGCGCTC GTSTEPSEGSAP CAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA GTSTEPSEGSAP CCAGGTAGCCCCTGCTGGCTCTCCGACTTCTACTGA GSPAGSPTSTEE GGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTG GSPAGSPTSTEE AGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGC GTSTEPSEGSAP GCTCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACT GSSPSASTGTGP GGCCCAGGTAGCTCTACCCCTTCTGGTGCTACCGG GSSTPSGATGSP CTCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGG GSSTPSGATGSP CTCTCCAGGTAGCGAACCGGCAACTTCCGGCTCTG GSEPATSGSETP AAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAG GTSESATPESGP TCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTC TGAAACCCCA GSEPATSGSETP LCW462 g GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCC GTSTEPSEGSAP 72 AGGTACCCTACTGAACCTTCCGAGGGCAGCGCTC GTSTEPSEGSAP CAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCA GTSTEPSEGSAP CCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCC GSSTPSGATGSP CCAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCT GASPGTS STGSP CCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCT GSSTPSGATGSP CCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG GTSESATPESGP TCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGA GSEPATSGSETP CTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC GTSTEPSEGSAP

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GCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACT GCACCAGGTTCTACCAGCGAATCTCCGTCTGGCAC TGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCG STTSTSCA GSTSESPSGTAP GSTSESPSGTAP GTSTPESGSASP LCW462 g GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCA GTSTPESGSASP 73 GGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCC AGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCC AGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCC AGGTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCC AGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCC AGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCC CAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGC CCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGA GGAAGGTTCTACTAGCGAATCTCCTTCTGGCACTG CACCAGGTTCTACCAGCGAATCTCCGTCTGGCACT GCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGC ТТСТССС GSTSSTAESPGP GSTSSTAESPGP GSSPSASTGTGP GSSTPSGATGSP GASPGTSSTGSP GSEPATSGSETP GTSESATPESGP GSPAGSPTSTEE GSTSESSPSGTAP GSSTSESPSGTAP GTSTPESGSASP LCW462 g GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGA GSPAGSPTSTEE 78 AGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTC CAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT CCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCT CCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGC ACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCG CACCAGGTACCTCTACCGAACCTTCCGAAGGTAGC GCTCCAGGTAGCCCGGCAGGTTCTCCTACTTCCACT GAGGAAGGTACTTCTACCGAACCTTCTGAGGGTAG CGCACCAGGTAGCGAACCTGCAACCTCTGGCTCTG AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGG CAGCGCACCA GTSESATPESGP GTSTEPSEGSAP GSTSESPSGTAP GSTSESPSGTAP GTSPSGESSTAP GTSTEPSEGSAP GSPAGSPTSTEE GTSTEPSEGSAP GSEPATSGSETP GTSESATPESGP GTSTEPSEGSAP LCW462 g GGTACCTCTACCGAACCTTCCGAAGGTAGCCGCTCC GTSTEPSEGSAP 79 AGGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGG AAGGTACTTCTACCGAACCTTCTGAGGGTAGCGCA CCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCT CCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCT CCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGC ACCAGGTTCTACCAGCGAATCCCCTTCTGGTACTG CTCCAGGTTCTACCAGCGAATCCCCTTCTGGCACC GCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGC TTCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTG AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGG CAGCGCACCA GSPAGSPTSTEE GTSTEPSEGSAP GTSPSGESSTAP GTSPSGESSTAP GTSPSGESSTAP GSTSESPSGTAP GSTSESPSGTAP GTSTPESGSASP GSEPATSGSETP GTSESATPESGP GTSTEPSEGSAP LCW462 g GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCC GSEPATSGSETP 87 AGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTC CAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGT CCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGC ACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGG GCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACT GTSESATPESGP GTSESATPESGP GTSPSGESSTAP GSTSSTAESPGP GTSPSGESSTAP

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LCW462_r 88 LCW462_r 89 GCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACT GCTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACT GCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCC GGGTCCAGGTAGCTCTACTCCGTCTGGTGCAACCG GTTCCCCAGGTAGCTCTACCCCTTCTGGTGCAACCG GCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAAACT GGCTCTCC GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGA AGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGG AAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCT CCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGC TCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCG CACCAGGTACTTCTGAAAGCGCAACCCCTGAATCC GGTCCAGGTGCATCTCCTGGTACCAGCTCTACCGG TTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGG CTCTCCAGGTGCTTCCCCGGGTACCAGCTCTACCG GTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTG GTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTT CCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTG GCTCTCCA GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCA GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA GGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCA GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGA AGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTC CAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT CCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGA CTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCT GGTCCAGGTACTTCTACTGAACCGTCTGAAGGTAG CGCACCAGGTACTTCTGAAAGCGCAACCCCGGAAT CCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAG TCCGGCCCA GSTSESPSGTAP GTSPSGESSTAP GSTSSTAESPGP GSSTPSGATGSP GSSTPSGATGSP GSTPSGANWL S GSPAGSPTSTEE GSPAGSPTSTEE GTSTEPSEGSAP GTSTEPSEGSAP GTSTEPSEGSAP GTSESATPESGP GASPGTSSTGSP GSSTPSGATGSP GASPGTSSTGSP GSSTPSGATGSP GTPGSGTASSSP GSSTPSGATGSP GSSTPSGATGSP GTPGSGTASSSP GSSTPSGATGSP GSPAGSPTSTEE GTSESATPESGP GTSTEPSEGSAP GTSESATPESGP GSEPATSGSETP GTSESATPESGP GTSTEPSEGSAP GTSESATPESGP GTSESATPESGP

Example 7. Building XTEN_AM288.

The entire LCW0462 library was dimerized as described in Example 6, resulting in an XTEN_AM288 clone library designated LCW0463. 1512 isolates from the LCW0463 library were examined using the protocol described in Example 6. The 176 highly expressed clones were sequenced and 40 preferred XTEN_AM288 segments were selected to construct multifunctional proteins that contain multiple 288 amino acid residue XTEN segments.

Example 8. Building XTEN_AM432.

The XTEN_AM432 segment library is obtained using recombination segments from the LCW0462 segment library XTEN_AM144 and segments from the LCW0463 segment library XTEN_AM288. This new XTEN_AM432 segment library is referred to as LCW0464. Plasmids were isolated from E. coli cultures carrying LCW0462 and LCW0463, respectively. 1512 isolates from the LCW0464 library were examined using the protocol described in Example 6. The 176 highly expressed clones were sequenced and 39 preferred XTEN_AM432 segments were selected to construct a longer XTEN and to construct multifunctional proteins that contain multiple 432 amino acid residue XTEN segments.

At the same time, an LMS0100 library of XTEN_AM432 segments is built using the preferred XTEN_AM144 and XTEN_AM288 segments. Testing of this library yielded 4 isolates, which were selected for further construction.

Example 9. Building XTEN_AM875.

Spacer vector pCW0359 was digested with BsaI and KpnI to remove the spacer segment, and the resulting vector fragment was isolated by agarose gel purification.

The phosphorylated BsaI-AscI-KpnIforP oligonucleotide is renatured:

AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC(SEQIDNO: 1652) and unphosphorylated BsaI-AscI-KpnIrev oligonucleotide:

CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCCCGCTTGCGCTTGC (SEQ ID NO: 1653) to introduce a sequencing island A (SI-A) that encodes the amino acids GASASGAPSTG (SEQ ID NO: 1654) and has internally the restriction enzyme AscI recognizing the nucleotide sequence GGCGCGCC. The renatured BsaI and KpnI oligonucleotide pairs are ligated with the pCW0359 cleavable spacer vector obtained above to obtain pCW0466 which contains SI-A. The XTEN_AM443 segment library is then prepared by recombining the 43 preferred XTEN_AM432 segments in Example 8 and the SI-A segments from pCW0466 at the C-terminus using the same dimerization method described in Example 5. This new XTEN_AM443 segment library is designated LCW0479. Obtain XTEN_AM875 segment library using recombination segments from LCW0479 segment library XTEN_AM443 and 43 preferred segments

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XTEN_AM432 from example 8 using the same dimerization method described in example 5.

This new XTEN_AM875 segment library is referred to as LCW0481.

Example 10. Building XTEN_AM1318.

Renature the phosphorylated BsaI-FseI-KpnIforP oligonucleotide:

AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC(SEQIDNO: 1655) and unphosphorylated BsaI-FseI-KpnIrev oligonucleotide:

CCTCGAGTGAAGACGAACCTCCGCTTGGGCCGGCCCCGTTGGTTCTGG(SEQIDNO: 1656) to introduce sequencing island B (SI-B) that encodes the amino acids GPEPTGPAPSG (SEQ ID NO: 1657) and has the restriction enzyme FseI recognition of the nucleotide sequence GGCCGGCC inside. Renatured oligonucleotide pairs of BsaI and KpnI are ligated to be cleaved with the pCW0359 spacer vector as used in Example 9 to obtain pCW0467 which contains SI-B. An XTEN_AM443 segment library is then prepared by recombining the 43 preferred XTEN_AM432 segments from Example 8 and the SI-B segments from pCW0467 at the C-terminus using the same dimerization method described in Example 5. This new XTEN_AM443 segment library is designated LCW0480.

The XTEN_AM1318 segment library was prepared using recombination segments from the XTEN_AM443 segment library LCW0480 and segments from the XTEN_AM875 segment library LCW0481 using the same dimerization method as in Example 5. This new XTEN_AM1318 segment library is designated LCW0487.

Example 11: Building XTEN_AD864.

Using several successive rounds of dimerization, a collection of XTEN_AD864 sequences was assembled, starting with the XTEN_AD36 segments shown in Example 1. These sequences were collected as described in Example 5. Some XTEN_AD864 isolates were evaluated and determined to demonstrate good expression and excellent solubility under physiological conditions. The XTEN_AD576 intermediate construct is sequenced. This clone was evaluated in the PK experiment on cynomolgus monkeys and a half-life of about 20 hours was measured.

Example 12. Building XTEN_AF864.

Using several successive rounds of dimerization, a collection of XTEN_AF864 sequences was assembled, starting with the XTEN_AF36 segments shown in Example 3. These sequences were collected as described in Example 5. Some XTEN_AF864 isolates were evaluated and determined to show good expression and excellent solubility under physiological conditions. The XTEN_AF540 intermediate construct is sequenced. This clone was evaluated in the PK experiment on cynomolgus monkeys and a half-life of about 20 hours was measured. The full-length clone XTEN_AF864 has excellent solubility and exhibits a half-life that is in excess of 60 hours for cynomolgus monkeys. A second set of XTEN_AF sequences is assembled which contains the sequencing islet as described in Example 9.

Example 13. Building XTEN_AG864.

Using several successive rounds of dimerization, a collection of XTEN_AG864 sequences was assembled, starting with the XTEN_AD36 segments shown in Example 1. These sequences were collected as described in Example 5. Some XTEN_AG864 isolates were evaluated and determined to show good expression and excellent solubility under physiological conditions. The full-length clone XTEN_AG864 has excellent solubility and exhibits a half-life that is in excess of 60 hours for cynomolgus monkeys.

Example 14. Methods for obtaining and evaluating CFXTEN with internal and terminal XTEN.

Creation, construction and evaluation of CFXTEN, which contains FVIII and one or more XTEN, carried out using a systematic approach. Regions suitable for XTEN insertion sites include, but are not limited to, regions at or close to known FVIII domain boundaries, exon boundaries, known outer loops, low order regions, and hydrophilic regions. Through the analysis of the above, various regions throughout the sequence of the FVIII sequence without B domain (BDD) are identified as XTEN insertion sites, non-limiting examples of which are given in Table. 5-8 and schematically illustrated in FIG. 8 and 9. First, separate constructs are created (using the methods described below) in which the DNA encodes a unique XTEN or an XTEN fragment with a length ranging from 6 to 2004 amino acid residues, inserted into the FVIII sequence corresponding to or close to (for example , within 6 amino acids) to each of the unique insertion sites, which are defined in Table. 5, tab. 6, tab. 7, tab. 8 and tab. 9, and the resulting constructs that express and regenerate the protein are then evaluated for their effect on maintaining the use of procoagulant activity, for example, one of the in vitro assays from Table. 49. For example, using the methods described below, constructs are created in which the XTEN sequence is introduced within the sequences of the FVIII A1, A2, B, A3, C1, and C2 domains, as well as associated with the C-terminus, and the resulting express fusion proteins are evaluated using chromogenic analysis from table. 49 compared to FVIII not associated with XTEN. The CFXTEN fusion proteins can be further subdivided into high, medium, and low categories based on the actions they exhibit. In cases where CFXTEN exhibits activity that is comparable or slightly lower than FVIII, the insertion site is considered appropriate. In cases where the activity is intermediate, the insertion site can be changed 1-6 amino acids towards the N- or C-terminus of the insertion site and/or the length or net charge of the XTEN can be changed and the resulting construct(s) re-evaluated. to determine if activity is improving. In addition, XTEN is introduced into a construct with flanking cleavage sites; preferably, sites that are susceptible to cleavage by proteases are provided in coagulation activity assays such that XTEN is released during activation of the FVIII component, thereby providing further information on the usefulness of the XTEN insertion site in the fusion protein.

Once all individual insertion sites have been evaluated and favorable insertion sites have been identified, libraries of constructs are created with two, three, four, five or more XTENs administered in favorable site permutations. The length and net charge of the XTEN (eg, XTEN family AE versus AG) are modified to determine the effects of these variables on FVIII activity and the physicochemical properties of the fusion protein. CFXTEN constructs that retain the desired degree of FVIII procoagulant activity in vitro are then evaluated in vivo using a murine and/or canine hemophilia A model as described in the examples below, or other models known in the art. In addition, constructs are analyzed in the presence of FVIII inhibitors and other anti-FVIII antibodies to determine constructs that retain activity. In addition, CFXTEN constructs are designed that insert cleaved sequences at or near the FVIII and XTEN junction region(s) (e.g., sequences from Table 8) designed to secrete XTEN and are evaluated for increased FVIII activity and end-time effects. half life. Using a cyclical method for obtaining constructs combining different insertion sites, different lengths and qualities and evaluating the composition of XTEN (for example, different families of XTEN), the specialist receives, using the above methods, CFXTEN with the desired properties, such as, but without limitation, procoagulant FVIII activity, reduced binding to FVIII inhibitors, improved pharmacokinetic properties, the ability to administer to a subject in a variety of ways, and/or improved pharmaceutical properties.

Example 15 Methods for Producing and Evaluating CFXTEN Which Contains FVIII and AE_XTEN.

The general scheme for preparing and evaluating CFXTEN compositions is illustrated in FIG. 15 and forms the basis for a general description of this Example. Using the disclosed methods and those known to any person skilled in the art, together with the guidance provided in the illustrative Examples, a person skilled in the art can design and evaluate CFXTEN fusion proteins that contain XTEN and FVIII, or FVIII variants known in the art. this field of technology. The example is therefore to be construed as illustrative only and not as limiting in any way; numerous variations will be apparent to those skilled in the art. In this Example, factor VIII BDD CFXTEN is generated, linked to the XTEN family of AE motifs.

The general scheme for the production of polynucleotides encoding XTEN is illustrated in FIG. 11 and 12. FIG. 14 is a schematic block diagram of representative assembly steps for an XTEN polynucleotide construct in certain embodiments of the present invention. Individual 501 oligonucleotides are annealed into 502 sequence motifs, such as a 12-amino acid motif (12-mer), which are ligated with additional sequence motifs from the library, which can be multimerized to create a pool that spans the desired length of XTEN 504, and also ligated with a lower concentration of oligo-containing restriction sites BbsI and KpnI 503. Motif libraries contain a specific sequence of the XTEN family; for example, the sequence AD, AE, AF, AG, AM or AQ from the table. 3, as illustrated in FIG. 14, the length of XTEN in this case is 36 amino acid residues, but longer lengths are achieved by this general method. For example, multimerization is carried out by ligation, PCR with overlapping primers, assembly PCR, or similar cloning techniques known in the art, which in this case result in a 288 amino acid residue construct. The resulting pool of ligation products is gel purified and the XTEN band is cut to the desired length, resulting in an isolated XTEN gene with a stopper sequence of 505. The XTEN gene can be cloned into a spacer vector. In this case, the vector encodes an additional CBD 506 sequence and a GFP 508 gene. BbsI/HindIII cleavage is then performed to remove 507 and 508 and place a stop codon. The resulting product is then cloned into a BsaI/HindIII cleavable vector that contains the gene encoding FVIII, resulting in the 500 gene encoding a fusion protein CFXTEN with 288 amino acid XTEN linked to the C-terminus of factor VIII. As should be apparent to any person skilled in the art, the methods are used to generate constructs in alternative conformations, with different XTEN lengths, or in multiple locations.

FVIII-encoding DNA sequences are usually obtained using standard methods known in the art from a cDNA library obtained from an appropriate glue.

- 173 041874 accurate source, from a genomic library, or can be created synthetically (eg, automated nucleotide synthesis) using a DNA sequence obtained from public databases, patents, or literary sources. In the present example, a FVIII variant without a B domain (BDD) is prepared as described in Example 17. The gene or polynucleotide encoding a portion of the FVIII protein or its complement is then cloned into a construct such as described herein, which may be a plasmid or other vector under the control of appropriate transcription and translation sequences at a high level of protein expression in a biological system. The second gene or polynucleotide encoding the XTEN portion or its complement is genetically fused to nucleic acids encoding the end of the FVIII gene by cloning it into a construct adjacent to and in frame with the gene encoding CF through a ligation or multimerization step. Thus, within the construct, a DNA fusion molecule encoding (or complementary to) the CFXTEN fusion protein is obtained. Optionally, the gene encoding the second XTEN is inserted and ligated within a reading frame internal to the nucleic acids encoding the FVIII coding region. Constructs create in different conformations to encode different insertion sites XTEN in sequences FVIII, which contains those of the table. 5, tab. 6, tab. 7, tab. 8 and tab. 9 or those illustrated in FIG. 8-9., optionally, the fused DNA molecule is transported or cloned into another construct that is a more appropriate expression vector; for example, a vector corresponding to a mammalian host cell such as CHO, BHK and the like. At this point, a host cell capable of expressing the fused DNA molecule is transformed with the fused DNA molecule described more fully below, or by well-known methods, depending on the type of host cell, as described above.

Host cells that contain XTEN-FVIII expressing the vector are cultured in conventional nutrient media modified to match promoter activation. Culture conditions such as temperature, pH, and the like, which have previously been used in the host cell selected for expression, will be apparent to one skilled in the art. After expression of the fusion protein, the culture broth is collected and separated from the cell mass and a crude extract is obtained, which is stored for purification of the fusion protein.

Gene expression is measured directly in the sample, for example, using conventional Southern blot, Northern blot to quantify mRNA transcription [Thomas, Proc. Natl. Acad. sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization using an appropriately labeled probe, based on the sequence provided herein. In addition, gene expression is measured using immunological fluorescent methods such as immunohistochemical staining of cells to directly quantify the gene expression product. Antibodies useful for immunohistochemical staining and/or analysis of sample fluids can be either monoclonal or polyclonal and can be obtained from any mammal. For convenience, antibodies can be generated against a FVIII sequence polypeptide using a synthetic peptide based on the sequences provided herein, or against an exogenous sequence fused to FVIII and encoding a specific antibody epitope. Examples of selectable markers are well known to those skilled in the art and contain reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal), or chloramphenicol acetyltransferase (CAT).

The CFXTEN polypeptide product is purified using methods known in the art. Methods such as gel chromatography, affinity purification, salt fractionation, ion exchange chromatography, gel permeation chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography or gel electrophoresis are all methods that can be used in purification. Specific purification methods are described in Robert K. Scopes, Protein Purification: Principles and Practice, Charles R. Castor, ed., Springer-Verlag 1994, and Sambrook, et al., supra. Multi-stage purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994).

As illustrated in FIG. 15, the isolated CFXTEN fusion proteins are characterized by their chemical and activity properties. An isolated fusion protein is characterized, for example, by sequence, purity, average molecular weight, solubility, and stability using standard methods known in the art. A fusion protein that meets the intended standards is evaluated for activity that can be measured in vitro or in vivo by measuring one of the Factor VIII related parameters described herein, using one or more of the assays disclosed herein, or with using analyzes from examples or table. 49.

In addition, the FVIII CFXTEN fusion protein is administered with one or more animal species to determine standard pharmacokinetic parameters and pharmacodynamic properties as described in Examples 25 and 26.

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Using a cyclic method for the production, expression and extraction of CFXTEN constructs, followed by their characterization using the methods disclosed in this document or others known in the art, CFXTEN compositions that contain CF and XTEN are generated and evaluated to confirm the expected properties, such as improved solubility, improved stability, improved pharmacokinetic properties and reduced immunogenicity, which lead to an overall improved therapeutic activity compared to the corresponding unfused FVIII. These fusion proteins that do not have the desired properties are designed in a different sequence or conformation, expressed, isolated and evaluated using these methods in order to obtain compositions with similar properties.

Example 16 Construction of BDD FVIII expression plasmids.

I. Construction of FVIII expression vectors without B domain (BDD FVIII).

An expression vector that encodes the FVIII BDD was created by cloning the FVIII BDD open reading frame into the pcDNA4 vector (Invitrogen, CA) which contains polyA for optimal expression of the mammalian FVIII gene, resulting in the construct designated pBC0100. Several natural sites are defined within this construct for use in cloning which contains BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI 4500 , PmeI 4527. To facilitate analysis, nucleic acids encoding Myc and histidine tags are introduced into the open reading frame of FVIII. pBC0100 is amplified by the PCR method using the following primers: 1) F8-BsiWI-F: tattccCGTACGgccgccaccATGCAAATAGAGCTCTCCACCT (SEQ ID NO: 1658); 2) F8-nostop-XhoI-R1: GGTGACCTCGAGcgtagaggtcctgtgcctcg (SEQ ID NO: 1659) to introduce BsiWI and XhoI to the respective sites. The PCR product is digested with BsiWI and XhoI. PcDNA4-Myc-His/C was digested with Acc65I and XhoI from which two products were obtained 5003 and 68 bps. The 5003bps product was ligated with a cleavable FVIII fragment, which was subjected to PCR and used to transform DH5alpha. The Acc65I and BsiWI enzymes create compatible ends, but this ligation suppresses the site if further cleavage occurs. The resulting construct is designated pBC0102 (pcDNA4-FVIII_3-Myc-His). To facilitate the design and implementation of future cloning strategies, especially those involving the generation of BDD FVIII expression constructs containing multiple XTEN inserts, additional unique restriction enzyme sites are selected for inclusion that contain BsiWI 908, NheI 1829, and ClaI 3281. Introduction of these sites is accomplished by QuikChange method (Agilent, CA) individually. The resulting construct is designated pBC0112 (pcDNA4-FVIII_4Myc-His). To avoid problems that can occur with linker peptides that connect between Myc/His and FVIII/Myc, and to remove restriction enzyme sites that are preferred for further XTEN insertion, the sequences encoding the peptide sequences from ARGHPF (SEQ ID NO : 1660) to GAGSPGAETA (SEQ ID NO: 178) (between FVIII and Myc), NMHTG (SEQ ID NO: 1661) to SPATG (SEQ ID NO: 1662) (between Myc and His) via the QuikChange method. The construct is designated pBC0114 (pcDNA4-FVIII_4GAGSPGAETA-Myc-SPATG-His (GAGSPGAETA and SPATG, disclosed as SEQ ID NOS 178 and 1662, respectively)) (sequence in Table 21), which is used as the main vector for the construction and design of other expressing vectors incorporated into the XTEN sequence. FVIII expression and activity data for this construct are presented.

II. Construction of FVIII expression vectors without the B domain (BDD FVIII).

The gene encoding BDD FVIII was synthesized by GeneArts (Regensburg, Germany) in a pMK cloning vector (pMK-BDD FVIII). The FVIII BDD proteins contain 1457 amino acids with a total molecular weight of 167539.66. Within the wild-type FVIII protein are 6 domains, domains A1, A2, B, A3, C1 and C2. In the FVIII BDD protein, most of the B domain is deleted, as it has been shown that the unstructured domain and domain deletion do not alter the critical functions of this protein. The pMK vector used by GeneArts does not contain a promoter and cannot be used as an expression vector. Restriction enzyme sites NheI at the 5' end and SfiI, SalI and XhoI at the 3' end are introduced to facilitate subcloning of the DNA sequence encoding BDD FVIII in expression vectors such as CET1019-HS (Millipore). Some unique restriction enzyme sites are also introduced into the FVIII sequence in order to allow further manipulation (eg insertion, mutagenesis) of DNA sequences. Unique sites listed with their incision site include, but are not limited to: SacI 391, AfiII 700, SpeI 966, PshAI 1417, Acc65I 2192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, ApaI 3893, Bsp1201 3893 , SwaI 4265, OliI 4626, XbaI 4644, and BstBI 4673. The HindIII site is located at the very end of the A2 domain and can potentially be used to modify the B domain. The synthesized pMK-BDD FVIII from GeneArts does not contain a stop codon. The stop codon is introduced by amplifying the 127 bp FVIII fragment using the following primers: 5'-GTGAACTCTCTAGACCCACCG-3' (SEQ ID NO: 1663); 5'CTCCTCGAGGTCGACTCAGTAGAGGTCCTGTGCCTCG-3' (SEQ ID NO: 1664). The fragment was digested with XbaI and SalI and ligated with XbaI/SalI cleavable pMK-BDD FVIII. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened from

- 175 041874 with the power of minipreparative DNA, and the desired constructs are confirmed by DNA sequencing. The construct, referred to as pBC0027 (pMK-BDD FVIII-STOP), contains coding sequences that code for the FVIII BDD protein. The pBC0027 construct was then digested with NheI/SalI and ligated with NheI/SalI cleavable vector CET1019-HS (Millipore). The CET1019-HS vector contains the human CMV promoter and the UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with miniprep DNA and the desired constructs are confirmed by DNA sequencing. The final construct, which encodes the FVIII BDD protein under the control of the human CMV promoter, is designated pBC0025 (CET1019-HS-BDD FVIII-STOP). Introduction of construct pBC0025 into mammalian cells is expected to achieve expression of the FVIII BDD protein with procoagulant activity.

Example 17 Construction of an expression plasmid for XTEN containing BDD FVIII.

1. Insert AE42 domain B.

Two PCR reactions are performed simultaneously in order to insert XTEN_AE42 into the remaining region of the B domain of the FVIII BDD constructs. PCR reactions contain the following primers:

cgaaagcgctacgcctgagaGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCTGAAACCCCT

GCCTCGAGCccaccagtcttgaaacgcc (SEQ ID NO: 1665);

TGATATGGTATCATCATAATCGATTTCCTCTTGATCTGACTG (SEQ ID NO: 1666);

agcttgaggatccagagttc (SEQ ID NO: 1667);

tctcaggcgtagcgctttcgCTTGTCCCCTCTTCTGTTGAGGTGGGGGAGCCAGCAGGAGAACC

TGGCGCGCCgttttgagagaagcttcttggt (SEQ ID NO: 1668).

The PCR products then serve as templates and a second PCR is performed to introduce XTEN_AE42 into FVIII encoding nucleotide sequences flanked by BamHI and ClaI. This PCR product was digested with BamHI and ClaI at the same time with PBC0114 digestion with the same two enzymes. The PCR product is ligated with a cleavable vector. This construct is designated pBC0135 (pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA-Myc-SPATG-His) (GAGSPGAETA and SPATG disclosed as SEQ ID NOS 178 and 1662, respectively) and encodes FVIII BDD with AE42 XTEN inserted within the residual B domain .

2. Insert AE42 and mutation R1648A.

The QuikChange method (Agilent, CA) was used to introduce the R1648A mutation in PBC0135. This construct is designated pBC0149 (pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA-Myc-SPATG-His_R1648A) (GAGSPGAETA and SPATG disclosed as SEQ ID NOS 178 and 1662, respectively), which excludes this FVIII processing site.

3. Insert AE288 domain B.

XTEN_AE288 is amplified by the PCR method using the following primers: tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1669) and tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1670). PBC0075 is used as template for this PCR reaction. The PCR product is cleaved by AscI and XhoI, and PBC0135 is cleaved by the same enzymes. The PCR product is ligated with the PBC0135 fragment. This construct is designated pBC0136 (pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA-Myc-SPATG-His) (GAGSPGAETA and SPATG disclosed as SEQ ID NOS 178 and 1662, respectively) and encodes FVIII BDD with AE288 XTEN inserted within the residual B domain.

4. Insert AE288 and mutation R1648A.

XTEN_AE288 is amplified by the PCR method using the following primers: tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1671) and tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1672). The pBC0075 construct is used as a template for this PCR reaction. The PCR product is cleaved by AscI and XhoI, and pBC0149 is cleaved by the same enzymes. The PCR product is ligated with the pBC0149 fragment. This construct is designated pBC0137 (pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA-MycSPATG-His R1648A) (GAGSPGAETA and SPATG disclosed as SEQ ID NOS 178 and 1662, respectively) and contains the AE288 XTEN sequence, internal to the B domain, with the mutation R1648A, which excludes this FVIII processing site.

3. Inserts AE144, AG144, AG288 of domain B with and without R1648A mutations.

Selected XTEN fragments are amplified by PCR to introduce AscI and XhoI sites at the 5' and 3' ends, respectively. The PCR product is digested with AscI and XhoI, and pBC0135 (in the case of R1648) or pBC0149 (in the case of A1648) is digested with the same enzymes. The PCR product is ligated with the vector pBC0135 or pBC0149. These constructs are designated pSD0005, 6, 7, 8, 17, and 18.

Construction of expression plasmids in the case of FVIII BDD with an XTEN insert at the C-terminus.

1. C end insert AE288.

XTEN_AE288 is amplified by the PCR method using the following primers: ggggccgaaacggccggtacctcagagtctgctacc (SEQ ID NO: 1673) and tgttcggccgtttcggcccctggcgcactgccttc (SEQ ID NO: 1674). The pBC0075 construct is used as a template for this PCR reaction. PCR product

- 176 041874 are cleaved with SfiI and pBC0114 is cleaved with the same enzyme. The PCR product is ligated with a cleavable fragment of pBC0114. This construct is designated pBC0145 (pcDNA4-FVIII_4-XTEN_AE288-GAGSPGAETA-Myc-SPATG-His) (GAGSPGAETA and SPATG disclosed as SEQ ID NOS 178 and 1662, respectively) and encodes the AE288 sequence at the C-terminus of BDD FVIII.

2. C end insert AG288.

XTEN_AG288 is designed and synthesized with DNA2.0 (Menlo Park, CA). The synthesized gene is amplified by PCR using the following primers: ggggccgaaacggccccgggagcgtcacc (SEQ ID NO: 1675) and tgttcggccgtttcggcccctgacccggttgcccc (SEQ ID NO: 1676). The PCR product was digested with SfiI and the PBC0114 vector was digested with the same enzyme. The PCR product is ligated with a cleavable PBC0114 fragment. This construct is designated pBC0146 (pcDNA4-FVIII_4XTEN_AG288-GAGSPGAETA-Myc-SPATG-His) (GAGSPGAETA and SPATG disclosed as SEQ ID NOS 178 and 1662, respectively) and encodes the AG288 sequence at the C-terminus of the FVIII BDD.

3. C end inserts AE/AG144, 288, 864.

The AscI and XhoI sites are introduced into the PBC0114-based vector by QuikChange methods using primers: 5O37-PBC0114-AscI-XhoI-F:

CAGGACCTCTACGGCGCgccagcctcgaGCGAACAAAAACTCATCTCAGAAGAGG (SEQ ID NO: 1677); 5O38-PBC0114-AscI-XhoI-R:

CCTCTTCTGAGATGAGTTTTTGTTTCGCtcgaggctggcGCGCCGTAGAGGTCCTG (SEQ ID NO: 1678). Various XTEN fragments are amplified by the PCR method with AscI and XhoI introduced at the 5' and 3' end, respectively. The PCR product is ligated with the PBC0114 cleavable vector. These constructs are designated pSD0013, pSD0014, pSD0015, pSD0016, pSD0019 and pSD0020.

Construction of expression plasmids in the case of FVIII BDD with inter- and intra-domain inserts XTEN 1. Inserts AE7, AE42 and AE144.

Four different strategies are used to insert AE42 at designated sites (e.g., natural or introduced restriction sites BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449 , AgeI 4500, PmeI 4527, BsiWI 908, NheI 1829 and ClaI 3281) within the FVIII BDD encoding each sequence involved in the creation of some constructs. By design, these AE42 inserts create AscI and XhoI sites on either side of the insert, allowing the insertion/replacement of longer XTEN, as well as XTEN with different sequences or built-in cleavages, as required. In particular, designs that contain XTEN_144 inserts are shown in Table. 21. These inserts are created by replacing either AE7 or AE42 with a PCRed XTEN_144 fragment flanked by AscI and XhoI sites.

2. Dual PCR mediated method.

Two PCR reactions are carried out simultaneously to insert XTEN_AE42 at the designated site. Two PCR reactions introduce XTEN at either the 3' or 5' end by using a long primer that contains partial XTEN. The PCR products then serve as templates and a second PCR is performed to introduce XTEN_AE42 into FVIII encoding nucleotide sequences flanked by selected restriction enzyme sites. This PCR product is digested with the respective enzymes at the same time as PBC0114 digestion using the same two enzymes. The PCR product is ligated with a cleavable vector. Using this method, constructs designated pBC0126, pBC0127, pBC0128, and pBC0129 are generated, resulting in AE42 inserts at locations R3, R3, P130, L216, respectively. The sequences are given in table. 21. Selected XTEN_144 sequences can then be subjected to PCR to introduce AscI and XhoI sites at either end of the fragment, and ligated to cleave the FVIIIXTEN_AE42 construct. For example, pSD0053 is created by replacing AE42 pBC0129 with XTEN_AE144. Other XTEN_144 constructs were created using the same strategy and are shown in Table. 21.

3. OuikChange-mediated two-step cloning method.

The QuikChange method is used to introduce XTEN_AE7 coding sequences that are flanked by AscI and XhoI at designated sites. The resulting intermediate construct is then cleaved with AscI and XhoI. XTEN_AE42 or XTEN_AE144 is amplified by the PCR method to introduce two sites and cleaved, respectively. The vector and insert are then ligated to create the final constructs. The sequences are given in table. 21.

4. Three PCR type II ligation method mediated by restriction enzyme.

Three PCR reactions are performed to generate two portions of the FVIII coding fragments flanked by one type I restriction enzyme that correlates to a unique site within the FVIII4 gene and one type II enzyme (e.g. BsaI, BbsI, BfuAI), a third PCR reaction generates XTEN_AE42 flanked by two type II restriction enzyme sites. The three PCR fragments are digested with the appropriate enzymes and ligated into one linear piece that contains the XTEN_AE42 insert within the FVIII fragment of the coding sequences. This product is then cleaved with the appropriate unique enzymes within the FVIII coding sequences and ligated with construct PBC0114 cleaved by the same enzymes, resulting in constructs designated pBC0130 (with XTEN insert at P333), pBC0132 (with XTEN insert at D403). ), pBC0133 (with XTEN insert on residue R490). The sequences are given in table. 21. Selected XTEN_144 sequences can then be subjected to PCR to introduce AscI and XhoI sites at either end of the fragment, and ligated to cleave the FVIII-XTEN_AE42 construct. For example, pSD0001 and pSD0003 are created by replacing AE42 pBC0132 with XTEN_AE144 and XTEN_AG144, respectively. Other designs XTEN_144, given in table. 21 are created by the same strategy.

5. Original gene synthesis.

The original gene synthesis was carried out using GeneArt (Regensburg, Germany). The genes are designed such that they contain nucleic acids encoding XTEN_AE42 introduced at the designated site(s) and the genes are flanked by two unique restriction enzyme sites selected within the FVIII_4 gene. The synthesized genes and PBC0114 are digested with the appropriate enzymes and ligated to create the final product with FVIII BDD incorporating XTEN_AE42 between restriction sites. Selected XTEN_144 sequences can then be subjected to PCR to introduce AscI and XhoI sites at either end of the fragment, and ligated to cleave the FVIII-XTEN_AE42 construct.

Construction of an expression plasmid with double XTEN inserts at the B domain and at the C end.

Construct pBC0136, which encodes FVIII BDD with AE288 XTEN inserted within the residual B domain, was digested with BamHI and ClaI and the resulting 1372bps fragment was inserted. The pBC0146 construct is digested with BamHI and ClaI and the 9791bps portion of this digest is a vector. The vector and insert are ligated together to create pBC0209, which contains the AE288 insert at the B domain and AG288 at the C-terminus. The same strategy is used to generate a construct that contains two AE288 inserts at the B domain and at the C-terminus, respectively, using PBC0145 as the vector.

Construction of an expression plasmid with multiple XTEN inserts.

Construct pBC0127, which encodes AE42 XTEN at the R3 position of FVIII, is cleaved with BsiWI and AflII, and the resulting 468bps fragment is an insert. The pBC0209 construct is digested with BsiWI and AflII, the 10830 bps portion of this digest is the vector. The vector and insert are ligated together to create a construct, designated pBC0210, which contains an AE42 insert at the A1 domain, an additional three ATR amino acids to restore the cleavage signal sequence, an AE288 XTEN insert at the B domain, and an AG288 at the C terminus. The same methodology is used to generate constructs encoding multiple XTENs at natural and introduced restriction sites; e.g., BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI 4527, BsiI32 ClaI8 908 and

Construction of BDD expression vectors FVIII-Internal-XTEN_AE288.

Two BsaI restriction enzyme sites were introduced into PBC0027 of the pMK-BDD FVIII construct between base pairs 2673 and 2674 using the QuikChange method according to the manufacturer's protocol (Agilent Technologies, CA). The DNA sequences introduced are gggtctcccgcgccagggtctccc and the resulting construct is pBC0205 (sequence in Table 21). The DNA sequence encoding AE288 (or other XTEN variants and lengths; eg, AE42, AG42, AG288, AM288) is then subjected to PCR, with primers that introduce BsaI sites at both 5' and 3'. The pBC0205 vector and insert (XTEN288) are then digested with BsaI and ligated to generate pBC0206, which encodes the FVIII gene with the XTEN_AE288 insert in the B domain (sequence in Table 21). The pBC0206 construct was then digested with NheI/SalI and ligated with the NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct, which encodes the FVIII BDD protein under the control of the human CMV promoter (sequence in Table 21), is designated pBC0207 (CET1019-HS-BDD FVIII-STOP). Introduction of construct pBC0207 into mammalian cells is expected to achieve expression of the FVIII BDD protein with internal XTEN_AE288. The same protocol is used to introduce, transform, and express constructs that contain other XTEN variants and lengths; e.g. AE42, AG42, AG288, AM288, AE864, AG864 or other XTENs from tab. 4.

Construction of FVIII-/-XTEN_AE864 BDD expression vectors.

A FVIII BDD fragment with NheI and SfiI flanking the 5' and 3' ends was generated by digestion of construct pBC0025. The cleaved fragment is then ligated with NheI/SfiI cleaving the pSecTag vector (pBC0048 pSecTag-FVIII-/-XTEN_AE864) that encodes FVIII followed by the XTEN_AE864 sequence. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with minipreparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct is pBC0060, which encodes the FVIII-/-XTEN_AE864 BDD protein under the control of the CMV promoter.

- 178 041874 people. The introduction of construct pBC0060 into mammalian cells is expected to express the FVIII protein with a C-terminal XTEN fusion (BDD FVIII-/-XTEN_AE864) with procoagulant activity.

Construction of FVIII-/FXI/-XTEN_AE864 BDD expression vectors.

A FVIII BDD fragment with NheI and SfiI flanking the 5' and 3' ends was generated by digestion of construct pBC0025. The cleaved fragment is then ligated with the NheI/SfiI digested pSecTag vector (pBC0047 pSecTag-FVIII-/FXI/-XTEN_AE864) encoding FVIII followed by the cleavage sequence (/FXI/) of FXI and XTEN_AE864. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct is pBC0051, which encodes the FVIII-/FXI/-XTEN_AE864 BDD protein under the control of the human CMV promoter. Introduction of construct pBC0051 into mammalian cells is proposed to express the FVIII protein with the XTEN C-terminal fusion (BDD FVIII-/FXI/XTEN_AE864), which can be subsequently cleaved by FXI, thus releasing the FVIII BDD protein with procoagulant activity.

Construction of FVIII-/FXI/-XTEN BDD expression vectors that contain AE288 or AG288.

The fused AE864 XTEN sequence in pBC0060 was replaced by cleaving the XTEN_AE288 and AG288 sequences with BsaI and HindIII. The next ligation step using the appropriate AE288 or AG288 XTEN fragment and BsaI/HindIII digested pBC0051 allows the replacement of the AE288 or AG288 sequences into the FVIII BDD expression vector. The resulting final constructs are pBC0061 for BDD FVIII-AE288, and pBC0062 for BDD FVIII-AG288. The introduction of construct pBC0061 into mammalian cells is expected to express the FVIII protein with the AE288 XTEN C-terminal fusion (BDD FVIII-/-XTEN_AE288) with procoagulant activity. Introduction of construct pBC0062 into mammalian cells for expression suggests a FVIII protein with a C-terminal AG288 XTEN fusion (BDD FVIII-/-XTEN_AG288) with procoagulant activity.

Construction of BDD expression vectors FVIII-/FXI/-XTEN with alternative XTEN.

The fused XTEN sequence in pBC0051 is replaced by cleaving DNA encoding other XTEN sequences (eg, other XTEN variants and lengths; eg, AE42, AG42, AG288, AM288) with BsaI and HindIII. Ligation using the XTEN fragment and BsaI/HindIII cleaving pBC0051 allows the exchange of different XTEN coding sequences in the FVIII BDD expression vector, providing alternative constructs. The introduction of alternative constructs into mammalian cells is proposed to express the FVIII protein with C-terminal XTEN (BDD FVIII-/FXI/-XTEN), which can be subsequently cleaved by FXI, releases FVIII, resulting in the fusion of procoagulant FVIII with procoagulant activity.

Example 18 Construction of an expression plasmid in the case of FVIII signal sequence XTEN-/FXI/-BDD.

Construction of expression vectors in the case of FVIII signal sequence XTEN_AE864.

Coding sequences in the case of the FVIII signal sequence are obtained by renaturation of the following two oligomers: 5'CTAGCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTT

TAGTGGGTCTCC-3' (SEQ IDNO: 1679); 5'ACCTGGAGACCCACTAAAGCAGAATCGCAAAAGGCACAGAAAGAAGCAGGTGGAGAGCTTATTTGCATG-3' (SEQIDNO: 1680).

The renatured oligomers flank the NheI and BsaI restriction enzyme sites at either end and are ligated with the NheI/BsaI digested pCW0645 vector that encodes FVII-XTEN_AE864. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct is designated pBC0029, which encodes the protein signal sequence-XTEN_AE864 under the control of the human CMV promoter. This construct is used as an intermediate construct to generate an expression construct with XTEN fused at the N-terminus of the FVIII protein and can also be used as a master plasmid to generate expression constructs that allow XTEN to be fused at the N-terminus of the secreted protein.

Construction of FVIII expression vectors signal sequence-XTEN_AE864-/FXI/BDD.

The 1800bp fragment within the FVIII coding region is amplified using primers that introduce NheI-BbsI-/FXI/-AgeI sites at the 5' end and endogenous restriction enzyme KpnI at the 3' end. The FVIII fragment digested with NheI/KpnI was ligated with the vector pBC0027 digested with NheI/KpnI. The ligated DNA mixture is used to transform DH5a bacterial cells.

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Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The resulting construct, which contains the /FXI/-FVIII protein coding sequences without the FVIII signal sequence, is designated pBC0052. This construct is used as an intermediate construct to create an expression construct with XTEN fused at the N-terminus to the FVIII protein.

Vector pBC0052 is digested with BbsI/XhoI enzymes and used to ligate pBC0029 digested with Bbsi/XhoI. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct, which encodes the FVIII protein signal sequence-XTEN_AE864-/FXI/-BDD under the control of the human CMV promoter, is designated pBC0053. Introduction of construct pBC0053 into mammalian cells is proposed to express the FVIII protein with the N-terminal XTEN fusion (XTEN_AE864-/FXI/-BDD FVIII signal sequence), which can be subsequently cleaved by FXI, thus releasing the FVIII BDD protein.

Construction of expression vectors FVIII signal sequence-XTEN-/FXI/-BDD.

The fused XTEN sequence in pBC0053 can be replaced by cleaving other XTEN fragments (eg, AM, AF, AG) with BsaI and BbsI. Ligation using the XTEN fragment and pBC0053 cleaved with BsaI/BbsI allows the exchange of different parts of XTEN (eg AM, AF, AG) in the FVIII BDD expression vector. Different XTEN fusions can increase the half-lives of these proteins in different ways, allowing modification of the properties (eg potency, specific activity) of these proteins. Introduction of any of these fusion constructs into mammalian cells is contemplated to express the FVIII protein with an N-terminal XTEN fusion (FVIII signal sequence-XTEN-/FXI/-BDD) in which the XTEN fusion peptide can be subsequently cleaved by FXI, generating the FVIII BDD protein.

Example 19 Building an FVIII BDD with an XTEN Interdomain Insert.

Construction of FVIII BDD expression vectors with an XTEN insert within the boundaries of the A2-B domain.

The construct pBC0027 (pMK-BDD FVIII-STOP) is a cloning vector designed to contain sequences encoding the FVIII BDD protein rather than a promoter positioned to initiate expression of the FVIII BDD. This construct is used to manipulate the FVIII BDD coding sequences as a vector backbone that contains very few restriction enzyme sites, thus allowing easy cloning strategies. The BDD FVIII proteins contain 1457 amino acids with a total molecular weight of 167539.66. There are 6 domains within the wild-type FVIII protein, domains A1, A2, B, A3, C1 and C2. In the FVIII BDD protein, most of the B domain is deleted because the unstructured domain and domain deletion are not believed to alter the critical functions of this protein. Despite this, the boundaries of the B domain appear to be excellent positions for creating XTEN fusions in order to achieve extended protein half-lives.

Within the pBC0027 construct, there is a unique restriction enzyme HindIII site at the border of the A2-B junction region. XTENs (eg, sequences from Table 4, or 13-17) are amplified using primers that introduce HindIII and a cleaved FXI site at either end of the XTEN-coding sequence. The fused XTEN sequence can be altered by amplifying different XTEN fragments. Different XTEN fusions can increase the half-lives of these proteins in different ways, allowing modification of the properties (eg potency, specific activity) of these proteins. The HindIII-/FXI/-XTEN/FXI/-HindIII fragment was digested with HindIII and ligated with HindIII-cleavable pBC0027. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct, which encodes the FVIII BDD protein with an XTEN interdomain fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)) rather than a promoter to initiate gene expression, is designated pBC0054.

The pBC0054 construct was digested with NheI/SalI and ligated with NheI/SalI digestible CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct that encodes the FVIII BDD protein with an interdomain (between the A2/B domains) XTEN fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)) under the control of the human CMV promoter, denote pBC0055 (CET1019-HS-FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)). The introduction of construct pBC0055 into mammalian cells is proposed to express the FVIII BDD protein with an XTEN cross-domain fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)), which can be subsequently cleaved by FXI, such thus releasing the FVIII BDD protein.

Construction of FVIII BDD expression vectors with an XTEN insert at the borders of the A1-A2 domains.

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The pBC0027 construct was built as a template for two PCR reactions using the following four primers:

(Reaction I) 5'-ATGATGGCATGGAAGCCTAT-3' (SEQ ID NO: 1681); 5’ATCCCTCACCTTCGCCAGAACCTTCAGAACCCTCACCTTCAGAACCTTCACCAGAAC

CTTCACCATCTTCCGCTTCTTCATTATTTTCAT-3' (SEQ W NO: 1682).

(Reaction II) 5' TTCTGGCGAAGGTGAGGGATCTGAAGGCGGTTCTGAAGGTGAAGGTGGCTCTGAGG

GTTCCGAATATGATGATGATCTTACTGATTCTGAAAT-3' (SEQ W NO: 1683); 5'TATTCTCTGTGTGAGGTACCAGC-3' (SEQ W NO: 1684).

The resulting PCR products are 150 bps and 800 bps, respectively. The 800 bp product is used as a template for the next step of the PCR reaction with the 150 bp product as one primer and 5'-TATTCTCTGTGTGAGGTACCAGC-3' (SEQ ID NO: 1685) as the other. The product for the second step PCR is 930 bps and digested with restriction enzymes PshAI and ACC65I. This PshAI/Acc65I surrounding the DNA fragment is ligated with pBC0027 cleaved with PshAI/Acc65I. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct that encodes the FVIII BDD protein with an interdomain (between the A1/A2 domains) XTEN fusion after residue D345 is designated pBC0058 (pMK-BDD FVIII-D345-XTENY36).

The pBC0058 construct was digested with NheI/SalI and ligated with NheI/SalI digestible CET1019-HS vector (Millipore). The CET1019-HS vector contains the human CMV promoter and the UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct, which encodes the FVIII BDD protein with an interdomain (between A1/A2 domains) fusion of XTEN after residue D345 under the control of the human CMV promoter, is designated pBC0059 (CET1019-HS-BDD FVIII D345-XTENY36). Introduction of construct pBC0059 into mammalian cells is proposed to express the FVIII BDD protein with an XTEN interdomain fusion (BDD FVIII D345-XTEN_Y36).

Example 20 Construction of FVIII with intradomain insertion XTEN.

Construction of FVIII BDD expression vectors with XTEN insert after P598 (in the A2 domain).

The coding sequences for XTEN_Y36 are amplified using PCR methods with the following primers:

' - GAAGCTGGT ACCTC AC AGAGAATAT AC AACGCTTTCTCCCC AATCC AGGTGAAGGT

TCTGGTGAAGG-3' (SEQ W NO: 1686)

5'-AACTCTGGATCCTCAAGCTGCACTCCAGCTTCGGAACCCTCAGAGCC-3' (SEQ W NO: 1687).

The 184 bp PCR product is flanked with KpnI and BamHI restriction enzyme sites at either end and ligated with the KpnII/BamHI cleavable vector pBC0027 which encodes the FVIII BDD gene. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct, which contains the DNA sequences encoding the FVIII protein with the XTEN_Y36 fusion after the P598 residue, is designated pBC0056. This cloning strategy is used to introduce various forms of XTEN into the FVIII BDD protein by changing the PCR reaction template and changing the primers, respectively.

The pBC0056 construct was digested with NheI/SalI and ligated with NheI/SalI digestible CET1019-HS vector (Millipore). The CET1019-HS vector contains the human CMV promoter and the UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened with mini-preparative DNA and constructs of interest are confirmed by DNA sequencing. The final construct, which encodes the FVIII BDD protein with an intradomain (in the A2 domain) fusion of XTEN under the control of the human CMV promoter, is designated pBC0057 (CET1019-HS-FVIII P598-XTEN_Y32). The introduction of construct pBC0057 into mammalian cells is proposed to express the FVIII BDD protein with an intradomain XTEN fusion (FVIII P598-XTENY32).

Construction of FVIII BDD expression vectors with other intradomain XTEN inserts.

To introduce different XTEN segments into other intradomain sites within the FVIII BDD (eg, XTEN from Table 4 or 13-17), primers are designed that extend sticky-end XTEN that can bind to the FVIII BDD. The FVIII coding sequence (pMK-BDD FVIII) is constructed with various unique restriction enzyme sites in order to achieve these specific inserts. Unique restriction enzymes are listed below with their truncated site: NheI 376, SacI 391, AfiII 700, SpeI 966, PshAI 1417, Acc6512192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, ApaI8 Swapl 3823, , OliI 4626, XbaI 4644, BstBI 4673,

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SalI4756 and Xhol 4762. The Nhel and Sall sites at either end of the coding sequence are used to insert the DNA fragment into the human CMV promoter vector, CET1019-HS (Millipore) for expression in mammalian cells. These constructs express the FVIII BDD protein with an XTEN fusion with the sequences shown in Table 1. 21.

Example 21 Construction of FVIII with XTEN inserts.

CFXTEN with two XTEN.

To obtain CFXTEN with two XTEN inserts in different regions (from N-terminus to C-terminus: Al-Rl, A1-R2, A2-R1, A2-R2, domain B, a3, A3-R1, A3-R2, C -end) use constructs that express fusions with single-XTEN inserts that retain FVIII activity. The FVIII coding sequence (pBC0114 pcDNA4-FVIII_4-X10-Myc-SPATG-His extra RE) ('SPATG' disclosed as SEQ ID NO: 1662) was created with various unique restriction enzyme sites in order to achieve these specific combinations. Unique restriction enzymes are given in table. 18 below with their relative sites between different regions: BsiWI (between N-terminals and Al-Rl), ANP (between A1-R1 and A1-R2), Nhel (between A1-R2 and A2-R1), Krp! (between A2-R1 and A2-R2), BamHI (between A2-R2 and domain B), Clal (between a3 and A3-R1), ΡΑΜΙ (between A3-R1 and A3-R2), Xbal (between A3-R2 and C-terminus), Agel (between FVIII C-termini and stop codon). Select building blocks and restriction enzymes for cloning libraries, as shown in table. below. Selected components in each region are mixed at a 1:1 molar ratio and two sets of DNA mixtures are digested with unique restriction enzymes. The DNA fragments are separated on a 1% agarose gel and purified using a Qiagen gel-based extraction kit. DNA with an XTEN insert in the first target region is treated as an insert (smaller DNA fragment in agarose gel), while DNA with an XTEN insert in the second target region is regarded as a vector (larger DNA fragment in agarose gel). The insert and vector are ligated to resuspend the plasmid. The ligated DNA mixture is used to transform competent E. coli DH5a host cells. Transformants are screened by rolling ring amplification (RCA) and Sanger sequencing to cover the potential library size by approximately 3-4 times. Unique clones are identified and miniprepared. Then, to further confirm the integrity of XTEN, two different restriction cleavages are used in each region. The amino acid and DNA encoding sequences for the resulting CFXTEN fusion proteins are shown in Table 1. 21.

CFXTEN with one or two XTEN inserts within the B/a3 and C terminus domains.

The a3 B/domain and C-terminus of FVIII are unstructured regions that tolerate XTEN inserts well. The B/a3 domain further promotes interaction with other cofactors that contain the von Willebrand factor. To explore optimal insertions of XTEN into the B/a3 domain, selected deletions and mutations of the region are carried out by PCR-based mutagenesis methods. Selected PCR reactions and vectors cleave the unique restriction enzymes shown in Table 1. 18. DNA fragments are isolated on a 1% agarose gel and purified on a Qiagen gel extraction kit. DNA with an ΧΤΕΝ insert in the first target region is treated as an insert (smaller DNA fragment on agarose gel), while DNA with an XTEN insert in the second target region is regarded as a vector (larger DNA fragment on agarose gel). The insert and vector are ligated in order to resuspend the plasmid. The ligated DNA mixture is used to transform competent E. coli DH5a host cells. Transformants are screened by molecular colony and Sanger sequencing to cover approximately eight times the potential library size. Unique clones are identified and miniprepared. Restriction digestion with three enzymes is then used to further confirm the integrity of the XTEN in each region. The amino acid and DNA encoding sequences of the resulting CFXTEN fusion proteins are shown in Table 1. 21.

Table 18

Plan cloning in case of FVIII libraries with two ΧΤΕΝ inserts

Library ID Insert Components (XTEN area) Vector components (XTEN area) Restriction enzymes LSD0001 pSD0005, pSD0006, pSD0007, pSD0008, pSD0017, pSD0018, pBC0136, pBC0137 (B domain) pSD0013 (C-terminal) Nhel + Clal LSD0002 pSD0005, pSD0006, pSD0007, pSD0014 (C-terminal) Nhel + Clal

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pSD0008, pSD0017, pSD0018, pBC0136, pBC0137 (Vdomain) LSD0003 pSD0005, pSD0006, pSD0007, pSD0008, pSD0017, pSD0018, pBC0136, pBC0137 (Vdomain) pSD0019 (C-terminal) Nhel + Clal LSD0004 pSD0005, pSD0006, pSD0007, pSD0008, pSD0017, pSD0018, pBC0136, pBC0137 (Vdomain) pSD0020 (C-end) Nhel + Clal LSD0005 pSD0045, pSD0046, pSD0048, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) pSDOOOl (A2-R1) BsiWI+AflII LSD0006 pSD0045, pSD0046, pSD0048, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) pSD0002 (A2-R1) BsiWI+AflII LSD0007 pSD0045, pSD0046, pSD0048, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) pSD0003 (A2-R1) BsiWI+AflII LSD0008 pSD0045, pSD0046, pSD0048, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) pSD0004 (A2-R1) BsiWI+AflII LSD0037 pSD0045, pSD0046, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) pSD0032 (A2-R1) BsiWI+AflII LSD0038 PSD0039 (a3) pSD0045, pSD0046, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) BamHI+Clal LSD0039 PSD0039 (a3) pSD0032, pSDOOOl, pSD0003 (A2-R1) BamHI+Clal LSD0040 pSD0040, pSDOOlO, pSD0041 (A3-R1) pSD0045, pSD0046, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) Clal+Xbal LSD0041 pSD0040, pSDOOlO, pSD0041 (A3-R1) pSD0032, pSDOOOl, pSD0003 (A2-R1) Clal+Xbal LSD0042 pSD0062, pSD0063, pSD0043, pSD0044 (A3-R2) pSD0045, pSD0046, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) Clal+Xbal LSD0043 pSD0062, pSD0063, pSD0043, pSD0044 (A3-R2) pSD0032, pSDOOOl, pSD0003 (A2-R1) Clal+Xbal LSD0044 pSD0062, pSD0063, pSD0043, pSD0044 (A3-R2) pSD0040, pSDOOlO, pSD0041 (A3-R1) PflMI+Xbal LSD0045 PSD0039 (a3) pSD0040, pSDOOlO, pSD0041 (A3-R1) BamHI+Clal LSD0046 PSD0039 (a3) pSD0062, pSD0063, pSD0043, pSD0044 (A3-R2) BamHI+Clal LSD0047 pSD0046 (Al-Rl) pSDOOOl, pSD0003 (A2-R1) BsiWI+AflII LSD0048 pNL0006 pNL0007 pSD0045, pSD0051 (Al-Rl) PCR product PCR product pSD0003 (A2-R1) LSD0003.006 (domain B and C end) LSD0003.006 (domain B and C end) BsiWI+AflII BamHI+PflMI Clal+PflMI PNL0008 PCR product LSD0003.009 (domain B and C end) Clal+PflMI pNL0009 PCR product pSD0039 (a3 domain) BamHI+AscI pNLOOlO LSD0003.006 (domain B and C end) pNL0009 (a3 domain) Xbal+Agel

Example 22 Construction of FVIII BDD expression vectors with 3-5 XTEN inserts within sites 18/26, 403, 745/1656, 1720, 1900, or 2332.

FVIII fusion constructs with XTEN inserts within sites 18/26, 403, 745/1656, 1720, 1900, or 2332 are selected for recombination and generation of 3, 4, 5, or 6 XTEN insert constructs.

Construction of FVIII BDD expression vectors with 3-5 XTEN inserts within sites 26, 403, 1656, 1720, or 1900.

The selected constructs with unique XTEN at the desired sites are: pSD0050, pSD0001, pSD0039, pSD0010 and pSD0062. Double XTEN constructs at the desired sites contain LSD0005.002, LSD0038.001, LSD0040.002, LSD0042.013, LSD0039.010, LSD0041.008, LSD0043.008, LSD0045.002, LSD0046.002, and LSD0044.002. Were selected building blocks and restriction enzymes for cloning constructs, which are shown in table. 19 below. Selected Components

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Construction of FVIII BDD expression vectors with 4-6 XTEN inserts within sites 18, 403, 1656, 1720, 1900, or 2332.

Constructs pSD0077 to pSD0092 serve as building blocks to generate 4- to 6-XTEN constructs with inserts at 18, 403, 1656, 1720, 1900, and 2332. Building block constructs and restriction enzymes were selected for cloning. designs, which are given in table. 19 below. Selected components are cleaved with unique restriction enzymes. Insert and vector DNA is separated on a 1% agarose gel and purified on a Qiagen gel extraction kit. The insert and vector are ligated and then transformed into DH5a. E. coli competent host cells. Eight colonies for each of the constructs were analyzed by the molecular colony method and DNA sequencing. Clones with the desired XTEN inserts are mini-prepared. Restriction cleavages are then used to further confirm the integrity of the XTEN in each region. The amino acid and DNA coding sequences for the resulting CFXTEN fusion proteins are shown in Table 1. 21. The resulting constructs are numbered pBC0247 through pBC0257, pNL0022, 23, 24, 25 and 30.

Construction of FVIII BDD expression vectors with 4-6 XTEN inserts within sites 18, 403, 745, 1720, 1900, or 2332.

Constructs pBC0247 to pBC0252, pBC0255, pNL0022 to pNL0025 serve as building blocks to produce 4- to 6-XTEN constructs with inserts in 18, 403, 745, 1720, 1900, and 2332. The constructs were selected building block and restriction enzymes for cloning structures, which are given in table. 19 below. Selected components are cleaved with unique restriction enzymes. Insert and vector DNA is separated on a 1% agarose gel and purified on a Qiagen gel extraction kit. The insert and vector are ligated and then transformed into DH5a. E. coli competent host cells. Eight colonies for each of the constructs were analyzed by the molecular colony method and DNA sequencing. Clones with the desired XTEN inserts are mini-prepared. Restriction cleavages are then used to further confirm the integrity of the XTEN in each region. The amino acid and DNA coding sequences for the resulting CFXTEN fusion proteins are shown in Table 1. 21. The resulting constructs are numbered pBC0258 to pBC0268.

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Table 19

Cloning plan for FVIII libraries with 3-5 XTEN inserts within sites 26, 403, 1656, 1720, or 1900

The name of the structure Insert Components (XTEN area) Vector components (XTEN area) Restriction enzymes PSD0077 pSD0050 (Al-Rl) LSD0039.010 (A2-R1, a3) BsiWI+AflII PSD0078 pSDOOlO (A3-R1) LSD0005.002 (Al-Rl, A2- Ri) Clal+Xbal PSD0079 pSD0062 (A3-R2) LSD0005.002 (Al-Rl, A2- Ri) Clal+Xbal PSD0080 pSD0050 (Al-Rl) LSD0045.002 (a3, A3-R1) BsiWI+AflII PSD0081 pSD0050 (Al-Rl) LSD0046.002 (a3, A3-R2) BsiWI+AflII PSD0082 pSD0050 (Al-Rl) LSD0044.002 (A3-R1, AZ- R2) BsiWI+AflII PSD0083 pSDOOlO (A3-R1) LSD0039.010 (A2-R1, a3) Clal+Xbal PSD0084 pSD0062 (A3-R2) LSD0039.010 (A2-R1, a3) Clal+Xbal PSD0085 pSD0062 (A3-R2) LSD0041.008 (A2-R1, AZ- R1) PflMI+Xbal PSD0086 pSD0062 (A3-R2) LSD0045.002 (a3, A3-R1) PflMI+Xbal PSD0087 LSD0039.010 (A2-R1, a3) LSD0040.002 (A1-R1, AZ- R1) Nhel+Clal PSD0088 LSD0039.010 (A2-R1, a3) LSD0042.013 (A1-R1, AZ- R2) Nhel+Clal PSD0089 LSD0044.002 (A3-R1, AZ- R2) LSD0005.002 (A1-R1, A2- R1) Clal+Xbal PSD0090 LSD0044.002 (A3-R1, AZ- R2) LSD0038.001 (A1-R1, a3) Clal+Xbal PSD0091 LSD0044.002 (A3-R1, AZ- R2) LSD0039.010 (A2-R1, a3) Clal+Xbal PSD0092 LSD0044.002 (A3-R1, AZ- R2) pSD0077 (Al-Rl, A2-R1, a3) Clal+Xbal pBC0247 PSD0077 LSD0050.003 Nhel+BstBI pBC0248 PSD0078 LSD0050.003 Nhel+BstBI pBC0249 PSD0079 LSD0050.003 Nhel+BstBI pBC0250 PSD0080 LSD0050.003 Nhel+BstBI pBC0251 PSD0082 LSD0050.003 Nhel+BstBI pBC0252 PSD0080 LSD0050.003 Nhel+BstBI

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pBC0253 PSD0087 LSD0050.003 Nhel+BstBI pBC0254 PSD0088 LSD0050.003 Nhel+BstBI pBC0255 PSD0089 LSD0050.003 Nhel+BstBI pBC0256 PSD0090 LSD0050.003 Nhel+BstBI pBC0257 PSD0092 LSD0050.003 Nhel+BstBI PNL0022 LSD0003.009 PSD0083 Xbal+Agel pNL0023 LSD0003.009 PSD0084 Xbal+Agel pNL0024 LSD0003.009 PSD0085 Xbal+Agel pNL0025 LSD0003.009 PSD0086 Xbal+Agel pNL0030 LSD0003.009 PSD0091 Xbal+Agel pBC0258 LSD0003.006 pBC0247 BamHI+Clal pBC0259 LSD0003.006 pBC0248 BamHI+Clal pBC0260 LSD0003.006 pBC0249 BamHI+Clal pBC0261 LSD0003.006 pBC0250 BamHI+Clal pBC0262 LSD0003.006 pBC0251 BamHI+Clal pBC0263 LSD0003.006 pBC0252 BamHI+Clal pBC0264 LSD0003.006 pBC0255 BamHI+Clal pBC0265 LSD0003.006 pNL0022 BamHI+Clal pBC0266 LSD0003.006 pNL0023 BamHI+Clal pBC0267 LSD0003.006 pNL0024 BamHI+Clal pBC0268 LSD0003.006 pNL0025 BamHI+Clal

Example 23 Construction of CFXTEN expression vectors with three or four XTENs: the first XTEN in the B domain, the second XTEN at the C-terminus, and the third or fourth XTEN insert within the A1 or A2 or A3 domains.

Libraries of CFXTEN fusion proteins are built with three XTEN inserts by combining coagulation-active clones with XTEN inserts in the A1, A2 or A3 domains and clones with XTEN introduced in the B domain and at the C-terminus. Additional libraries are built with a fourth XTEN added to domains A1, A2, or A3 in order to select members of the 3 XTEN libraries. The structure of the cloning scheme is summarized in table. below. DNA is obtained in the case of inserts and vectors by restriction enzyme digestion and purification on agarose gel. After ligating the insert with the appropriate vectors, the ligated DNA mixture is used to transform DH5a competent E. coli host cells. Transformants are screened by RCA and sequencing to cover approximately 3-4 potential library sizes. Unique clones are identified and miniprepared. Then, to further confirm the integrity of each XTEN, three different restriction cleavages are used. The amino acid and DNA encoding sequences of the resulting CFXTEN fusion proteins are shown in Table 1. 21.

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Table 20

Plan cloning in case of FVIII libraries with 3 XTEN inserts within domain B sites, C-terminus and domain A1/A2/a3

Library ID Insert Components (XTEN area) Vector components (XTEN area) Restriction enzymes LSD0049 LSD0003.006 (Domain B and C-terminal) pSD0045, pSD0046, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) BamHI+Agel LSD0050 LSD0003.009 (Domain B and C-terminal) pSD0045, pSD0046, pSD0049, pSD0050, pSD0051, pSD0052 (Al-Rl) BamHI+Agel LSD0051 LSD0003.006 (Domain B and C-terminal) pSD0032, pSDOOOl, pSD0003 (A2-R1) BamHI+Agel LSD0052 LSD0003.009 (Domain B and C-terminal) pSD0032, pSDOOOl, pSD0003 (A2-R1) BamHI+Agel LSD0053 pSD0040, pSDOOlO, pSD0041 (A3-R1) LSD0003.006 (domain B and C end) Clal+Xbal LSD0054 pSD0040, pSDOOlO, pSD0041 (A3-R1) LSD0003.009 (Domain B and End) Clal+Xbal LSD0055 pSD0062, pSD0063, pSD0043, pSD0044 (A3-R2) LSD0003.006 (Domain B and End) Clal+Xbal LSD0056 pSD0062, pSD0063, pSD0043, pSD0044 (A3-R2) LSD0003.009 (Domain B and End) Clal+Xbal LSD0057 pSDOOOl (A2-R1) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Nhel+BamHI LSD0058 pSD0003 (A2-R1) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Nhel+BamHI LSD0059 pNL0005 (A2-R1) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Nhel+BamHI LSD0060 pBC0246 (A2-R1) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Nhel+BamHI LSD0061 pSD0009 (A3-R1) LSD0049.021, LSD0049.002, Clal+Xbal

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LSD0049.011, LSD0049.012 (Al-RI, domain B and C-terminus) LSD0062 pSDOOlO (A3-R1) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (Al-R1, domain B and C-terminus) Clal+Xbal LSD0063 pNL0004 (A3-R2) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Clal+Xbal LSD0064 pSD0063 (A3-R2) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Clal+Xbal LSD0065 pNL0002 (A3-R2) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Clal+Xbal LSD0066 pSD0043 (A3-R2) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Clal+Xbal LSD0067 pNL0003 (A3-R2) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Clal+Xbal LSD0068 pSD0044 (A3-R2) LSD0049.021, LSD0049.002, LSD0049.011, LSD0049.012 (A1-R1, domain B and C-terminus) Clal+Xbal LSD0069 pSD0009 (A3-R1) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0070 pSDOOlO (A3-R1) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0071 pNL0004 (A3-R2) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0072 pSD0063 (A3-R2) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0073 pNL0002 (A3-R2) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0074 pSD0043 (A3-R2) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0075 pNL0003 (A3-R2) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal LSD0076 pSD0044 (A3-R2) LSD0051.002, pBC0244, pBC0245 (A2-R1, domain B and Cend) Clal+Xbal PSD0093 pNL0004 (A3-R2) LSD0053.022 (A3-R1, domain B and C-terminus) PflMI+Xbal PSD0094 pSD0063 (A3-R2) LSD0053.022 (A3-R1, domain B and C-terminus) PflMI+Xbal

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PSD0095 pNL0002 (A3-R2) LSD0053.022 (A3-R1, domain B and C-terminus) PflMI+Xbal PSD0096 pSD0043 (A3-R2) LSD0053.022 (A3-R1, domain B and C-terminus) PflMI+Xbal PSD0097 pNL0003 (A3-R2) LSD0053.022 (A3-R1, domain B and C-terminus) PflMI+Xbal PSD0098 pSD0044 (A3-R2) LSD0053.022 (A3-R1, domain B and C-terminus) PflMI+Xbal pCSOOOl pBC0168 (Al) LSD0055.021 (A3-R1, domain B and C-terminus) BsiWI+BamHI pCS0002 pBC0134 (A2_R2) LSD0055.021 (A3-R1, domain B and C-terminus) BsiWI+BamHI pCS0003 pBC0179 (Cl) LSD0055.021 (A3-R1, domain B and C-terminus) Apal+Xbal pCS0004 pBC0143(Cl) LSD0055.021 (A3-R1, domain B and C-terminus) Apal+Xbal pCS0005 pBC0182 (C2) LSD0055.021 (A3-R1, domain B and C-terminus) Apal+Xbal pCS0006 pBC0144(C2) LSD0055.021 (A3-R1, domain B and C-terminus) Apal+Xbal pBC0269 pBC0165 (AI RI) LSD0003.006 (Domain B and End) BsiWI+BamHI pBC0270 pBC0132 (A2_R1) LSD0003.006 (Domain B and End) BsiWI+BamHI pBC0271 pBC0138 (A3 R1) LSD0003.006 (Domain B and End) Clal+Xbal pBC0272 pBC0176 (A3 R2) LSD0003.006 (Domain B and End) Clal+Xbal pBC0273 pSDOOOl(A2_R1) LSD0003.006 (Domain B and End) BsiWI+BamHI pBC0274 pSD0009 (A3 R1) LSD0003.006 (Domain B and End) Clal+Xbal pBC0275 pNL0004 (A3 R2) LSD0003.006 (Domain B and End) Clal+Xbal pBC0276 pBC0280 (AI RI) LSD0003.006 (Domain B and End) BsiWI+BamHI pBC0277 pBC0281 (A2_R1) LSD0003.006 (Domain B and End) BsiWI+BamHI pBC0278 pBC0282 (A3_R1) LSD0003.006 (Domain B and End) Clal+Xbal pBC0279 pBC0283 (A3_R2) LSD0003.006 (Domain B and End) Clal+Xbal L0901 pBC0284 (CT) pBC0285, 286, 287, 288, 289, 290, 291, 292, 293 (domain B and AZ R2) Xbal+Agel L0901 pSD0014 (CT) pBC0285, 286, 287, 288, 289, 290, 291, 292, 293 (domain B and AZ R2) Xbal+Agel L0901 pSD0020 (CT) pBC0285, 286, 287, 288, 289, 290, 291,292,293 (domain B and A3 R2) Xbal+Agel

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Table 21

FVIII-XTEN DNA and amino acid sequence constructs

Design name Amino acid sequence disclosed as SEQ ID NO: The DNA sequence disclosed as SEQ ID NO: pBC0114 595 596 pBC0126 597 598 pBC0127 599 600 pBC0165 601 602 pBC0183 603 604 pBC0184 605 606 pBC0166 607 608 pBC0185 609 610 pBC0167 611 612 pBC0128 613 614 pBC0168 615 616 pBC0129 617 618 pBC0169 619 620 pBC0130 621 622 pBC0131 623 624 pBC0132 625 626 pBC0170 627 628 pBC0133 629 630 pBC0171 631 632 pBC0134 633 634 pBC0172 635 636 pBC0135 637 638 pBC0149 639 640 pBC0136 641 642 pBC0137 643 644 pBC0138 645 646 pBC0139 647 648 pBC0140 649 650 pBC0173 651 652 pBC0174 653 654 pBC0175 655 656 pBC0176 657 658 pBC0177 659 660 pBC0178 661 662 pBC0141 663 664 pBC0179 665 666 pBC0180 667 668 pBC0142 669 670 pBC0143 671 672 pBC0181 673 674 pBC0182 675 676 pBC0144 677 678 pBC0145 679 680 pBC0146 681 682

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pSDOOOl 683 684 PSD0002 685 686 PSD0003 687 688 PSD0004 689 690 PSD0005 691 692 PSD0006 693 694 PSD0007 695 696 PSD0008 697 698 PSD0009 699 700 pSDOOlO 701 702 pSDOOll 703 704 PSD0012 705 706 PSD0013 707 708 PSD0014 709 710 PSD0017 711 712 PSD0018 713 714 PSD0019 715 716 PSD0020 717 718 PSD0015 719 720 PSD0016 721 722 PSD0021 723 724 PSD0022 725 726 PSD0023 727 728 PSD0024 729 730 PSD0025 731 732 PSD0026 733 734 PSD0027 735 736 PSD0028 737 738 PSD0029 739 740 PSD0030 741 742 PSD0031 743 744 PSD0032 745 746 PSD0033 747 748 PSD0034 749 750 PSD0035 751 752 PSD0036 753 754 PSD0037 755 756 PSD0038 757 758 PSD0039 759 760 PSD0040 761 762 PSD0041 763 764 PSD0042 765 766 PSD0043 767 768 PSD0044 769 770 PSD0062 771 772 PSD0063 773 774 PSD0045 775 776 PSD0046 777 778 PSD0047 779 780 PSD0048 781 782

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PSD0049 783 784 PSD0050 785 786 PSD0051 787 788 PSD0052 789 790 PSD0053 791 792 PSD0054 793 794 PSD0055 795 796 PSD0056 797 798 PSD0057 799 800 PSD0058 801 802 PSD0059 803 804 PSD0060 805 806 PSD0061 807 808 LSD0001.002 809 810 LSD0001.005 811 812 LSD0001.006 813 814 LSD0001.011 815 816 LSD0001.012 817 818 LSD0001.013 819 820 LSD0001.016 821 822 LSD0001.021 823 824 LSD0002.001 825 826 LSD0002.002 827 828 LSD0002.014 829 830 LSD0003.004 831 832 LSD0003.006 833 834 LSD0003.009 835 836 LSD0003.014 837 838 LSD0004.010 839 840 LSD0004.011 841 842 LSD0004.014 843 844 LSD0004.016 845 846 LSD0004.022 847 848 LSD0003.016 849 850 LSD0005.002 851 852 LSD0005.004 853 854 LSD0005.005 855 856 LSD0005.011 857 858 LSD0005.018 859 860 LSD0006.002 861 862 LSD0006.005 863 864 LSD0006.007 865 866 LSD0006.011 867 868 LSD0007.002 869 870 LSD0007.004 871 872 LSD0007.013 873 874 LSD0008.001 875 876 LSD0008.002 877 878 LSD0008.006 879 880 LSD0008.009 881 882

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LSD0008.017 883 884 LSD0002.025 885 886 LSD0002.013 887 888 LSD0003.025 889 890 LSD0004.025 891 892 LSD0003.005 893 894 LSD0007.008 895 896 LSD0044.002 897 898 LSD0044.005 899 900 LSD0044.039 901 902 LSD0044.022 903 904 LSD0044.003 905 906 LSD0044.001 907 908 LSD0038.001 909 910 LSD0038.003 911 912 LSD0038.008 913 914 LSD0038.012 915 916 LSD0038.013 917 918 LSD0038.015 919 920 LSD0039.001 921 922 LSD0039.003 923 924 LSD0039.010 925 926 LSD0045.001 927 928 LSD0045.002 929 930 LSD0042.014 931 932 LSD0042.023 933 934 LSD0042.006 935 936 LSD0042.013 937 938 LSD0042.001 939 940 LSD0042.039 941 942 LSD0042.047 943 944 LSD0042.003 945 946 LSD0042.004 947 948 LSD0042.008 949 950 LSD0042.038 951 952 LSD0042.082 953 954 LSD0042.040 955 956 LSD0037.002 957 958 LSD0037.009 959 960 LSD0037.011 961 962 LSD0047.002 963 964 LSD0047.005 965 966 LSD0048.007 967 968 LSD0046.001 969 970 LSD0046.002 971 972 LSD0046.003 973 974 LSD0040.011 975 976 LSD0040.042 977 978 LSD0040.002 979 980 LSD0040.008 981 982

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LSD0040.021 983 984 LSD0040.037 985 986 LSD0040.046 987 988 LSD0040.003 989 990 LSD0040.006 991 992 LSD0040.007 993 994 LSD0040.010 995 996 LSD0040.039 997 998 LSD0040.052 999 1000 LSD0041.001 1001 1002 LSD0041.004 1003 1004 LSD0041.006 1005 1006 LSD0041.008 1007 1008 LSD0041.010 1009 1010 LSD0041.014 1011 1012 LSD0041.016 1013 1014 LSD0041.035 1015 1016 LSD0043.001 1017 1018 LSD0043.002 1019 1020 LSD0043.005 1021 1022 LSD0043.006 1023 1024 LSD0043.007 1025 1026 LSD0043.008 1027 1028 LSD0043.015 1029 1030 LSD0043.029 1031 1032 LSD0043.043 1033 1034 PSD0077 1035 1036 PSD0078 1037 1038 PSD0079 1039 1040 PSD0080 1041 1042 PSD0081 1043 1044 PSD0082 1045 1046 PSD0083 1047 1048 PSD0084 1049 1050 PSD0085 1051 1052 PSD0086 1053 1054 PSD0087 1055 1056 PSD0088 1057 1058 PSD0089 1059 1060 PSD0090 1061 1062 PSD0091 1063 1064 PSD0092 1065 1066 LSD0049.002 1067 1068 LSD0049.008 1069 1070 LSD0049.011 1071 1072 LSD0049.012 1073 1074 LSD0049.020 1075 1076 LSD0049.021 1077 1078 LSD0050.002 1079 1080 LSD0050.003 1081 1082

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LSD0050.007 1083 1084 LSD0050.010 1085 1086 LSD0050.012 1087 1088 LSD0050.014 1089 1090 LSD0051.002 1091 1092 LSD0051.003 1093 1094 LSD0052.001 1095 1096 LSD0052.003 1097 1098 LSD0053.021 1099 1100 LSD0053.022 1101 1102 LSD0053.024 1103 1104 LSD0054.021 1105 1106 LSD0054.025 1107 1108 LSD0054.026 1109 1110 LSD0055.021 1111 1112 LSD0055.022 1113 1114 LSD0055.026 1115 1116 LSD0056.021 1117 1118 LSD0056.024 1119 1120 LSD0056.025 1121 1122 pNLOOOl 1123 1124 pNL0002 1125 1126 pNL0003 1127 1128 pNL0004 1129 ISO pNL0005 1131 1132 pNL0006 1133 1134 pNL0007 1135 1136 pNL0008 1137 1138 pNL0009 1139 1140 pNLOOlO 1141 1142 pBC0244 1143 1144 pBC0245 1145 1146 pBC0246 1147 1148 pBC0247 1149 1150 pBC0248 1151 1152 pBC0249 1153 1154 pBC0250 1155 1156 pBC0251 1157 1158 pBC0252 1159 1160 pBC0253 1161 1162 pBC0254 1163 1164 pBC0255 1165 1166 pBC0256 1167 1168 pBC0257 1169 1170 pBC0259 1171 1172 pBC0260 1173 1174 pBC0262 1175 1176 pBC0263 1177 1178 pBC0264 1179 1180 pBC0266 1181 1182

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pBC0267 1183 1184 pBC0268 1185 1186 pNL0016 1187 1188 pNL0017 1189 1190 pNL0018 1191 1192 pNL0022 1193 1194 pNL0023 1195 1196 pNL0024 1197 1198 pNL0025 1199 1200 pNL0030 1201 1202 LSD0057.001 1203 1204 LSD0057.004 1205 1206 LSD0057.005 1207 1208 LSD0057.010 1209 1210 LSD0058.003 1211 1212 LSD0058.005 1213 1214 LSD0058.006 1215 1216 LSD0059.002 1217 1218 LSD0059.003 1219 1220 LSD0059.005 1221 1222 LSD0059.006 1223 1224 LSD0060.001 1225 1226 LSD0060.003 1227 1228 LSD0060.004 1229 1230 LSD0061.002 1231 1232 LSD0061.007 1233 1234 LSD0061.008 1235 1236 LSD0061.012 1237 1238 LSD0062.001 1239 1240 LSD0062.002 1241 1242 LSD0062.006 1243 1244 LSD0062.007 1245 1246 LSD0063.001 1247 1248 LSD0063.003 1249 1250 LSD0063.011 1251 1252 LSD0064.017 1253 1254 LSD0064.018 1255 1256 LSD0064.020 1257 1258 LSD0064.021 1259 1260 LSD0065.001 1261 1262 LSD0065.007 1263 1264 LSD0065.014 1265 1266 LSD0066.001 1267 1268 LSD0066.002 1269 1270 LSD0066.009 1271 1272 LSD0066.011 1273 1274 LSD0067.004 1275 1276 LSD0067.005 1277 1278 LSD0067.006 1279 1280 LSD0067.008 1281 1282

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LSD0068.001 1283 1284 LSD0068.002 1285 1286 LSD0068.005 1287 1288 LSD0068.010 1289 1290 LSD0069.004 1291 1292 LSD0069.008 1293 1294 LSD0070.003 1295 1296 LSD0070.004 1297 1298 LSD0070.005 1299 1300 LSD0071.001 1301 1302 LSD0071.002 1303 1304 LSD0071.008 1305 1306 LSD0072.001 1307 1308 LSD0072.002 1309 1310 LSD0072.003 1311 1312 LSD0073.002 1313 1314 LSD0073.004 1315 1316 LSD0073.006 1317 1318 LSD0074.007 1319 1320 LSD0074.010 1321 1322 LSD0074.011 1323 1324 LSD0075.003 1325 1326 LSD0075.004 1327 1328 LSD0075.007 1329 1330 LSD0076.002 1331 1332 LSD0076.003 1333 1334 PSD0093 1335 1336 PSD0094 1337 1338 PSD0095 1339 1340 PSD0096 1341 1342 PSD0097 1343 1344 PSD0098 1345 1346 PSD0099 1347 1348 pSDOlOO 1349 1350 pSDOlOl 1351 1352 PSD0102 1353 1354 PSD0103 1355 1356 PSD0104 1357 1358 pCSOOOl 1359 1360 pCS0002 1361 1362 pCS0003 1363 1364 pCS0004 1365 1366 pCS0005 1367 1368 pCS0006 1369 1370 pBC0269 1371 1372 pBC0270 1373 1374 pBC0271 1375 1376 pBC0272 1377 1378 pBC0273 1379 1380 pBC0274 1381 1382

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pBC0275 1383 1384 pBC0276 1385 1386 pBC0277 1387 1388 pBC0278 1389 1390 pBC0279 1391 1392 pBC0280 1393 1394 pBC0281 1395 1396 pBC0282 1397 1398 pBC0283 1399 1400 pBC0284 1401 1402 pBC0285 1403 1404 pBC0286 1405 1406 pBC0287 1407 1408 pBC0288 1409 1410 pBC0289 1411 1412 pBC0290 1413 1414 pBC0291 1415 1416 pBC0292 1417 1418 pBC0293 1419 1420 pBC0294 1421 1422 pBC0295 1423 1424 pBC0296 1425 1426 pBC0297 1427 1428 pBC0298 1429 1430 pBC0299 1431 1432 rVSZOO 1433 1434 pBC0301 1435 1436 pBC0302 1437 1438 rTHPHOS 1439 1440 pBC0304 1441 1442 pBC0305 1443 1444 rVSOZob 1445 1446 pBC0307 1447 1448 pBC0308 1449 1450 pBC0309 1451 1452 pWSOZU 1453 1454 rWAPO 1455 1456 rWAPO 1457 1458 pBC0313 1459 1460 pBC0314 1461 1462 pBC0315 1463 1464 pBC0316 1465 1466 rWAPO 1467 1468 pBC0318 1469 1470 pBC0319 1471 1472 pBC0320 1473 1474 pBC0321 1475 1476 РВС0322 1477 1478 РВС0323 1479 1480 pNL0040 1481 1482 pNL0041 1483 1484 pNL0042 1485 1486 pNL0043 1487 1488

Example 24 Transfection of mammalian cells, expression of FVIII-ΧΤΕΝ and evaluation of FVIII activity.

Mammalian cells that contain, but are not limited to, CHO, BHK, COS, and HEK293 are suitable for transformation with the vectors of the examples above in order to express and restore the FVIII-ΧΤΕΝ fusion protein. Below are the details of the methods used to express the FVIII BDD and constructs pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0146, and pBC0149 of the FVIII-ΧΤΕΝ fusion protein by transient transfection, which includes electroporation and chemical (PEI) transfection methods.

Adherent HEK293 cells purchased from ATCC are reconstituted in the manufacturer's recommended medium and subcultured several generations before several tubes are frozen in 5% DMSO medium. One tube is reconstituted and subcultured again before transfection. HEK293 cells were seeded 1-2 days prior to transfection at a density of approximately 7x10 ⁵ per ml in T175 per transfection using 35 ml of media. On the day of transfection, cells are trypsinized, separated and counted, then washed in media until a cell suspension is reached. The cells are counted and the appropriate cell volume (based on the number of cells above) is transferred into a 50 ml centrifuge tube so that they are approximately 4 x 10 ⁶ cells per transfection. The cells are centrifuged for 5 min at 500 RCF, the supernatant is removed and the cells are resuspended in 10 ml D-PBS.

Electroporation: In the case of electroporation, an appropriate volume of resuspension buffer is added using a micropipette (provided in the Neon™ Transfection System 100μl Kit) so that 110 μl of buffer per transfection is available. Divide volumes of 110 µl of cell suspension, add to each Eppendorf tube which contains 11 µl of plasmid DNA for each of the individual FVIII-XTEN constructs for a total of 6 µg (DNA volume can be smaller, amounts up to 11 µl with sterile H2O). For transfection use Neon™ Transfection Device. The program is set for electroporation at 1100 volts with a pulse duration of 20 ms, for a total of two pulses. The Neon™ Tube (provided in the Neon™ Transfection System 100 µL Kit) is placed in the Neon™ Pipette Station. A volume of 3 ml of Electrolytic Buffer E2 (provided in the Neon™ Transfection System 100 μL Kit) is added to the Neon™ Tube. Neon™ Pipettes and 100 μL Neon™ Tips are used to electroporate 100 μl of cell-plasmid DNA mixture using the Neon™ Pipette Station. Electroporation is performed, and when completed, the Neon™ Pipettes are removed from the Station and the pipette with the transfected cells is used to transport the cells, in a circular motion, into a 100 mm x 20 mm petri dish that contains 10 ml of Opti-MEM I Reduced-Serum Medium (IX, Invitrogen ) so that the transfected cells are evenly distributed in the dish. For expression, the cells for each transfection are incubated at 37°C. On the 3rd day after transfection, 10% volume of saline solution of 10 mM Hepes, 5 mM CaCl ₂ and 4M NaCl was added to each cell culture and mixed gently for 30 minutes. Each cell culture is transported into a 50 ml conical centrifuge tube and centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatants of each culture are placed in a new 50 ml conical tube and then aliquoted into 5x1 ml Eppendorf and 2x15 ml conical assay tubes or flash frozen before testing for FVIII-XTEN expression in an ELISA and properties in the FVIII activity assay as described in this document.

Chemical transfection: Chemical transfection can be performed using standard methods known in the art. In the present example, PEI is used as described.

The 293 cell suspension is seeded the day before transfection at 7x105 cells/ml in sufficient Freestyle 293 medium (Invitrogen) to provide at least 30 ml working volume and incubated at 37°C. On the day of transfection, a 1.5 ml aliquot of transfection medium is kept at room temperature, to which 90 μl of 1 mg/ml PEI is added and briefly vortexed. A volume of 30 μl of DNA encoding the FVIII-XTEN_AE288 construct (at a concentration of 1 mg/ml) was added to the PEI solution, which was vortexed for 30 sec. The mixture is kept at room temperature for 5-15 minutes. The DNA/PEI mixture is added to HEK293 cells and the suspension is incubated at 37° C. using pre-set shake flask conditions. Approximately 4 hours after the addition of the DNA/PEI mixture, 1x volume of aged medium is added and the cells are incubated at 37° C. for 5 days. On the day of collection, 10% volume of saline solution of 10mM Hepes, 5mM CaCl2 and 4M NaCl is added to the cell culture and mixed gently for 30 minutes. The cell culture is transported into a 50 ml conical centrifuge tube and centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant is placed in a new 50 ml conical tube and then aliquoted into 5x1 ml Eppendorf and 2x15 ml conical assay tubes or flash frozen prior to testing for FVIII-XTEN expression in an ELISA and/or properties in the FVIII activity assay as described in this document.

Generation of stable pools and cell lines that receive FVIII-XTEN.

Stable pools are obtained by culturing the transfected cells for 3-5 weeks in a medium containing a choice of antibiotics such as pyromycin, with a medium change every 23 days. Stable cells can be used to either obtain or generate stable clones. If a stable cell line is selected during initial testing, cells from stable pools, either from ongoing passages or recovered from frozen tubes, are plated in 96-well plates at a target density of 0.5 cells/well. About 1 week after inoculation, used media from wells with the same cell cluster, as observed under a microscope, is tested for FVIII expression by activity assay or antigen measurement.

For additional testing cycles, normalized cell numbers are plated in multi-well plates. The used medium is collected and the FVIII concentration is checked by ELISA and FVIII activity assay. Cells can also be harvested from plates and counted using Vi-Cell. Clones are ordered by (1) FVIII titers according to ELISA and activity; (2) ELISA titer/cell count and activity titer/cell count ratios; and (3) the integrity and homogeneity of the cloned products as measured by Western blotting. The number of clones for each of the constructs is selected from the primary testing in case of additional testing cycles.

For the second round of testing, cells in 96-well plates for the best clones selected from the primary test are first developed in T25 flasks and then plated in identical 24-well plates. Used media is harvested from FVIII activity quantification plates and antigen, and cells are harvested and counted with Vi-Cell. Clones are ordered and then selected according to titers by ELISA and activity assay, titer/cell ratio and activity titer/number of cells in the ELISA assay. Frozen tubes are prepared in the case of at least 5-10 clones and again these clones are examined and sorted according to titers in the case of ELISA and activity assay, and titer/cell count ratio and activity titer/cell count of the ELISA assay, and product integrity and homogeneity by western blotting, and 2-3 clones are selected for shake flask productivity evaluation.

The resulting clones are selected based on specific productivity and product quality. Obtaining FVIII-XTEN secreted in cell culture medium using suspension stable clones 293.

Stable HEK293 cell clones selected using the above methods are seeded in shake flasks at 1-2 x 105 cells/ml in expression medium. Cell count, cell viability, FVIII activity and antigen expression titers are monitored daily. On a day when FVIII activity, antigen titers and production quality are optimal, the culture is harvested either by centrifugation/sterile filtration or depth filtration/sterile filtration. The filtrate is either used directly for tangential flow filtration (TFF) processing and purification, or stored in a freezer at 80° C. for TFF processing and purification later.

Example 25 Cleaning and characterization of CFXTEN structures.

Exemplary methods for cleaning and characterizing CFXTEN structures with one or more XTENs are provided below.

Purification of FVII-XTEN_AE864 by FVIII affinity chromatography.

CFXTEN, which contains the supernatant, is filtered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssure capsule and subsequently concentrated by tangential flow filtration using a 30,000 Da MWCO Millipore Pellicon 2 Mini cartridge. Using the same tangential flow filtration cartridge, the sample was diafiltered into 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride and 0.02% Tween 80 at pH 7.0. The FVIII selection resin (GE 17-5450-01) selectively binds FVIII or FVIII without a B domain using a 13 kDa recombinant protein ligand in combination with a chromatography resin. The resin is equilibrated with 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride and 0.02% Tween 80 at pH 7.0 and the supernatant is loaded. The column was washed with 20 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride and 0.02% Tween 80 at pH 7.0, then washed with 20 mM histidine, 20 mM calcium chloride, 1.0 M sodium chloride and 0.02% Tween 80 at pH 7.0 and eluted with 20 mM histidine, 20 mM calcium chloride, 1.5 M sodium chloride and 0.02% Tween 80 dissolved in 50% ethylene glycol at pH 7.0.

Concentration and buffer exchange using tangential flow filtration and diafiltration.

Series of supernatants with a total volume of at least 10 L from stable CHO cell lines expressing CFXTEN are filtered using a Cuno ZetaPlus Biocap filter and Cuno BioAssure capsules. They are subsequently concentrated approximately 20 times by tangential flow filtration using a 30,000 Da MWCO Millipore Pellicon 2 Mini cartridge. Using the same tangential flow filtration cartridge, the sample is diafiltered with 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride and 0.02% Tween 80 at pH 7.0 10 mM Tris pH 7.5, 1 mM EDTA with 5 volumes of complete buffer replacement. Samples are divided into 50 ml aliquots and frozen at -80°C.

Purification of CFXTEN by anion exchange chromatography.

Using the Akta FPLC system, the sample is purified using a SuperQ-650M column. The column is equilibrated in buffer (0.02 mol/l imidazole, 0.02 mol/l glycine ethyl ester hydrochloride, 0.1-5 mol/l, NaCl, 2.5% glycerol, pH 6.9) and load sample, sample diluted with buffer B (5 mmol/l histidine HCl (His/HCI), 1.15 mol/l NaCl, pH 7.0). The 215 nmol chromatogram is used to monitor the elution profile. The eluted fractions are analyzed for FVIII by ELISA, SDS-PAGE or activity assay. Peak fractions are collected and stored or subjected directly to thrombin activation (O'Brien et al., Blood (1990) 75:1664-1672). Fractions are analyzed for FVIII activity using aPTT-based factor analysis. The Bradford method is carried out to determine the total amount of protein in the loading and eluate fractions.

Purification of CFXTEN by hydrophobic interaction chromatography.

Samples of CFXTEN in buffer A (50 mmol/l histidine, 1 mmol/l CaCl 2 , 1 M NaCl and 0.2 g/l Tween 80®, pH 7.0) are loaded onto toyopearl 650M ether resin equilibrated in buffer A. Column 200 041874 wash with buffer A 10 times the column volume to remove DNA, misfolded forms and FVIII, and other contaminating proteins. CFXTEN is eluted with buffer B (25 mmol/l histidine, 0.5 mmol/l CaCl and 0.4 mol/l NaCl, pH 7.0) as a separate elution step (US Pat. No. 6,005,082). Fractions are analyzed for FVIII activity using aPTT-based factor analysis. The Bradford method is carried out to determine the total amount of protein in the loading and eluate fractions.

Removal of aggregated protein from monomeric CFXTEN by anion exchange chromatography.

Using the Akta FPLC system, the sample was purified using a macrocap Q column. The column was equilibrated in buffer (20 mM MES, 1 mM CaCl ₂ , pH 7.0) and the sample loaded. The sample is diluted using a linear gradient from 30% to 80% buffer B (20 mM MES, 1 mM CaCl ₂ , pH 7.0+500 mM NaCl) over 20 column volumes. The 215 nm chromatogram is used to monitor the elution profile. The fractions corresponding to the initial part of the elution contain primarily the monomeric protein, while the last part of the elution contains primarily the aggregated species. Fractions from the macrocapQ column are analyzed by gel permeation chromatography with a 60 cm BioSep G4000 column to determine which of the pools creates a non-aggregated sample.

FVIII activation by thrombin.

Purified FVIII in 5 mmol/l histidine HCl (His/HCI), 1.15 mol/l NaCl, pH 7.0 is treated with thrombin at a ratio of 1:4 human thrombin units to FVIII units and the sample is incubated at 37°C for up to 2 hours. In order to control the mode of activation, aliquots of this sample are then taken, and the acetone is precipitated by adding 4.5 volumes of very cold acetone. The sample is incubated on ice for 10 minutes and the pellet is collected by centrifugation at 13,000 g in a microcentrifuge for 3 minutes. The acetone is removed and the pellet is resuspended in 30 µl SDS-PAGE, reducing sample buffer, and boiled for 2 minutes. Samples are then analyzed by SDSPAGE or Western blotting. FVIII to FVIIIa conversion is verified by looking for heavy chain conversion to 40 and 50 kDa fragments and light chain conversion to 70 kDa fragment (O'Brien et al., Blood (1990) 75:1664-1672).

SEC analysis of CFXTEN.

FVII-XTEN purified by affinity and anion exchange chromatography was analyzed by gel permeation chromatography with a 60 cm BioSep G4000 column. A monodisperse population with a hydrodynamic radius of ~10 nmol/apparent MW ~1.7 MDa (fused XTEN-288) or ~12 nmol/apparent MW 5.3 MDa (fused XTEN-864) is evidence of a sample without aggregation. CFXTEN is expected to have an average molecular weight factor of up to or about 8 (in the case of a fusion of XTEN-288 with FVIII) or up to or about ~15 (in the case of a fusion of XTEN-864 with FVIII).

Determination of CFXTEN concentration based on ELISA.

Quantification of factor VIII/CFXTEN antigen concentrations using an enzyme linked double antibody immunosorbent assay (ELISA) was performed using validated antibody bindings (FVIII antigen kit from VisuLize™, Affinity Biologicals, Ontario, Canada). The strip wells are precoated with a sheep polyclonal antibody to human FVIII. Plasma samples are diluted and used in wells. The FVIII antigen present is bound to the coated antibody. After washing out the unbound material, a peroxidase-labeled sheep-identifying antibody is used and allowed to bind to the captured FVIII. The wells are washed again and a TMB (peroxidase-specific tetramethylbenzidine) solution is used and allowed to react for a fixed period of time. A blue color develops, which changes to yellow when the reaction is quenched with acid. The color formed is measured spectrophotometrically in a plate reader at 450 nm. The absorbance at 450 nm is directly proportional to the amount of FVII,I antigen captured in the well. The assay is calibrated using either the supplied plasma calibrator or by replacing the standard CFXTEN in the appropriate matrix.

Evaluation of CFXTEN activity by testing with a chromogenic substrate associated with FXa.

Using Chromogenix Coamatic Factor VIII (Chromogenix, cat# 82258563), the activity of FVIII or CFXTEN which contains FVIII is evaluated as follows. In the presence of calcium ions and phospholipids, factor X is activated to factor Xa by factor IXa. This activation is greatly stimulated by factor VIII, which acts as a cofactor in this reaction. By using the optimal amount of Ca ²⁺ , phospholipid, factor IXa, and excess factor X, the rate of factor X activation is linearly related to the amount of factor VIII. Factor Xa hydrolyzes the chromogenic substrate S-2765, thus releasing the chromophore group, pNA. The color is then read spectrophotometrically at 405 nm. Factor Xa is obtained and thus the intensity of the color is proportional to the factor VIII activity in the sample. Hydrolysis of S-2765 by the generated thrombin is prevented by adding the synthetic thrombin inhibitor I-2581 along with the substrate. The activity of an unknown sample is determined by comparing the final A405 of that sample with a standard curve constructed from known amounts of FVIII. Also

- 201 041874 to determine the amount of FVIII antigen present in samples (using A280 or

ELISA) determine the specific activity of a sample to understand the specific activity of a particular FVIII preparation. This allows one to evaluate for activity and classify the relative effectiveness of various isolation strategies or structures of CFXTEN fusion constructs.

aPTT-based assays to determine CFXTEN activity.

CFXTEN acts to replace FVIII in the intrinsic or contact pathway of blood clotting activation. The activity of this coagulation pathway is assessed using an activated partial thromboplastin time (aPTT) assay. FVIII activity is specifically measured as follows: a standard curve is prepared by diluting a normal control plasma (Pacific Hemostasis cat# 100595) 2-fold with FVIII-deficient plasma (cat# 100800) and then making 6.4-fold serial dilutions again with plasma with factor VIII deficiency. This creates a standard curve with points at 500, 130, 31, 7.8, 2.0, 0.5 and 0.1 IU/ml of activity, where one unit of activity is defined as the amount of FVIIIC activity in 1 ml of normal human plasma. To determine the background level of activity in the original plasma, FVIII-deficient plasma is also covered. The sample is prepared by adding CFXTEN to FVIII deficient plasma in a ratio of 1:10 by volume. Samples are tested using the aPTT assay as follows. Samples are incubated at 37° C. in a Molecular Devices plate reader for 2 minutes, at the point where an equivalent volume of aPTT reagent (Pacific Hemostasis cat# 100402) is added and an additional 3 minutes of incubation at 37° C. is performed. After incubation, the assay is activated by adding one volume of calcium chloride (Pacific Hemostasis cat# 100304). Turbidity is measured at 450 nm for 5 minutes to generate reaction profiles. The aPTT time or time to start clotting activity is defined as the first time where OD405 nm is increased by 0.06 from baseline. Log - A linear standard curve is generated with log activity linearly related to aPTT time. From this, the activity of the sample in the well of the plate is determined, and then the activity in the sample is determined by multiplying by 11 to account for dilution in FVIII deficient plasma. Also by determining the amount of FVIII antigen present in the samples (by A280 or ELISA), the specific activity of the sample can be determined to understand the relative specific activity of a particular FVIII preparation. This makes it possible to classify the relative effectiveness of different allocation strategies or structure constructs in the case of CFXTEN mergers.

Western blot analysis of expressed FVIII/FVIII-XTEN proteins.

Samples are placed on 8% homogenous SDS gel and subsequently transferred to a PVDF membrane. Samples in lanes 1-15 are: MM standards, FVIII (42.5 ng), pBC0100B, pBC0114A, pBC0100, pBC0114, pBC0126, pBC0127 (8/5/11; #9), pBC0128, pBC0135, pBC0136, pBC0137, pBC0145, pBC0149 and pBC0146, respectively. The membrane is first blocked with 5% milk, then labeled with an anti-FVIII monoclonal antibody, GMA-012, specific for the A2 domain of the heavy chain (Ansong C, Miles SM, Fay PJ. J Thromb Haemost. 2006 Apr;4(4):842-7 ). Insertion of XTEN288 into the B domain is observed for pBC0136 (lane 8, Fig. 22) and pBC0137 (lane 9, Fig. 22), while insertion of XTEN288 at the C-terminus is observed for pBC0146 (lane 12, Fig. 22 ). All analyzed FVIIIXTEN proteins show the presence of a single chain protein with a molecular weight of at least 21 kDa more than in the case of the main construct pBC0114 or the FVIII standard. In addition, the AE42 insert is observed in the case of pBC0135 (lane 7, Fig. 22) and pBC0149 (lane 11, Fig. 22) with a single chain has ~5 kDa more than in the case of the main protein pBC0114 and the heavy chain has ~5 kDa greater than the 90 kDa core protein band.

Analysis of Expressed FVIII by ELISA.

To test and quantify the expression of the FVIII-XTEN construct fusion proteins, an ELISA assay is performed in cell culture. Immobilized antibodies, either SAF8C-AP (Affinity Biologicals) or GMA-8002 (Green Mountain Antibodies), or GMA011 antibodies (Green Mountain Antibodies) in the case of FVIII-LC ELISA), or GMA016 antibody, are immobilized in the wells of the ELISA plate. The wells are then incubated with blocking buffer (1 x PBS/3% BSA) to prevent non-specific binding of other proteins to the anti-FVIII antibody. Dilutions of the FVIII standard (range ~50 ng-0.024 ng), quality controls and cell culture media samples are then incubated for 1.5 h in the wells to allow binding of the expressed FVIII protein to the coated antibody. The wells are then thoroughly washed and the bound protein is incubated with an anti-FVIII identifying antibody, SAF8C-Biotinylated (Affinity Biologicals). Then, streptavidin-HRP, which binds biotin conjugated to the FVIII-identifying antibody, is added to the well and incubated for 1 hour. Finally, the OPD substrate is added to the wells and its hydrolysis with the HRP enzyme is monitored by a plate reader at a wavelength of 490 nm. The concentrations of samples containing FVIII are then calculated by comparing the colorimetric response to each culture dilution with a standard curve. The results in the table. 22 below demonstrate that various FVIII-XTEN constructs are expressed at 0.4-1 μg/ml in cell culture growth medium. The ELISA results and activity data indicate that the FVIII-XTEN fusion proteins were very well expressed using the described transfection methods. In addition, under experimental conditions, the results demonstrate that the specific activity values of the FVIII-ΧΤΕΝ proteins are similar or higher than those of the main pBC0114 construct (expression of FVIII BDD) and the support provided by insertion of XTEN into the C-terminus or B domain of FVIII. to preserve the function of the FVIII protein.

Chromogenic analysis of the activity of the fusion protein CFXTEN.

BDD FVIII and constructs pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0146 and pBC0149 of the fusion protein CFXTEN in various conformations, which contain XTEN AE288 and AG288, introduced at the C-terminus of the FVIII BDD sequence and the FVIII-ΧΤΕΝ fusion proteins with AE42 and AE28, introduced after residue 745 (or residue 743) and before residue 1640 (or residue 1638) of the B domain (which contains constructs with a P1648 processing site mutated to alanine), expressed in transiently transfected Freestyle 293 cells as described above and tested for procoagulant activity. The procoagulant activity of each of the FVIII-ΧΤΕΝ proteins present in the cell culture medium is assessed using the Chromogenix Coamatic® factor VIII assay, an assay in which factor X activation is linearly related to the amount of factor VIII in the sample. The analysis is carried out according to the manufacturer's instructions using the endpoint method, which is determined spectrophotometrically at OD405 nm. A standard curve was created using purified FVIII protein at concentrations of 250, 200, 150, 100, 75, 50, 37.5, 25, 12.5, 6.25, 3.125, and 1.56 mU/mL. Factor VIII standard dilutions, quality controls, and samples are prepared with assay buffer and PEI culture media to account for media influence when performing the assay. Positive controls consist of purified factor VIII protein at concentrations of 20, 40, and 80 mU/ml and cell culture growth medium of the basic pBC0114 FVIII construct lacking XTEN inserts. Negative controls consist separately of assay buffer or PEL culture medium. Cell culture growth medium for the FVIII-ΧΤΕΝ constructs is prepared as described above and tested in duplicate at 1:50, 1:150 and 1:450 dilutions and the activity of each is calculated in units/. ml. Each FVIII-ΧΤΕΝ construct exhibits procoagulant activity that is at least similar to, and in some cases higher than, the positive control of the main construct, and support that, under experimental conditions, binding of XTEN, which contains AE288 or AG288, at the C-terminus FVIII, or an XTEN insert that contains AE42 or AE288 within the B domain, results in the maintenance or even an increase in the procoagulant activity of FVIII.

Table 22

Results of ELISA and chromogenic assays of FVIII activity

FVIII-XTEN construction Activity (IU/ml) Concentration (µg/ml) Specific activity (IU/mg) Design description pBC0114 h, o 0.6 5000 main BDD FVIII design used for XTEN inserts pBC0146 7.4 0.6 12759 FVIII construct with AG288 XTEN introduced at the C-terminus of FVIII pBC0145 3.1 0.6 4844 FVIII construct with AE288 XTEN introduced at the C-terminus of FVIII pBC0135 4.0 1.0 4124 FVIII construct with AE42 XTEN inserted between residues 745 and 1640 pBC0149 4.9 0.9 5581 FVIII construct with AE42 XTEN introduced between residues 745 and 1640 and with an Argl648 mutation in Ala pBC0136 2.7 0.4 7670 FVIII construct with AE288 XTEN inserted between residues 745 and 1640 pBC0137 1.9 oh, s 6013 FVIII construct with AE288 XTEN introduced between residues 745 and 1640 and with an Argl648 mutation in Ala

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Coatest assay in the case of an activity assay of a cell culture sample that contains the CFXTEN fusion protein.

Using the Coatest assay, the activity of FVIII or CFXTEN that contains FVIII is evaluated as follows.

Matrix Analysis: All wells in the same plate are adjusted to the same percentage of medium to monitor for matrix effects. The test samples are diluted so that the reading at OD405 falls within the linear range of the standard. The concentration range for the FVIII standard is from 100 mU/mL to 0.78 mU/mL obtained by four-fold serial dilution of the FVIII standard in 1X Coatest buffer (DiaPharma) plus a predetermined percentage of culture medium.

Coatest SP FVIII Reagent Kit (DiaPharma) contains 10x Coatest buffer stock solution, factor IXa + factor X, phospholipid, CaCl2 and substrate. A 1x Coatest solution is made by adding 9X cold ddH2O to 1X the mixture. Cell culture medium is then added to make a 1X solution at a predetermined ratio to normalize the percentage of matrix in all test wells. Factor IXa + factor X, phospholipid and substrate are reconstituted according to the manufacturer's recommendations.

Coatest analysis technique.

The substance assay is obtained and kept on ice until needed. 25 µl of diluted test samples and standards are added to a 96-well plate in duplicate. 50 µl of phospholipid/factor IXa/factor X is added to each well and mixed by lightly pressing on the side of the plate. The plates are incubated at 37°C for 5 min on a 37°C plate heater. 25 µl CaCl ₂ is added to each well and mixed. The plates are incubated at 37° C. for 5 minutes in a plate heater. Then, 50 µl of substrate is added to each well, mixed, and the plates are incubated at 37° C. for an additional 5-10 minutes until the upper standard at OD405 reaches a reading of about 1.5. To stop the reaction, 25 µl of 20% acetic acid was added to each well with stirring, and the wells were read at OD405 using a SpectraMAX® plus spectrophotometer (Molecular Devices). Data analysis is carried out using the SoftMax program (version 5.2). The LLOQ in the assay varies, but is generally 0.0039 IU/mL.

Results. the data are presented in table. 23-26. Tab. 23 represents the results for CFXTEN fusion proteins with XTEN introduced at unique sites selected based on the criteria described herein, including Example 34. The pBC00114 FVIII positive control shows good FVIII expression and activity. Of the 106 XTEN single fusion proteins analyzed, 68% retain measurable FVIII activity, with 30% showing 3+ to 4+ activity in the coagulation activity assay. Thirty-one percent of the analyzed fusion proteins have results below the limits of quantitation (which may be due to poor expression reflected in the corresponding ELISA expression results). All four insert constructs in the B domain show good activity, as do the constructs associated with the C-terminus, indicating that these regions are likely to be favorable insertion sites.

The results of the data of a single insertion site are the reason for the creation of XTEN constructs with 2 XTEN inserts, the results of which are presented in Table. 24. Overall, the percentage of positive assays is 67%, with 31% of fusion proteins showing 3+ to 4+ activity in the coagulation activity assay.

The results of the above data are the reason for the creation of XTEN designs with 3 XTEN inserts, the results of which are presented in table. 25. Overall, 92% of samples have measurable FVIII activity, with a total of 79% showing 3+ to 4+ activity in the coagulation activity assay.

A limited number of constructs with 4 XTENs introduced into the A1, A2 and A3 domains were generated and analyzed, with 4 of 5 showing FVIII activity (Table 26), suggesting that the insertion of multiple XTENs does not compromise the ability of the resulting fusion proteins to maintain activity. FVIII.

Conclusions: Under experimental conditions, the results confirm that the criteria for selecting the site of XTEN insertion are appropriate, that insertion of one or more XTENs at the selected FVIII sites is preferable to result in retention of the procoagulant activity of the resulting CFXTEN molecule, and that the insertion of three XTEN appears to result in a higher proportion of fusion proteins, while maintaining high levels of FVIII procoagulant activity, when compared to single or double XTEN insert constructs.

- 204 041874

Table 23 Results of clotting activity assays in the case of CFXTEN ____________ which contains one XTEN _________

Insertion site Domain Design Activity ELISA expression pBC0114 +++ +++ 3 A1 pBC0126 LLOQ* LLOQ 3 A1 pBC0127 + + 18 A1 pBC0165 ++ ++ 22 A1 pBC0183 +++ ++ 26 A1 pBC0184 ++ ++ 40 A1 pBC0166 ++ ++ 60 A1 pBC0185 LLOQ LLOQ 116 A1 pBC0167 LLOQ LLOQ 130 A1 pBC0128 LLOQ LLOQ 188 A1 pBC0168 ++ ++ 216 A1 pBC0129 ++ ++ 230 A1 pBC0169 LLOQ LLOQ 333 A1 pBC0130 ++ ++ 375 A2 pBC0131 LLOQ +++ 403 A2 pBC0132 ++ ++ 442 A2 pBC0170 ++ ++ 490 A2 pBC0133 + ++ 518 A2 pBC0171 LLOQ + 599 A2 pBC0134 ++ ++ 713 A2 pBC0172 + +++ 745 IN pBC0135 +++ +++ 745 IN pBC0149 +++ +++ 745 IN pBC0136 ++ ++ 745 V pBC0137 +++ +++ 1720 AZ pBC0138 +++ +++ 1796 AZ pBC0139 + ++ 1802 AZ pBC0140 + ++ 1827 AZ pBC0173 LLOQ LLOQ 1861 AZ pBC0174 LLOQ LLOQ 1896 AZ pBC0175 LLOQ LLOQ 1900 AZ pBC0176 +++ +++ 1904 AZ pBC0177 + + 1937 AZ pBC0178 LLOQ LLOQ 2019 AZ pBC0141 LLOQ + 2068 C1 pBC0179 ++ ++ 2111 C1 pBC0180 LLOQ LLOQ 2120 C1 pBC0142 LLOQ + 2171 C2 pBC0143 ++ +++ 2188 C2 pBC0181 LLOQ LLOQ 2227 C2 pBC0182 ++ +++ 2277 C2 pBC0144 ++ ++ 2332 ST pBC0145 +++ +++ 2332 st pBC0146 +++ +++ 403 A2 pSDOOOl +++ +++ 599 A2 PSD0002 + + 403 A2 PSD0003 +++ +++ 599 A2 PSD0004 + + 745 IN PSD0005 +++ ++ 745 IN PSD0006 +++ +++ 745 V PSD0007 +++ ++ 745 V PSD0008 +++ +++ 1720 AZ PSD0009 + + 1720 AZ pSDOOlO ++ ++ 2171 C2 pSDOOll + ++ 2171 C2 PSD0012 + ++ 2332 ST PSD0013 +++ ++ 2332 st PSD0014 +++ +++ 745 V PSD0017 +++ +++

- 205 041874

745 IN PSD0018 +++ +++ 2332 ST PSD0019 +++ +++ 2332 st PSD0020 +++ +++ 2332 st PSD0015 ++ ++ 2332 st PSD0016 +++ +++ 0 N term PSD0021 + + 32 Al PSD0022 +++ +++ 65 Al PSD0023 LLOQ LLOQ 81 Al PSD0024 LLOQ LLOQ 119 Al PSD0025 LLOQ LLOQ 211 Al PSD0026 + + 220 Al PSD0027 + + 224 Al PSD0028 + + 336 Al PSD0029 ++ +++ 339 Al PSD0030 ++ +++ 378 A2 PSD0031 LLOQ ++ 399 A2 PSD0032 ++ ++ 409 A2 pSDOO33 ++ ++ 416 A2 PSD0034 + + 487 A2 pSDOO35 LLOQ + 494 A2 PSD0036 LLOQ + 500 A2 PSD0037 LLOQ + 603 A2 PSD0038 + + 1656 A3 PSD0039 +++ +++ 1656 A3 pNL009** ++++ ND 1711 A3 PSD0040 ++ + 1725 A3 PSD0041 LLOQ ++ 1749 A3 PSD0042 LLOQ LLOQ 1905 A3 PSD0043 ++ ++ 1910 A3 PSD0044 + + 1900 A3 PSD0062 ++ ++ 1900 A3 PSD0063 +++ ++ 18 Al PSD0045 +++ +++ 18 Al PSD0046 +++ +++ 22 Al PSD0047 LLOQ LLOQ 22 Al PSD0048 LLOQ LLOQ 26 Al PSD0049 +++ +++ 26 Al PSD0050 +++ +++ 40 Al PSD0051 +++ +++ 40 Al PSD0052 +++ +++ 216 Al pSDOO53 LLOQ LLOQ 216 Al PSD0054 LLOQ LLOQ 375 A2 pSDOO55 LLOQ + 442 A2 PSD0056 LLOQ LLOQ 442 A2 PSD0057 LLOQ LLOQ 1796 A3 PSD0058 LLOQ LLOQ 1796 A3 PSD0059 + + 1802 A3 PSD0060 + + 1802 A3 PSD0061 LLOQ LLOQ

* LLOQ: below the limits of quantitation.

** pNL009 contains a deletion 745-1656.

- 206 041874

Table 24 Results of clotting activity assays for CFXTEN containing two XTENs

Box 1 Box 2 Design Activity Insertion site Domain Insertion site Domain 745 IN 2332 ST LSD0001.002 +++ 745 IN 2332 ST LSD0001.005 +++ 745 IN 2332 st LSD0001.006 +++ 745 V 2332 st LSD0001.011 +++ 745 V 2332 st LSD0001.012 +++ 745 V 2332 st LSD0001.013 +++ 745 V 2332 st LSD0001.016 +++ 745 V 2332 st LSD0001.021 +++ 745 V 2332 st LSD0002.001 +++ 745 V 2332 st LSD0002.002 +++ 745 V 2332 st LSD0002.014 +++ 745 V 2332 st LSD0003.004 +++ 745 V 2332 st LSD0003.006 +++ 745 V 2332 st LSD0003.009 +++ 745 V 2332 st LSD0003.014 + 745 V 2332 st LSD0004.010 +++ 745 V 2332 st LSD0004.011 LLOQ 745 V 2332 st LSD0004.014 +++ 745 V 2332 st LSD0004.016 +++ 745 V 2332 st LSD0004.022 +++ 745 V 2332 st LSD0003.016 +++ 0745 V 2332 st pNL006 +++ 0745 V 2332 st pNL007 +++ 0745 V 2332 st pNL008 ++ 1656 a3 2332 st pNLOlO +++ 26 A1 403 A2 LSD0005.002 ++ 26 A1 403 A2 LSD0005.004 ++ 40 A1 403 A2 LSD0005.005 ++ 40 A1 403 A2 LSD0005.011 ++ 18 A1 403 A2 LSD0005.018 ++ 26 A1 599 A2 LSD0006.002 + 40 A1 599 A2 LSD0006.005 ++ 40 A1 599 A2 LSD0006.007 ++ 40 A1 599 A2 LSD0006.011 +++ 40 A1 403 A2 LSD0007.002 + 40 A1 403 A2 LSD0007.004 +

- 207 041874

26 Al 403 A2 LSD0007.013 ++ 26 Al 599 A2 LSD0008.001 4-4- 40 Al 599 A2 LSD0008.002 ++ 26 Al 599 A2 LSD0008.006 + 18 Al 599 A2 LSD0008.009 ++ 40 Al 599 A2 LSD0008.017 + 745 IN 2332 ST LSD0002.025 +++ 745 IN 2332 ST LSD0002.013 +++ 745 V 2332 st LSD0003.025 +++ 745 V 2332 st LSD0004.025 +++ 745 V 2332 st LSD0003.005 ++ 26 Al 403 A2 LSD0007.008 ++ 1720 AZ 1900 AZ LSD0044.002 LLOQ 1725 AZ 1900 AZ LSD0044.005 LLOQ 1720 AZ 1900 AZ LSD0044.039 LLOQ 1711 AZ 1905 AZ LSD0044.022 LLOQ 1720 AZ 1905 AZ LSD0044.003 LLOQ 1725 AZ 1905 AZ LSD0044.001 LLOQ 1656 AZ 26 A1 LSD0038.001 4-4- 1656 AZ 18 A1 LSD0038.003 ++ 1656 AZ 18 A1 LSD0038.008 +++ 1656 AZ 40 A1 LSD0038.012 ++ 1656 AZ 40 A1 LSD0038.013 ++ 1656 AZ 26 A1 LSD0038.015 ++ 1656 AZ 399 A2 LSD0039.001 + 1656 AZ 403 A2 LSD0039.003 4-4- 1656 AZ 403 A2 LSD0039.010 ++ 1656 AZ 1725 AZ LSD0045.001 + 1656 AZ 1720 AZ LSD0045.002 ++ 1900 AZ 18 A1 LSD0042.014 + 1900 AZ 18 A1 LSD0042.023 + 1900 AZ 26 A1 LSD0042.006 + 1900 AZ 26 A1 LSD0042.013 4-4- 1900 AZ 40 A1 LSD0042.001 + 1900 AZ 40 A1 LSD0042.039 + 1900 AZ 26 A1 LSD0042.047 + 1905 AZ 18 A1 LSD0042.003 + 1905 AZ 40 A1 LSD0042.004 LLOQ 1905 AZ 26 A1 LSD0042.008 LLOQ 1905 AZ 26 A1 LSD0042.038 LLOQ 1905 AZ 40 A1 LSD0042.082 LLOQ 1910 AZ 26 A1 LSD0042.040 LLOQ 18 A1 399 A2 LSD0037.002 ++ 26 A1 399 A2 LSD0037.009 + 40 A1 399 A2 LSD0037.011 4-4- 18 A1 403 A2 LSD0047.002 ++ 18 A1 403 A2 LSD0047.005 +

- 208 041874

18 Al 403 A2 LSD0048.007 + 1656 AZ 1900 AZ LSD0046.001 ++ 1656 AZ 1900 AZ LSD0046.002 + 1656 AZ 1905 AZ LSD0046.003 + 1711 AZ 40 A1 LSD0040.011 LLOQ 1711 AZ 26 A1 LSD0040.042 LLOQ 1720 AZ 26 A1 LSD0040.002 + 1720 AZ 40 A1 LSD0040.008 + 1720 AZ 18 A1 LSD0040.021 + 1720 AZ 26 A1 LSD0040.037 LLOQ 1720 AZ 18 A1 LSD0040.046 + 1725 AZ 26 A1 LSD0040.003 LLOQ 1725 AZ 40 A1 LSD0040.006 LLOQ 1725 AZ 26 A1 LSD0040.007 LLOQ 1725 AZ 18 A1 LSD0040.010 LLOQ 1725 AZ 40 A1 LSD0040.039 LLOQ 1725 AZ 18 A1 LSD0040.052 + 1720 AZ 403 A2 LSD0041.001 + 1720 AZ 399 A2 LSD0041.004 LLOQ 1711 AZ 403 A2 LSD0041.006 LLOQ 1720 AZ 403 A2 LSD0041.008 LLOQ 1725 AZ 403 A2 LSD0041.010 LLOQ 1725 AZ 403 A2 LSD0041.014 LLOQ 1725 AZ 399 A2 LSD0041.016 LLOQ 1711 AZ 403 A2 LSD0041.035 LLOQ 1900 AZ 399 A2 LSD0043.001 LLOQ 1900 AZ 403 A2 LSD0043.002 LLOQ 1905 AZ 403 A2 LSD0043.005 LLOQ 1900 AZ 399 A2 LSD0043.006 LLOQ 1900 AZ 403 A2 LSD0043.007 LLOQ 1900 AZ 403 A2 LSD0043.008 LLOQ 1905 AZ 399 A2 LSD0043.015 LLOQ 1905 AZ 403 A2 LSD0043.029 LLOQ 1910 AZ 403 A2 LSD0043.043 LLOQ

- 209 041874

Table 25

Clotting activity assay results for CFXTEN containing three XTENs

Box 1 Box 2 Box 3 Insertional home Insertional home Insertional home constructs Active th site n th site n th site n AND I be 26 A1 403 A2 1656 AZ PSD0077 +++ 26 A1 403 A2 1720 AZ PSD0078 ++ 26 A1 403 A2 1900 AZ PSD0079 ++ 26 A1 1656 AZ 1720 AZ PSD0080 +++ 26 A1 1656 AZ 1900 AZ PSD0081 LLOQ 26 A1 1720 AZ 1900 AZ PSD0082 + 403 A2 1656 AZ 1720 AZ PSD0083 +++ 403 A2 1656 AZ 1900 AZ PSD0084 +++ 403 A2 1720 AZ 1900 AZ PSD0085 + 1656 AZ 1720 AZ 1900 AZ PSD0086 +++ 18 A1 745 IN 2332 ST LSD0049.0 02 +++ 26 A1 745 IN 2332 st LSD0049.0 08 +++ 26 A1 745 V 2332 st LSD0049.0 AND +++ 40 A1 745 V 2332 st LSD0049.0 12 +++ 40 A1 745 V 2332 st LSD0049.0 20 +++ 18 A1 745 V 2332 st LSD0049.0 21 +++ 40 A1 745 V 2332 st LSD0050.0 02 +++ 18 A1 745 V 2332 st LSD0050.0 03 +++ 26 A1 745 V 2332 st LSD0050.0 07 LLOQ 18 A1 745 V 2332 st LSD0050.0 10 +++ 26 A1 745 V 2332 st LSD0050.0 12 +++ 40 A1 745 V 2332 st LSD0050.0 14 +++ 403 A2 745 V 2332 st LSD0051.0 02 +++ 399 A2 745 V 2332 st LSD0051.0 03 +++ 403 A2 745 V 2332 st LSD0052.0 01 +++ 399 A2 745 V 2332 st LSD0052.0 03 +++ 1725 AZ 745 V 2332 st LSD0053.0 21 LLOQ 1720 AZ 745 V 2332 st LSD0053.0 22 +++ 1711 AZ 745 V 2332 st LSD0053.0 24 +++ 1720 AZ 745 V 2332 st LSD0054.0 21 +++ 1711 AZ 745 V 2332 st LSD0054.0 25 ++ 1725 AZ 745 V 2332 st LSD0054.0 26 +++

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1900 AZ 745 IN 2332 ST LSD0055.0 21 +++ 1905 AZ 745 IN 2332 st LSD0055.0 22 +++ 1900 AZ 745 IN 2332 st LSD0055.0 26 +++ 1900 AZ 745 V 2332 st LSD0056.0 21 +++ 1900 AZ 745 V 2332 st LSD0056.0 24 +++ 1910 AZ 745 V 2332 st LSD0056.0 25 +++ 0745 IN 1900 AZ 2332 st pBC0294* 0745 IN 1900 AZ 2332 st pBC0295* 0745 V 1900 AZ 2332 st pBC0296* 0745 V 1900 AZ 2332 st pBC0297* 0745 V 1900 AZ 2332 st pBC0298* 0745 V 1900 AZ 2332 st pBC0299* 0745 V 1900 AZ 2332 st rVSOZOO* 0745 V 1900 AZ 2332 st pWSOZO!* 0745 V 1900 AZ 2332 st pBC0302* 0745 V 1900 AZ 2332 st rTHPHO* 0745 V 1900 AZ 2332 st pBC0304* 0745 V 1900 AZ 2332 st pBC0305* 0745 V 1900 AZ 2332 st pVSOZob* 0745 V 1900 AZ 2332 st pBC0307* 0745 V 1900 AZ 2332 st pBC0308* 0745 V 1900 AZ 2332 st pBC0309* 0745 V 1900 AZ 2332 st pBSOZU* 0745 V 1900 AZ 2332 st rWSOZI* 0745 V 1900 AZ 2332 st pBC0312* 0745 V 1900 AZ 2332 st pBC0313* 0745 V 1900 AZ 2332 st rWHOSM* 0745 V 1900 AZ 2332 st pBC0315* 0745 V 1900 AZ 2332 st pBC0316* 0745 V 1900 AZ 2332 st pBC0317* 0745 V 1900 AZ 2332 st pBC0318* 0745 V 1900 AZ 2332 st pBC0319* 0745 V 1900 AZ 2332 st pBC0320* 0018 A1 0745 IN 2332 st pBC0269* 0403 A2 0745 IN 2332 st pBC0270* 1720 AZ 0745 V 2332 st pBC0271* 1900 AZ 0745 V 2332 st pBC0272* 0403 A2 0745 V 2332 st pBC0273* 1720 AZ 0745 V 2332 st pBC0274* 1900 AZ 0745 V 2332 st pBC0275* 0018 A1 0745 V 2332 st pBC0276* 0403 A2 0745 V 2332 st pBC0277* 1720 AZ 0745 V 2332 st pBC0278* 1900 AZ 0745 V 2332 st pBC0279*

* Construct with mutation R1648A.

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Table 26 Results of clotting activity assays for CFXTEN containing four XTENs

XTEN Box 1 XTEN Box 2 XTEN Insert XTEN Box 4 XTEN Box 5 XTEN Box 6 Design ID Activity 26 403 1656 1720 - - PSD0087 +++ 26 403 1656 1900 - - PSD0088 +++ 26 403 1720 1900 - - PSD0089 LLOQ 26 1656 1720 1900 - - PSD0090 ++ 403 1656 1720 1900 - - PSD0091 ++ 0040 0403 745 2332 - - LSD0058.006* ++ 0018 0409 745 2332 - - LSD0059.002* + 0040 0409 745 2332 - - LSD0059.006* + 0040 0409 745 2332 - - LSD0060.001* + 0018 0409 745 2332 - - LSD0060.003* + 0040 1720 745 2332 - - LSD0061.002* + 0026 1720 745 2332 - - LSD0061.007* ++ 0018 1720 745 2332 - - LSD0061.008* ++ 0018 1720 745 2332 - - LSD0061.012* ++ 0018 1720 745 2332 - - LSD0062.001* ++ 0026 1720 745 2332 - - LSD0062.002* ++ 0018 1720 745 2332 - - LSD0062.006* ++ 0018 1900 745 2332 - - LSD0063.001* ++ 0018 1900 745 2332 - - LSD0064.017* ++ 0026 1900 745 2332 - - LSD0064.020* ++ 0040 1900 745 2332 - - LSD0064.021* ++ 0040 1905 745 2332 - - LSD0065.001* + 0018 1905 745 2332 - - LSD0065.014* + 0040 1905 745 2332 - - LSD0066.001* + 0026 1905 745 2332 - - LSD0066.002* + 0018 1905 745 2332 - - LSD0066.009* ++ 0018 1905 745 2332 - - LSD0066.011* ++ 0018 1910 745 2332 - - LSD0067.004* ++ 0018 1910 745 2332 - - LSD0067.005* + 0040 1910 745 2332 - - LSD0067.006* + 0026 1910 745 2332 - - LSD0067.008* + 0018 1910 745 2332 - - LSD0068.001* + 0026 1910 745 2332 - - LSD0068.002* +

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0040 1910 745 2332 - - LSD0068.005* + 0018 1910 745 2332 - - LSD0068.010* ++ 0409 1720 745 2332 - - LSD0069.004* + 0403 1720 745 2332 - - LSD0069.008* + 0409 1720 745 2332 - - LSD0070.003* + 0403 1720 745 2332 - - LSD0070.004* ++ 0403 1720 745 2332 - - LSD0070.005* ++ 0403 1900 745 2332 - - LSD007L001* ++ 0403 1900 745 2332 - - LSD0071.002* + 0409 1900 745 2332 - - LSD0071.008* ++ 0403 1900 745 2332 - - LSD0072.001* ++ 0403 1900 745 2332 - - LSD0072.002* + 0409 1900 745 2332 - - LSD0072.003* + 0409 1905 745 2332 - - LSD0073.002* + 0403 1905 745 2332 - - LSD0073.004* + 0403 1905 745 2332 - - LSD0073.006* + 0403 1905 745 2332 - - LSD0074.007* ++ 0409 1905 745 2332 - - LSD0074.010* + 0403 1905 745 2332 - - LSD0074.011* + 0409 1910 745 2332 - - LSD0075.004* + 0403 1910 745 2332 - - LSD0075.007* + 0403 1910 745 2332 - - LSD0076.002* + 0403 1910 745 2332 - - LSD0076.003* + 0403 1910 745 2332 - - PSD0093* + 1720 1900 745 2332 - - PSD0094* ++ 1720 1905 745 2332 - - PSD0095* + 1720 1910 745 2332 - - PSD0097* + 1720 1910 745 2332 - - PSD0098* + 0403 1656 1720 2332 - - pNL0022 + 0403 1656 1900 2332 - - pNL0023 + 0403 1720 1900 2332 - - pNL0024 LLOQ 1656 1720 1900 2332 - - pNL0025 + 0018 0403 1656 2332 - - pBC0247 ++ 0018 0403 1720 2332 - - pBC0248 + 0018 0403 1900 2332 - - pBC0249 + 0018 1656 1720 2332 - - pBC0250 + 0018 1656 1900 2332 - - pBC0251 ++ 0018 1720 1900 2332 - - pBC0252 LLOQ 0018 0403 0745 2332 - - LSD57.005 ++ 0018 0745 1720 2332 - - LSD62.001 ++

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0018 0745 1900 2332 - - pBC0262 ++ 0403 0745 1720 2332 - - LSD70.004 + 0403 0745 1900 2332 - - pBC0266 + 0745 1720 1900 2332 - - pBC0268 + 0188 1900 0745 2332 - - pCSOOOl* ND 0599 1900 0745 2332 - - pCS0002* ND 2068 1900 0745 2332 - - pCS0003* ND 2171 1900 0745 2332 - - pCS0004* ND 2227 1900 0745 2332 - - pCS0005* ND 2277 1900 0745 2332 - - pCS0006* ND 0403 1656 1720 1900 2332 - pNL0030 LLOQ 0018 0403 1656 1720 2332 - pBC0253 + 0018 0403 1656 1900 2332 - pBC0254 + 0018 0403 1720 1900 2332 - pBC0255 LLOQ 0018 1656 1720 1900 2332 - pBC0256 + 0018 0403 0745 1720 2332 - pBC0259* + 0018 0403 0745 1900 2332 - pBC0260* + 0018 0745 1720 1900 2332 - pBC0263 + 0403 0745 1720 1900 2332 - pBC0267 LLOQ 0018 0403 1656 1720 1900 2332 pBC0257 LLOQ 0018 0403 0745 1720 1900 2332 pBC0264 LLOQ

* Construct with R1648A mutation.

Example 26. Determination of XTEN radii and related parameters.

In order to quantify the hydrodynamic radii of the XTEN components of CFXTEN fusion proteins and how the magnitude of multiple XTENs varies depending on the unique XTEN, a series of formulas are created based on empirically derived data from gel permeation chromatography analyzes of various fusion proteins that contain one or more XTENs. . The incorporation of several XTENs into CFXTEN is believed to provide a larger overall hydrodynamic radius of the XTEN component, compared to CFXTEN with smaller XTEN, which has approximately the same total XTEN amino acid count. The maximum radius of an individual XTEN polypeptide is calculated (hereinafter XTEN radius) according to the formula given in Equation II:

Radius XTEN = (Uddina XTEN 0.2037) + 3.4627 II

The sum of the maximum XTEN radii of all XTEN segments in CFXTEN is calculated (hereinafter Sum of XTEN radii) according to the formula given in Equation III:

Radius; XTEN

Sum of XTEN radii = ^1-1 III where η = number of XTEN segments and i is an iterator.

The ratio of the sum of the XTEN radii of a CFXTEN that contains several XTENs, for which the XTEN radius is calculated in the case of a unique XTEN of equivalent length (in the total number of amino acid residues with the CFXTEN) (hereinafter, the XTEN Radius Ratio) according to the formula given in Equation IV:

ΣΡ ₌₁ Radius; XTEN n vtc\i (λ/ΣΡ—1 Length XTEN * 0.2037) + 3.4627

Radius ratio XTEN = (-ι lengths!im / IV where η = number of XTEN segments and i is an iterator.

Results. Equation II is applied to XTEN with lengths 144, 288, 576 and 864. The results are presented in table. 27. Equation IV is applied to the various CFXTEN fusion proteins described herein with two, three or four XTENs. The XTEN radius ratio has a value of 1 for all CFXTENs that contain a unique XTEN. The ratio of radii XTEN is presented in Table. 28. The XTEN radius ratio in the case of pSD0092, which contains 5 XTEN inserts, has a value of 3.31. Taken together, the results indicate that the inclusion of multiple XTENs increases the XTEN radius ratio to values above 2, resulting in higher values for four inserts than for three inserts.

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Table 27

Radius calculation results in case of CFXTEN which contains XTEN

XEN length Radius XTEN 42 4.8 144 5.9 288 6.9 576 8.4 864 9.5

Table 28

Radius calculation results in case of CFXTEN which contains XTEN

Box 1 Box 2 Box 3 Box 4

Embed site Domain Insert Site Domain Insert Site Domain Embed site Domain Design Radius ratio XTEN 40 A1 pBC0166 1.00 745 IN 2332 ST LSD0001.002 1.67 745 IN 2332 ST LSD0001.005 1.71 745 IN 2332 st LSD0001.006 1.71 745 IN 2332 st LSD0001.011 1.71 745 IN 2332 st LSD0001.012 1.71 745 IN 2332 st LSD0001.013 1.67 745 IN 2332 st LSD0001.016 1.67 745 IN 2332 st LSD0001.021 1.67 745 IN 2332 st LSD0002.001 1.67 745 IN 2332 st LSD0002.002 1.67 745 IN 2332 st LSD0002.004 1.71 745 V 2332 st LSD0002.008 1.67 745 V 2332 st LSD0002.014 1.67 745 V 2332 st LSD0003.001 1.67 745 V 2332 st LSD0003.004 1.66 745 V 2332 st LSD0003.006 1.67 745 V 2332 st LSD0003.009 1.67 745 V 2332 st LSD0003.014 1.66 745 V 2332 st LSD0003.018 1.67 745 V 2332 st LSD0004.010 1.66 745 V 2332 st LSD0004.011 1.67 745 V 2332 st LSD0004.014 1.66 745 V 2332 st LSD0004.016 1.66 745 V 2332 st LSD0004.022 1.66 745 V 2332 st LSD0003.016 1.67 26 A1 403 A2 LSD0005.002 1.71 26 A1 403 A2 LSD0005.004 1.71 40 A1 403 A2 LSDOOO5.OO5 1.71 40 A1 403 A2 LSD0005.011 1.71 18 A1 403 A2 LSD0005.018 1.71 26 A1 599 A2 LSD0006.002 1.71 40 A1 599 A2 LSD0006.005 1.71 40 A1 599 A2 LSD0006.007 1.71 40 A1 599 A2 LSD0006.011 1.71 40 A1 403 A2 LSD0007.002 1.71 40 A1 403 A2 LSD0007.004 1.71 26 A1 403 A2 LSD0007.013 1.71 26 A1 599 A2 LSD0008.001 1.71 40 A1 599 A2 LSD0008.002 1.71 26 A1 599 A2 LSD0008.006 1.71 18 A1 599 A2 LSD0008.009 1.71 40 A1 599 A2 LSD0008.017 1.71

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745 IN 2332 ST LSD0002.025 1.71 745 IN 2332 st LSD0002.013 1.67 745 V 2332 st LSD0003.025 1.67 745 V 2332 st LSD0004.025 1.67 745 V 2332 st LSDOOO3.OO5 1.66 26 A1 403 A2 LSD0007.008 1.71 1720 AZ 1900 AZ LSD0044.002 1.71 1725 AZ 1900 AZ LSD0044.005 1.71 1720 AZ 1900 AZ LSD0044.039 1.71 1711 AZ 1905 AZ LSD0044.022 1.71 1720 AZ 1905 AZ LSD0044.003 1.71 1725 AZ 1905 AZ LSD0044.001 1.71 1656 AZ 26 A1 LSD0038.001 1.71 1656 AZ 18 A1 LSD0038.003 1.71 1656 AZ 18 A1 LSD0038.008 1.71 1656 AZ 40 A1 LSD0038.012 1.71 1656 AZ 40 A1 LSD0038.013 1.71 1656 AZ 26 A1 LSD0038.015 1.71 1656 AZ 399 A2 LSD0039.001 1.71 1656 AZ 403 A2 LSD0039.003 1.71 1656 AZ 403 A2 LSD0039.010 1.71 1656 AZ 1725 AZ LSD0045.001 1.71 1656 AZ 1720 AZ LSD0045.002 1.71 1900 AZ 18 A1 LSD0042.014 1.71 1900 AZ 18 A1 LSD0042.023 1.71 1900 AZ 26 A1 LSD0042.006 1.71 1900 AZ 26 A1 LSD0042.013 1.71 1900 AZ 40 A1 LSD0042.001 1.71 1900 AZ 40 A1 LSD0042.039 1.71 1900 AZ 26 A1 LSD0042.047 1.71 1905 AZ 18 A1 LSD0042.003 1.71 1905 AZ 40 A1 LSD0042.004 1.71 1905 AZ 26 A1 LSD0042.008 1.71 1905 AZ 26 A1 LSD0042.038 1.71 1905 AZ 40 A1 LSD0042.082 1.71 1910 AZ 26 A1 LSD0042.040 1.71 18 A1 399 A2 LSD0037.002 1.71 26 A1 399 A2 LSD0037.009 1.71 40 A1 399 A2 LSD0037.011 1.71 18 A1 403 A2 LSD0047.002 1.71 18 A1 403 A2 LSD0047.005 1.71 18 A1 403 A2 LSD0048.007 1.71 1656 AZ 1900 AZ LSD0046.001 1.71 1656 AZ 1900 AZ LSD0046.002 1.71 1656 AZ 1905 AZ LSD0046.003 1.71

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1711 AZ 40 A1 LSD0040.011 1.71 1711 AZ 26 A1 LSD0040.042 1.71 1720 AZ 26 A1 LSD0040.002 1.71 1720 AZ 40 A1 LSD0040.008 1.71 1720 AZ 18 A1 LSD0040.021 1.71 1720 AZ 26 A1 LSD0040.037 1.71 1720 AZ 18 A1 LSD0040.046 1.71 1725 AZ 26 A1 LSD0040.003 1.71 1725 AZ 40 A1 LSD0040.006 1.71 1725 AZ 26 A1 LSD0040.007 1.71 1725 AZ 18 A1 LSD0040.010 1.71 1725 AZ 40 A1 LSD0040.039 1.71 1725 AZ 18 A1 LSD0040.052 1.71 1720 AZ 403 A2 LSD0041.001 1.71 1720 AZ 399 A2 LSD0041.004 1.71 1711 AZ 403 A2 LSD0041.006 1.71 1720 AZ 403 A2 LSD0041.008 1.71 1725 AZ 403 A2 LSD0041.010 1.71 1725 AZ 403 A2 LSD0041.014 1.71 1725 AZ 399 A2 LSD0041.016 1.71 1711 AZ 403 A2 LSD0041.035 1.71 1900 AZ 399 A2 LSD0043.001 1.71 1900 AZ 403 A2 LSD0043.002 1.71 1905 AZ 403 A2 LSD0043.005 1.71 1900 AZ 399 A2 LSD0043.006 1.71 1900 AZ 403 A2 LSD0043.007 1.71 1900 AZ 403 A2 LSD0043.008 1.71 1905 AZ 399 A2 LSD0043.015 1.71 1905 AZ 403 A2 LSD0043.029 1.71 1910 AZ 403 A2 LSD0043.043 1.71 26 A1 403 A2 1656 AZ PSD0077 2.30 26 A1 403 A2 1720 AZ PSD0078 2.30 26 A1 403 A2 1900 AZ PSD0079 2.30 26 A1 1656 AZ 1720 AZ PSD0080 2.30 26 A1 1656 AZ 1900 AZ PSD0081 2.30 26 A1 1720 AZ 1900 AZ PSD0082 2.30 403 A2 1656 AZ 1720 AZ PSD0083 2.30 403 A2 1656 AZ 1900 AZ PSD0084 2.30 403 A2 1720 AZ 1900 AZ PSD0085 2.30 1656 AZ 1720 AZ 1900 AZ PSD0086 2.30 26 A1 403 A2 1656 AZ 1720 AZ PSD0087 2.83 26 A1 403 A2 1656 AZ 1900 AZ PSD0088 2.83 26 A1 403 A2 1720 AZ 1900 AZ PSD0089 2.83 26 A1 1656 AZ 1720 AZ 1900 AZ PSD0090 2.83 403 A2 1656 AZ 1720 AZ 1900 AZ PSD0091 2.83

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26 A1 403 A2 1656 AZ 1720 AZ PSD0092 2.83 18 A1 745 IN 2332 ST LSD0049.002 2.24 26 A1 745 IN 2332 st LSD0049.008 2.24 26 A1 745 IN 2332 st LSD0049.011 2.24 40 A1 745 V 2332 st LSD0049.012 2.24 40 A1 745 V 2332 st LSD0049.020 2.24 18 A1 745 V 2332 st LSD0049.021 2.24 40 A1 745 V 2332 st LSD0050.002 2.24 18 A1 745 V 2332 st LSDOO5O.OO3 2.24 26 A1 745 V 2332 st LSD0050.007 2.24 18 A1 745 V 2332 st LSD0050.010 2.24 26 A1 745 V 2332 st LSD0050.012 2.24 40 A1 745 V 2332 st LSD0050.014 2.24 403 A2 745 V 2332 st LSD0051.002 2.24 399 A2 745 V 2332 st LSD0051.003 2.24 403 A2 745 V 2332 st LSD0052.001 2.24 399 A2 745 V 2332 st LSD0052.003 2.24 1725 AZ 745 V 2332 st LSD0053.021 2.24 1720 AZ 745 V 2332 st LSD0053.022 2.24 1711 AZ 745 V 2332 st LSD0053.024 2.24 1720 AZ 745 V 2332 st LSD0054.021 2.24 1711 AZ 745 V 2332 st LSD0054.025 2.24 1725 AZ 745 V 2332 st LSD0054.026 2.24 1900 AZ 745 V 2332 st LSD0055.021 2.24 1905 AZ 745 V 2332 st LSD0055.022 2.24 1900 AZ 745 V 2332 st LSD0055.026 2.24 1900 AZ 745 V 2332 st LSD0056.021 2.24 1900 AZ 745 V 2332 st LSD0056.024 2.24 1910 AZ 745 V 2332 st LSD0056.025 2.24

Example 27 Binding of FVIII-XTEN disruption to an anti-FVin antibody.

The ability of XTEN administered at different locations of the CFXTEN fusion proteins to influence the binding of anti-FVIII antibodies is determined using sandwich ELISA assays. Two anti-FVIII antibodies; e.g. GMA-8021 (Green Mountain Antibodies, Burlington, VT) and ESH8 (American Diagnostica Inc., Stamford, CT), which bind to the A2 and C2 domains, respectively, are used as capture antibodies. XTEN-free FVIII-His-Myc protein is used as a calibration standard and positive control for all ELISAs. Ten CFXTEN fusion proteins are created with single XTEN inserts in either the A1, A2 or A3 domain, which additionally contain His and Myc affinity tags. The protein concentrations of each test sample are normalized to 100% based on the use of ELISA detection of anti-His-immobilized anti-Myc simultaneously on the same plate as the ELISA to detect anti-FVIII antibody-immobilized anti-Myc.

Briefly, the appropriate wells in a 96-well plate are coated with GMA-8021, ESH8 or anti-His antibody overnight at 4°C, then washed and blocked with BSA. Equivalent volumes of the appropriate control or fusion proteins are injected into identical wells and allowed to react with coated GMA-8021, ESH8, or anti-His antibody for 2 hours at room temperature. After incubation, unbound material is washed out and rabbit anti-Myc identifying antibody is added and incubated for an additional 1 hour at room temperature. The plate is then washed, a peroxidase-conjugated donkey anti-rabbit secondary antibody is injected and incubated for 1 hour at room temperature. The plate is washed again, followed by the addition of TMB substrate and the reaction is allowed to proceed for 5-20 min. H ₂ SO ₄ is introduced to stop the reaction and the absorbance is read with a spectrophotometer at 450 nm.

Results. The results are presented in table. 29. Taken together, these results indicate that two antibodies against CFXTEN to XTEN fusion proteins introduced into the A2 domain show reduced FVIII binding compared to CFXTEN to XTEN introduced into the A1 or A3 domain when the anti-FVIII capture antibody is GMA-8021 (with binding affinity for the A2 domain). In contrast, there was no noticeable inhibition or increase in binding by any CFXTEN when the anti-FVIII capture antibody is ESH8, with binding affinity for the C2 domain.

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Table 29

Linking FVIII-XTEN disruption to anti-FVIII antibody

Test sample Insert XTEN (domain, site, XTEN) aFVIII/Myc concentration ± aHis/Myc concentration His/Myc GMA-8021/Myc (domain A2) ESH8/Myc (domain C2) FVIII-His-Myc None 100% 92% 104% FVIII-XTEN-HisMus A2, 403, AE144 100% 103%±1% 141%±24% A2, 403, AG144 100% 104%±6% 129%±12% A2, 399, AE144 100% 100%±8% 140%±18% AZ, 1656, AG144 100% 153% 158% Al, 18, AE144 100% 129% 130% Al, 18, AG144 100% 150% 131% Al, 26, AE144 100% 155% 87% Al, 26, AG144 100% 157% 147% Al, 40, AE144 100% 137% 147% Al, 40, AG144 100% 164%±0% 153%±18%

aFVIII/Myc = disease with GMA-8021/Myc or ESH8/Myc antibodies;

aHis/Myc = anti-His/Myc antibody disease.

Example 28 Activity Assay of CFXTEN Fusion Proteins in the Presence of FVIII Inhibitors.

Method for testing an inhibitor by titration.

Selected antibodies that inhibit the procoagulant activity of FVIII are purchased from commercial sources. The antibodies target selected FVIII domains (eg, A2, A3, C1, C2) and inhibit FVIII-dependent procoagulant activity. In order to establish the optimal concentration of FVIII inhibitors to use in the assay, an initial titration experiment is performed using different amounts of each inhibitory antibody incubated at 37° C. for 2 h with a major His/Myc double-tagged FVIII expression vector, and a second sample with the antibody and at least one CFXTEN fusion protein. The samples are then used in a coagulation activity assay to determine FVIII activity. Activity is measured using the Coatest quantitation method described herein. The concentration that results in optimal inhibition of FVIII activity is determined for each antibody individually.

Inhibitor testing method.

The FVIII inhibitor antibodies are then used at their optimum concentration to analyze the test samples. CFXTEN and positive control samples are individually incubated with each antibody at 37°C for 2 h, the samples are then collected and used in the Coatest activity assay, along with untreated aliquots of CFXTEN and positive control. In selected cases, CFXTEN constructs with the R1648A mutation are tested to determine the effect of this mutation, if any, on resistance to inhibitors as measured by retention of FVIII activity.

Results.

The results of the titration experiment are illustrated in FIG. 26. Data indicate an order of magnitude right shift of approximately 0.7 in the amount of antibody required to inhibit CFXTEN's LSD0049.002 procoagulant activity to a level of 50%, compared to a FVIII positive control indicating that CFXTEN with three XTEN inserts ( at the insertion points corresponding to BDD-FVIII amino acid residues 18, 745 and 2332) has lower antibody binding compared to FVIII, reflected in the retention of clotting activity.

The results of Coatest analyzes are presented in table. 30 and 31 for the FVIII inhibitor antibodies GMA8008 and GMA8021, respectively. All of the untreated, validated CFXTEN fusion protein constructs exhibited procoagulant activity, as did the pBC00114 FVIII positive control. The positive control sample is pre-incubated with FVIII inhibitor antibodies, resulting in a dramatic decrease in measured clotting activity to 0.05-0.15 (5-15%), relative to the untreated sample, as is the case with most CFXTEN constructs treated with GMA8008 antibody to domain C2. Despite this, the three CFXTEN fusion proteins retained at least twice the relative remaining activity as compared to the FVIII control; LSD0049.020, LSD0053.024 and LSD0056.025 each with three XTEN inserts.

CFXTEN samples showed a lower degree of inhibition by GMA8021 to the A2 domain, compared to untreated samples, which was further reduced with additional XTEN inserts (Table 30). Fig. 29 illustrates a graph of mean ratios for control of retained activity that demonstrates a linear relationship between the amount of XTEN administered and reduced inhibition with GMA8021 versus inhibition of the FVIII control. The same way,

- 219 041874 values + S.O. in relation to the control values is 2.26+0.12 in the case of 1 XTEN, 3.48+0.26 in the case of 2 XTEN and 5.70+0.29 in the case of 3 XTEN inserts. CFTXEN with at least three XTEN inserts treated with the GMA8021 antibody has at least 4.5 to 9.2 times greater retention of FVIII activity compared to the FVIII control. In addition, in those CFXTENs with three XTEN inserts, constructs with a higher degree of separation (number of amino acid residues) between any two inserts result in a higher degree of procoagulant activity and hence less FVIII inhibitor antibody binding, compared to inserts clustered more tight; for example, on the C-terminal sides of the B-domain. The results of the analysis of constructs with the R1648A mutation were comparable to those without mutations.

Conclusions: The results suggest that, under experimental conditions, insertion of XTEN into FVIII results in protection from binding by FVIII inhibitors while maintaining procoagulant activity, and that insertion of multiple XTEN inserts increases resistance to, in particular, an A2 domain inhibitor antibody. Finally, there appears to be an influence that the spatial separation between XTEN inserts has.

Table 30 Coagulation Activity Assay Results with CFXTEN Treated with Antibody GMA8008 to C2 Domain

The name of the structure Relative residual activity Relative to control Insert 1XTEN Box 2 XTEN Insert 3XTEN Mutations and pVS0114ST 0.05-0.15 1 pBC0149 OD 0.8 0745 AE42 1 PSD0045 oh, s 1.1 0018_AE144_5 A PSD0046 oh, s 1.0 0018AG144F PSD0050 0.2 0.9 0026AG144F PSD0051 oh, s 1.3 0040-AE144-5 A PSD0052 0.2 1.0 0040AG144F pSDOOOl 0.2 0.9 0403-AE 144_2 A pBC0136 0.2 1.2 0745 AE288 1 pBC0137 0.2 1.1 0745 AE288 1 R1648A PSD0013 0.1 0.9 2332_AE144_6 IN PSD0014 0.1 0.8 2332AG1441 pBC0145 0.1 0.6 2332 AE288 1 PSD0019 0.1 0.5 2332 AE288 1 pBC0146 0.1 0.7 2332AG2881 PSD0015 0.1 0.8 2332 AE864 LSD0038.008 0.1 0.9 0018AG144F 1656AG1 44 C LSD0038.013 0.1 0.6 0040AG144F 1656AG1 44 C LSD003.09 0.1 0.9 0745-AE144-3 IN 2332-AE2 88 1 LSD003.06 0.0 0.8 0745-AE 144-3 IN 2332-AE2 88 1 R1648A LSD0046.001 0.0 0.6 1656 AG144 C 1900_AGl 44 C PSD077 0.1 1.0 0026AG144F 0403_AE 1 44 2A 1656 A.G. 144 C PSD080 0.1 1.0 0026AG144F 1656AG1 44 C 1720_AG 144 C PSD083 0.1 0.8 0403-AE 144-2 A 1656-AG1 44 C 1720_AG 144 C PSD084 0.1 0.9 0403 AE144 2 1656AG1 1900 AE1

-220041874

A 44_С 44_4A LSD0050.010 0.1 0.7 0018_AE144_5 A 0745_AE1 44 SG 2332_AE2 88 1 LSD0049.021 0.0 0.6 0018_AE144_5 A 0745_AE1 44 SG 2332_AE2 88 1 R1648A LSD0049.002 0.1 0.9 0018AG144F 0745_AE1 44 SG 2332_AE2 88 1 R1648A LSD0049.008 0.1 0.9 0026_AE144_5 A 0745_AE1 44 SG 2332_AE2 88 1 R1648A LSD0049.011 0.1 0.9 0026AG144F 0745_AE1 44 SG 2332_AE2 88 1 R1648A LSD0049.020 0.2 2.6 0040_AE144_5 A 0745_AE1 44 SG 2332_AE2 88 1 R1648A LSD0050.002 oh oh 0.2 0040AG144F 0745_AE1 44 SG 2332_AE2 88 1 LSD0053.024 0.2 2.5 1711_AE144_4 A 0745_AE1 44 SG 2332_AE2 88 1 LSD0054.021 0.2 1.5 1720 AG144 C 0745_AE1 44 SG 2332_AE2 88 1 LSD0055.021 0.2 1.6 1900_AE144_4 A 0745_AE1 44 SG 2332_AE2 88 1 R1648A LSD0056.021 0.2 1.6 1900 AG144 C 0745_AE1 44 SG 2332_AE2 88 1 LSD0056,025 oh, s 2.0 1910 AG144 C 0745_AE1 44 SG 2332_AE2 88 1

the percentage of activity remaining in relation to the corresponding untreated sample;

the ratio of relative residual activity (relative to its own control), compared to the positive control pBC0114 FVIII.

Table 31

Coagulation activity assay results with CFXTEN treated with GMA8021 anti-A2 domain antibody

Design name Relative residual activity Relationship to control Box 1 XTEN Box 2 XTEN Box 3 XTEN Mutats nor pBC0114 0.05-0.15 1 pBC0149 0.2 1.3 0745 AE42 1 PSD0045 oh, s 2.7 0018_AE144_ 5A PSD0046 0.2 2.1 0018_AG144 F PSD0050 0.2 2.4 0026_AG144 F PSD0051 oh, s 3.1 0040_AE144_ 5A PSD0052 oh, s 2.7 0040_AG144 F pSDOOOl 0.2 1.6 0403_AE144_2A pBC0136 oh, s 2.4 0745_AE288_ 1 pBC0137 oh, s 2.4 0745_AE288_ 1 R1648A

- 221 041874

PSD0013 0.2 1.8 2332_AE144_ 6V PSD0014 0.2 2.1 2332_AG144 1 pBC0145 oh, s 2.1 2332_AE288_ 1 PSD0019 oh, s 2.3 2332_AE288_ 1 pBC0146 oh, s 2.1 2332_AG288 1 PSD0015 oh, s 2.8 2332 AE864 LSD0038.0 08 0.4 h, o 0018_AG144 F 1656_AG144 C LSD0038.0 13 0.4 h, o 0040_AG144 F 1656_AG144 C LSD003.09 oh, s 3.6 0745_AE144_ 3B 2332_AE288_ 1 LSD003.06 oh, s 3.4 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0046.0 01 0.2 4.4 1656_AG144 C 1900_AG144 C PSD077 0.4 5.8 0026_AG144 F 0403_AE144_2A 1656_AG144_C PSD080 0.4 5.7 0026_AG144 F 1656_AG144 C 1720_AG144_C PSD083 oh, s 5.0 0403_AE144_2A 1656_AG144 C 1720_AG144_C PSD084 oh, s 4.5 0403AE144 1656AG144 1900AE144 2A _C 4A LSD0050.0 10 0.4 6.7 0018_AE144_5A 0745_AE144_ 3B 2332_AE288_ 1 LSD0049.0 21 0.4 6.7 0018_AE144_5A 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0049.0 02 0.5 9.2 0018_AG144 F 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0049.0 08 0.4 5.9 0026_AE144_5A 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0049.0 AND 0.4 5.6 0026_AG144 F 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0049.0 20 oh, s 5.0 0040_AE144_5A 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0050.0 02 oh, s 6.2 0040_AG144 F 0745_AE144_ 3B 2332_AE288_ 1 LSD0053.0 24 oh, s 4.5 1711_AE144_4A 0745_AE144_ 3B 2332_AE288_ 1 LSD0054.0 21 0.5 5.2 1720_AG144 C 0745_AE144_ 3B 2332_AE288_ 1 LSD0055.0 21 0.5 5.4 1900_AE144_4A 0745_AE144_ 3B 2332_AE288_ 1 R1648A LSD0056.0 21 0.5 5.1 1900_AG144C 0745_AE144_ 3B 2332_AE288_ 1 LSD0056.0 25 0.5 4.8 1910_AG144C 0745_AE144_ 3B 2332_AE288_ 1

the percentage of activity remaining in relation to the corresponding untreated sample;

the ratio of relative residual activity (relative to its own control), compared to the positive control pBC0114 FVIII.

Example 29 Protein Purification of pBC0145 and pBC0146 CFXTEN Fusion Proteins.

Two CFXTEN constructs with a C-terminal XTEN are used to establish the purification method. For both pBC0145, with a C-terminal 288 amino acid XTEN of the AE family (see sequence in Table 21), and pBC0146 with a C-terminal XTEN of 288 amino acids of the AG family (see sequence in Table 21), a tangential flow filtration step (TFF) is used to replace the buffer clarified conditioned medium from cell culture. The products are then obtained using a strong anion exchange resin for chromatography, and then further purified using VIII selective affinity chromatography (GE Healthcare). Additionally, gel permeation chromatography (GE Healthcare) is applied to FVIII-pBC0146 as a third processing step to remove high molecular weight species. The purity of both fusion proteins was found to be acceptable after HPLC-SEC and further confirmed by SDS-PAGE analysis of two CFXTEN constructs,

- 222 041874 which show CFXTEN products in the expected sizes. The specific activity of both molecules is similar to FVIII without B domains as measured by aPTT coagulation assay and FVIII concentration by ELISA.

Example 30 Pharmacokinetic properties of CFXTEN pBC0145 and pBC0146 fusion proteins in HemA and FVIII/VWF DKO mice.

Male FVIII knockout (HemA) or double knockout (DKO) FVIII/VWF mice, 8-12 weeks old, are treated with a single intravenous injection of either recombinant BDD-FVIII, pBC0145CFXTEN, or pBC0146 fused purified proteins (from Example 23) at 200 IU/ kg dose (n=4/time point). At selected time points, blood samples are collected by vena cava. In HemA mice, blood samples are collected at 5 min, 1, 4, 8, 16, 20, 24, 32, and 48 h post-dose of rBDD-FVIII, and at 5 min, 8, 16, 24, 32, 48, 55 and 72 hours post dose of fusion proteins pBC0145 and pBC0146. In FVIII/VWF DKO mice, blood samples were collected 5 min, 30 min and 1 hour post-dose of rBDD-FVIII, and 5 min, 4, 8, 16 and 24 h post-dose of the pBC0145 and pBC0146 fusion proteins. Plasma FVIII activity is measured by FVIII chromogenic assay and PK profile is analyzed by WinNonlin software.

Results. As shown in Table. 32 and FIG. 24, CFXTEN with an AE insert at the C-terminus of XTEN (pBC0145) shows a 1.6 and 14.1-fold increase in FVIII half-life (T1/2), compared to rBDD FVIII in HemA mice and FVIII/VWF DKO mice, respectively. CFXTEN with an AG insert at the C-terminus of XTEN (pBC0146) has a 1.4-fold and 14.4-fold increase in half-life compared to rBDDFVIII in HemA mice and FVIII/VWF DKO mice, respectively. The magnitude of the increase in FVIII half-life provided by the XTEN insert is much more pronounced in FVIII/VWF DKO mice compared to HemA mice, as demonstrated by the 1.4-fold longer half-life of FVIII and FVIII-AE-XTEN. and FVIII-AG-XTEN, compared to rBDD-FVIII. In addition, compared to rBDD-FVIII, FVIII with C-terminal insert AE or AG-XTEN also have significantly improved FVIII recovery at 5 min, reduced clearance, volume of distribution, and increased AUC in DKO mice. Under experimental conditions, CFXTEN with XTEN C-terminal inserts shows great potential in increasing the half-life of FVIII, and, in combination with other intradomain FVIII inserts, can potentially further extend the half-life of FVIII.

Table 32

Pharmacokinetic parameters of CFXTEN in . mice HemA and FVIII/VWF DKO

M mouse strain Treatment Recovery 5 min (%) Т1/2 (h) MRT (h) C1 (ml/h/kg) Vss (ml/kg) AUC_ D (hkgm IU/ml/mIU) Growth T1/2 Mice strain Hema pBC0145 73 11.88 16.47 3.81 62.74 0.26 1.6 Hema pBC0146 64 10.54 13.31 5.66 75.34 0.18 1.4 rBDDFVIII 89 7.58 11.02 4.33 47.68 0.23 FVIII/ VWF DKO pBC0145 74 3.38 3.76 13.06 63.68 0.0765 13.9 FVIII/ VWF DKO pBC0146 61 3.45 3.61 17.40 86.63 0.0575 14.2 rBDDFVIII 23 0.24 0.24 460.62 161.51 0.0022

compared to rBDD-FVIII.

Example 31 Cell Culture and Cell Culture Medium Concentration for the CFXTEN pSD0050 and pSD0062 Fusion Proteins.

Variants pSD0050 of construct CFXTEN with intradomain AG XTEN of 144 amino acids introduced after amino acid residue 26 BDD FVIII, pSD0062 with intradomain AE XTEN of 144 amino acids introduced after residue 1900 of FVIII BDD (Note: amino acid is numbered based on the entire length of FVIII) and construct encoding rBDD-FVIII was transfected into HEK293F cells (Invitrogen, Carlsbad, CA) using polyethyleneimine (PEI, Polysciences Inc. Warrington, PA). Transiently transfected cells are grown in 293 Free Style medium (Invitrogen, Carlsbad, CA) for 4 days and 50-100 ml of cell culture medium is then concentrated 10-20 times with a Centricon Spin Column (100 kDa MM cut-off) for in order to achieve an FVIII activity of 10-30 IU/ml. The concentrated materials are then flash frozen and stored at -80°C for further in vitro analysis and in vivo pharmacokinetic studies.

Example 32 Pharmacokinetic properties of pSD0050 and pSD0062 CFXTEN fusion proteins in HemA and FVIII/VWF DKO mice.

Male HemA or FVIII/VWF double knockout (DKO) mice, 8-12 weeks old, are treated with a single intravenous injection of cell culture concentrates from Example 31, which contain either recombinant BDD-FVIII, CFXTEN pBD0050 or pBD062 at 100- 300 IU/kg (n=3/group). At selected time points, blood samples are collected by retroorbital sinus sampling from the same set of mice. In HemA mice, blood samples are collected at 5 min, 24 h, and 48 h post-dose, while in FVIII/VWF DKO mice, blood samples are collected at 5 min, 8 h, and 16 h. FVIII activity of plasma samples and culture concentrates cells are analyzed using FVIII chromogenic assay, rBDD FVIII PK profile and FVIII-XTEN variants are analyzed using WinNonlin software.

Results. the PK profiles of two CFXTEN intradomain insertion variants pSD0050 and pSD0062 and rBDD-FVIII in HemA mice and FVIII/VWF DKO mice are illustrated in FIG. 25 and in table. 33. In HemA mice, a similar initial recovery within 5 minutes was observed for the three test FVIII molecules. Both CFXTEN fusion proteins show twice the half-life as compared to wild-type BDD-FVIII. In FVIII-VWF DKO mice, due to loss of VWF protection, rBDD-FVIII only has a 15 min plasma half-life. In the case of two CFXTENs, despite this, the half-life is increased to 3.15 hours and 3.83 hours, respectively; values that are similar to CFXTEN with 288 C-terminal XTEN inserts (Example 24) suggest that an additional increase in XTEN length at this insertion point may not be necessary. Under experimental conditions, the results of the study clearly demonstrate that the intradomain insertion of XTEN with 144 amino acid residues not only retains FVIII activity, but also provides a similar FVIII half-life advantage as the 288 amino acid C-terminal insertion variants in XTEN, suggesting that the combination of intradomain and C-terminal inserts of FVIII may allow an additional increase in the half-life of FVIII.

Table 33

Pharmacokinetic Parameters of CFXTEN in HemA and FVIII/VWF DKO Mice

M mouse strain Treatment Recovery 5 min (%) Tl/2 (h) MRT (h) Cl (ml/h/kg) Vss (ml/kg) AUC D (hkgmIU / ml / mIU) Tl/2 Increase t Newsha PSD005 0 40 14.12 14.25 5.27 75.03 0.19 2.3 PSD006 2 43 12.96 14.79 4.24 62.67 0.24 2.1 rBDDFVIII 47 6.19 2.62 6.35 16.62 0.16 FVIII/ VWF DKO PSD005 0 34 3.15 2.59 21.73 56.28 0.05 -12 PSD006 2 35 3.83 3.71 18.51 68.69 0.05 -15 rBDDFVIII 23 -0.25

compared to rBDD-FVIII.

Example 33 Pharmacokinetic analysis of CFXTEN fusion polypeptides in rats.

The pharmacokinetic properties of the various CFXTEN fusion proteins compared to FVIII alone were tested in Sprague-Dawley rats. CFXTEN and FVIII are administered to female Sprague-Dawley rats (n=3) IV via a jugular catheter at 3-10 μg/rat. Blood samples (0.2 ml) are collected in pre-chilled pre-dose heparinized tubes at time points 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 h and processed in plasma. The quantitation of the test substances is carried out using an ELISA assay using an anti-FVIII antibody for both immobilization and detection. Non-compartmental analysis is carried out in WinNonLin with all time points that are contained in the set to determine the PK parameters. The results are expected to show an increase in terminal half-life and area under the curve, a reduced volume of distribution for CFXEN compared to FVIII alone, and the results are used in conjunction with the results of fold and pharmacodynamic assays to select fusion protein conformations with the desired properties.

Example 34 FVIII analysis for XTEN insertion sites.

The selection of XTEN insertion sites within the factor VIII molecule is carried out by predicting the localizations of permissive sites within loop structures or, in other cases, the conformationally labile surface of the located structural elements. These analyzes use the atomic coordinates of two independently determined crystallographic structures of FVIII obtained by X-ray diffraction analysis (Shen BW, et al. The tertiary structure and domain organization of coagulation factor VIII. Blood. (2008) Feb 1; 111(3): 1240 -1247 Ngo JC, et al Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex Structure (2008)16(4):597-606) and factor VIII and factor VIIIa obtained from simulation of molecular

- 224 041874 (MDS) (Venkateswarlu, D. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study. BMC Struct Biol. (2010) 10:7). Protein Data Bank (PDB) atomic coordinates are analyzed to identify FVIII/FVIIIa regions expected to have a highly solvent-accessible surface area using ASAView algorithms (Ahmad S, et al. ASAView: database and tool for solvent accessibility representation in proteins. BMC Bioinformatics (2004) 5:51) and GetArea (Rychkov G, Petukhov M. Joint neighbors approximation of macromolecular solvent accessible surface area. J Comput Chem (2007) 28(12): 1974-1989). The resulting set of sites is then further prioritized based on the high fluctuation of the predicted atomic positions based on the published results of the MDS study. Sites within the acidic peptide region flank the A1, A2, and A3 domains, and also deprioritize those that appeared by visual inspection, existing in other regions, and not on the surface of the located loops. The resulting set of potential sites are evaluated based on interspecies sequence conservatism, with those sites in regions of high sequence conservation among the 20 vertebrate species being favored in the evaluation. Additionally, putative exit receptor binding sites, FVIII interaction sites with other molecules (such as vWF, FIX), domain and exon boundaries are also considered in the selection of the fusion site. Finally, sites in the immediate vicinity of the mutations are implicated in hemophilia A, listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database eliminated (Kembal-Cook G, et al. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res. (1998) 26(1):216-219). Based on this criterion, in the case of XTEN inserts, the construction of 42 FVIII-XTEN variants is proposed. Of these, three are XTEN inserts within the B residual domain sequence, two are extensions of the factor VIII molecule at the C-terminus, and 37 are XTEN inserts within structurally defined inter- and intra-domain building blocks; those. residues 3, 18, 22, 26, 40, 60, 116, 130, 188, 216, 230, 333, 375, 403, 442, 490, 518, 599, 713, 745, 1720, 1796, 1802, 1827, 1861 , 1896, 1900, 1904, 1937, 2019, 2068, 2111, 2120, 2171, 2188, 2227, 2277 and 2332.

Example 35 Functional Analysis of FVIII-XTEN Constructs.

Two FVIII-XTEN fusion proteins, FVIII-AE288 (F8X-40) and FVIII-AG288 (F8X-41), contain AE288_1 XTEN or AG288_1 XTEN, respectively, fused at the C-terminus of the FVIII C2 domain. To determine if FVIII activity is retained after XTEN fusion, HEK293 cells are transfected separately with these two FVIII-XTEN fusion constructs using polyethyleneimine (PEI) in serum-free medium. 3 or 5 days after transfection, the cell culture supernatant is tested for FVIII activity using a two-step chromogenic assay. Purified recombinant FVIII, calibrated against the international WHO standard, is used to create a standard curve in the chromogenic assay. The fusion protein products of both constructs, F8X-40 and F8X-41, were expressed at levels similar to those of the wild-type BDD-FVIII constructs (Table 34).

Table 34

FVIII titer of FVIII-XTEN fusion proteins. in transient transfection cell culture

FVIII molecules FVIII 066 ^a pBC0114 ^a F8X-40 F8X-41 Activity FVIII (IU/ml) Sample A 6.42 6.68 7.47 3.32 ^b Sample B 7.13 7.61 8.25 Not done

^a Both FVIII 066 and pBC 0114 contain FVIII without B domains without XTEN fusion. ^b Sample F8X-41 was from a 3 day transfection while the other samples were from a 5 day transient transfection.

Example 36 Functional Analysis of FVIII-XTEN Constructs: FVIII Activity and PK Properties.

The potential for increased half-life of constructs F8X-40 and F8X-41 was assessed in mice with two knockout genes, FVIII and von Willebrand factor, by hydrodynamic DNA plasmid injection, with the FVIIIFc DNA construct serving as a positive control. The mice are randomly divided into 3 groups with 4 mice per group. The plasmid DNA encodes the FVIIIFc, F8X-40 or F8X-41 BDD fusion protein, all spreading the same DNA vector backbone as administered to the mice in the respective groups. Approximately 100 micrograms of the appropriate plasmid DNA is administered to each mouse by hydrodynamic injection, and plasma samples are collected 24 and 48 hours after injection. Plasma FVIII activity is measured by a two-step chromogenic assay using calibrated recombinant FVIII as a standard. As shown in FIG. 23, samples from groups F8X-40 and F8X-41 show higher plasma titers of FVIII than those from BDD FVIIIFc, suggesting that fusion of FVIII with XTEN prolongs the in vivo half-life of FVIII. Taken together, these data provide the conclusion that the FVIII-XTEN fusion proteins retain FVIII activity in transient transfection and, in an animal model, exhibit a prolonged circulating half-life.

Example 37 Pharmacodynamic evaluation of CFXTEN in animal models.

- 225 041874

The in vivo pharmacological activity of the CFXTEN fusion proteins is evaluated using a range of preclinical bleeding models, which include, but are not limited to, hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding, and hydrodynamic injection. These models are developed in several species that contain mice, rats, rabbits, and dogs using methods equivalent to those used and published for other FVIII approaches. Compositions of CFXTEN provide in an aqueous buffer compatible with in vivo administration (for example: phosphate-buffered saline or TRIS-buffered saline). The compositions are administered at appropriate doses, dosing frequency, dosing regimen and routes of administration that are optimized for the particular model. Efficacy definitions include FVIII activity measurement, one-step coagulation activity assay, FVIII chromogenic assay, prothrombin formation and activity time (aPTT), bleeding time, whole blood clotting time (WBCT), thromboelastography (TEG or ROTEM), among others.

In one example, the PD model, CFXTEN and FVIII are administered to genetically deficient or experimentally induced HemA mice. At various time points after administration, FVIII and CFXTEN levels are measured by ELISA, FVIII and CFXTEN activity is measured by commercially available FVIII activity kits, and clotting time is measured by aPTTa assay. Overall, the results may indicate that CFXTEN constructs may be more effective in inhibiting bleeding than FVIII and/or equivalent in specific activity with a similar dosage of FVIII at less frequent or more convenient dosing intervals.

In the case of bleeding in the CFXTEN mouse, the PD and FVIII models are administered to genetically deficient or experimentally induced HemA mice and the effect on the hemostatic problem is measured. A hemostatic problem may include a tail truncation problem, a hemarthrosis problem, articular bleeding, or a saphenous vein problem, among others. At various time points after administration, FVIII and CFXTEN levels are measured by ELISA, FVIII and CFXTEN activity is measured by a commercially available FVIII activity kit, bleeding time and clotting time are measured by aPTT assay. Overall, the results are expected to show that CFXTEN constructs are more effective in inhibiting bleeding than FVIII and/or equivalent in specific activity at a similar dosage of FVIII with less frequent or more convenient dosing intervals, and the results are used in conjunction with folding results and other analyses, in order to select fusion protein conformations with desired properties.

In the canine model, PD, CFXTEN and FVIII are administered to genetically deficient dogs with hemophilia. At various time points after administration, FVIII and CFXTEN levels are measured by ELISA, FVIII and CFXTEN activity is measured by a commercially available FVIII activity kit, and clotting time is measured by aPTT assay. Overall, the results indicate that CFXTEN constructs may be more effective in inhibiting bleeding than FVIII and/or equivalent in specific activity at a similar dosage of FVIII with less frequent or more convenient dosing, and the results are used in conjunction with clotting results and other assays. , in order to select fusion protein conformations with desired properties.

In the case of bleeding in a PD dog, the CFXTEN and FVIII model is administered to genetically deficient dogs with hemophilia and the impact on the hemostatic problem is measured. The hemostatic problem contains, among others, bleeding time in the cuticle. At various time points after administration, FVIII and CFXTEN levels are measured by ELISA, FVIII and CFXTEN activity is measured by a commercially available FVIII activity kit, bleeding time is measured, and clotting time is measured by aPTT assay. Overall, the results indicate that CFXTEN constructs may be more effective in inhibiting bleeding than FVIII and/or equivalent in specific activity at a similar dosage of FVIII with less frequent or more convenient dosing intervals, and the results are used in conjunction with the results of clotting and other assays in order to select fusion protein conformations with desired properties.

Additional preclinical bleeding models include, but are not limited to, hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding, and hydrodynamic injection. These models can be developed in multiple species containing mice, rats, rabbits and dogs using methods equivalent to those used and published for other FVIII approaches. Overall, the results indicate that CFXTEN constructs may be more effective in inhibiting bleeding than FVIII and/or equivalent in specific activity at a similar dosage of FVIII with less frequent or more convenient dosing intervals, and the results are used in conjunction with clotting and other results. analyzes in order to select fusion protein conformations with desired properties.

Example 38 CFXTEN with split sequences.

C-terminal XTEN secreted by FXIa.

The fusion protein CFXTEN, consisting of the XTEN protein fused to the C-terminus of FVIII, was created using

- 226 041874 XTEN secretion site cleavage sequence placed between FVIII and XTEN components as illustrated in FIG. 12. Illustrative sequences are shown in table. 51. In this case, the secretion site cleavage sequence is inserted into CFXTEN, which contains an amino acid sequence that is recognized and cleaved by the FXIa protease (EC 3.4.21.27, Uniprot P03951). Specifically, the amino acid sequence of KLTRAET (SEQ ID NO: 1688) is truncated after the arginine sequence with the FXIa protease. FXI is a procoagulant protease located just before FVIII in the intrinsic or intrinsic coagulation pathway. Active FXIa is obtained from FXI by proteolytic zymogen cleavage with FXIIa. The production of FXIa is tightly controlled and occurs only when coagulation is necessary for proper hemostasis. Thus, by incorporating the KLTRAET cleavage sequence (SEQ ID NO: 1688), the XTEN domain is removed from FVIII only, concurrently with activation of the intrinsic coagulation pathway and if coagulation is physiologically required. This creates a situation where the CFXTEN fusion protein is processed in an additional manner during intrinsic pathway activation.

C-terminal XTEN secreted by FIIa (thrombin).

The CFXTEN fusion protein, consisting of the XTEN protein fused to the C-terminus of FVIII, was created with the XTEN secretion site cleavage sequence placed between FVIII and the XTEN components, as illustrated in FIG. 12. In this case, the secretion site contains an amino acid sequence that is recognized and cleaved by the FIIa protease (EC 3.4.21.5, Uniprot P00734). In particular, the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320] is truncated after arginine at position 4 in the sequence. Active FIIa is obtained by cleavage of FII with FXa in the presence of phospholipids and calcium and further downstream from factor IX in the coagulation pathway. Once activated, its natural role is to coagulate to break down fibrinogen (Figure 2), which then in turn initiates clot formation. FIIa activity is tightly controlled and occurs only when coagulation is necessary for proper hemostasis. Thus, by inserting the sequence LTPRSLLV (SEQ ID NO: 1618), the XTEN domain is removed from FVIII only, concurrently with activation of either the extrinsic or intrinsic clotting pathways, and if clotting is physiologically required. This creates a situation where the CFXTEN merge is handled in a separate additional path during folding activation.

C-terminal XTEN secreted by elastase-2.

The CFXTEN fusion protein, consisting of the XTEN protein fused to the C-terminus of FVIII, was created with the XTEN secretion site cleavage sequence placed between FVIII and the XTEN components, as illustrated in FIG. 12. Illustrative sequences are provided in table. 51. In this case, the secretion site contains an amino acid sequence that is recognized and cleaved by elastase-2, a protease (EC 3.4.21.37, Uniprot P08246). In particular, the sequence LGPVSGVP (SEQ ID NO: 1689) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], truncate after position 4 in the sequence. Elastase is constitutively expressed by neutrophils and is present in the circulation at all times. Its activity is tightly controlled by serpins and is therefore minimally active most of the time. Thus, while long-lived CFXTEN is circulating, its fraction is cleaved, and the creation of a pool of short-lived FVIII will be used in clotting. In a desirable feature of the inventive composition, this creates a circulating depot of prodrug that continuously releases a prophylactic amount of FVIII.

C-terminal XTEN secreted by MMP-12.

The CFXTEN fusion protein, consisting of the XTEN protein fused to the C-terminus of FVIII, was created with the XTEN secretion site cleavage sequence placed between FVIII and the XTEN components, as illustrated in FIG. 12. Illustrative sequences are provided in table. 51. In this case, the secretion site contains an amino acid sequence that is recognized and cleaved by the MMP-12 protease (EC 3.4.24.65, Uniprot P39900). In particular, the sequence GPAGLGGA (SEQ ID NO: 1690) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], truncate after position 4 of the sequence. MMP-12 is constitutively expressed in whole blood. Thus, while long-lived CFXTEN is circulating, its fraction is cleaved, and the creation of a pool of short-lived FVIII will be used in clotting. In a desirable feature of the inventive composition, this creates a circulating depot of prodrug that continuously releases a prophylactic amount of FVIII.

C-terminal XTEN secreted by MMP-13.

The CFXTEN fusion protein, consisting of the XTEN protein fused to the C-terminus of FVIII, was created with the XTEN secretion site cleavage sequence placed between FVIII and the XTEN components, as illustrated in FIG. 12. Illustrative sequences are provided in table. 51. In this case, the secretion site contains an amino acid sequence that is recognized and cleaved by the MMP-13 protease (EC 3.4.24.-, Uniprot P45452). In particular, according to

- 227 041874 GPAGLRGA sequence (SEQ ID NO: 1691) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], truncate after position 4. MMP-13 is constitutively expressed in whole blood. Thus, while long-lived CFXTEN is circulating, its fraction is cleaved, and the creation of a pool of short-lived FVIII will be used in clotting. In a desirable feature of the inventive composition, this creates a circulating depot of prodrug that continuously releases a prophylactic amount of FVIII.

C-terminal XTEN secreted by MMP-17.

The CFXTEN fusion protein, consisting of the XTEN protein fused to the C-terminus of FVIII, was created with the XTEN secretion site cleavage sequence placed between FVIII and the XTEN components, as illustrated in FIG. 12. Illustrative sequences are provided in table. 51. In this case, the secretion site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot Q9ULZ9). In particular, the sequence APLGRLLR (SEQ ID NO: 1692) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], truncate after position 4 in the sequence. MMP-17 is constitutively expressed in whole blood. Thus, while long-lived CFXTEN is circulating, its fraction is cleaved, and the creation of a pool of short-lived FVIII will be used in clotting. In a desirable feature of the inventive composition, this creates a circulating depot of prodrug that continuously releases a prophylactic amount of FVIII.

C-terminal XTEN secreted by MMP-20.

The CFXTEN fusion protein, consisting of the XTEN protein fused to the C-terminus of FVIII, was created with the XTEN secretion site cleavage sequence placed between FVIII and the XTEN components, as illustrated in FIG. 12. Illustrative sequences are provided in table. 51. In this case, the secretion site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot 060882). In particular, the PALPLVAQ sequence (SEQ ID NO: 1693) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320] cut off after position 4 (shown by arrow). MMP-20 is constitutively expressed in whole blood. Thus, while long-lived CFXTEN is circulating, its fraction is cleaved, the creation of a pool of short-lived FVIII will be used in clotting. In a desirable feature of the inventive composition, this creates a circulating depot of prodrug that continuously releases a prophylactic amount of FVIII.

Optimization of XTEN secretion rate.

Variants of the above examples can be created in which the rate of secretion of XTEN inserted at the C-terminus, N-terminus, or within the XTEN is altered. Since the rate of secretion of XTEN by the XTEN secretion protease depends on the sequence of the XTEN secretion site, the rate of XTEN secretion can be controlled by changing the amino acid sequence at the XTEN secretion site. The sequence specificity of many proteases is well known in the art and is described in several databases. Here, protease amino acid specificity is displayed using combinatorial substrate libraries [Harris, J. L., et al. (2000) Proc Natl Acad Sci USA, 97: 7754] or using the following substrate mixture digestion as shown in [Schellenberger, V., et al. (1993) Biochemistry, 32: 4344]. An alternative is to identify optimal sequences for protease digestion using phage display [Matthews, D., et al. (1993) Science, 260: 1113]. Constructs are created with various sequences and analyzed for XTEN secretion using standard assays for the detection of XTEN polypeptides.

Example 39 Human clinical trial designs for the evaluation of CFXTEN which contains FVIII.

Kogenate® FS is a recombinant human coagulation factor VIII designed to promote hemostasis in subjects with hemophilia A. Due to its short half-life, Kogenate is dosed intravenously every other day during prophylaxis and 8 every 12 hours in the treatment of bleeding until hemostasis is achieved. The fusion of one or more XTENs with FVIII is believed to improve the half-life of the protein, thereby reducing the dosing frequency of such CFXTEN formulations containing the fusion protein.

Clinical trials are designed so that the efficacy and benefits of CFXTEN over Kogenate or other commercially available FVIII formulations can be tested in humans. Such studies include three stages. The first, Phase I safety and pharmacokinetic studies in adult patients are performed to determine the maximum tolerated dose, pharmacokinetic properties and pharmacodynamic properties in humans (either normal subjects or patients with hemophilia), as well as to determine potential toxic effects and side effects that are necessary. track in future research. Stage I studies are conducted in which single incremental doses of CFXTEN compositions are administered by route (eg, subcutaneously, intramuscularly, or intravenously) and biochemical, PK, and clinical parameters are measured at defined intervals. This allows you to determine the minimum effective dose, the maximum tolerance

- 228 041874 current dose and establish the threshold and maximum concentrations in the dosage and circulating drug, which represent a therapeutic window for the respective components, as well as bioavailability when administered intramuscularly or subcutaneously. Based on this information, a dose and dosing regimen is obtained that allows less frequent administration of CFXTEN compositions while maintaining a pharmacological response. Thereafter, clinical trials are conducted in diseased patients testing the efficacy of the CFXTEN formulations at a dose that can be administered compared to a positive control such as Kogenate to establish improved properties of the CFXTEN formulations.

Phase II and III clinical trials are conducted in patients suffering from any disease in which factor VIII is expected to provide a clinical benefit. For example, CFXTEN is used in clinical trials in the treatment of indications approved for the use of factor VIII; such indications include cases of bleeding in patients with hemophilia A, patients with factor VIII inhibitors, prevention of bleeding during surgery or invasive procedures in patients with hemophilia A with factor VIII inhibitors, cases of treatment of bleeding in patients with congenital factor VIII deficiency, and prevention of bleeding in surgical interventions or invasive methods in patients with congenital factor VIII deficiency. CFXTEN may also be indicated for use in additional patient populations. A stage II dosing study is conducted in patients with hemophilia, where pharmacodynamic property, clotting, bleeding and other parameters, physiological, PK, safety and clinical, and clinical results suitable for testing are evaluated as a function of dosing of fusion protein compositions, with information on determining the optimal dose of doses that are appropriate for the study in the next stage III, in addition to collecting data on safety associated with side effects. The PK parameters correlate with physiological, clinical and safety data setting the therapeutic window and therapeutic dosing regimen for the CFXTEN formulation, allowing the clinician to establish appropriate dosage ranges for the formulation. In a separate study, hemophilia A patients with factor VIII inhibitors can be evaluated in order to establish doses and dosing regimens of the CFXTEN pharmaceutical composition that result in the achievement and maintenance of hemostasis and the prevention or reduction of bleeding events. Finally, a Phase III efficacy study is conducted where patients are administered the pharmaceutical composition of CFXTEN and a positive control (such as commercially available Kogenate) is administered using a dosing regimen that will be considered appropriate given the pharmacokinetic and pharmacodynamic properties of the respective compositions based on the obtained results of Phase II, with all agents administered over an appropriate long period of time to achieve the expected results of the study. Parameters that are monitored include aPTT assay, one- or two-stage coagulation activity assays, control of bleeding events or the occurrence of spontaneous bleeding events; parameters that are monitored against placebo or positive control groups. Efficacy results are determined using standard statistical methods. Toxicity and adverse event markers will also be monitored in this study to verify that the compound is safe when used in the manner described. In an additional Phase III study, hemophilia A patients with factor VIII inhibitors may be evaluated to establish the effectiveness of CFXTEN pharmaceutical compositions in achieving and maintaining hemostasis and preventing or attenuating bleeding events.

Example 40 Analytical gel permeation chromatography of XTEN fusion proteins with different payloads.

GPC assays are performed on fusion proteins that contain various therapeutic proteins and extended unstructured recombinant proteins. The exemplary assay uses a TSKGel-G4000 SWXL (7.8 mm x 30 cm) column in which 40 μg of purified glucagon fusion protein at 1 mg/ml is separated at a flow rate of 0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profiles are monitored using OD214hm and OD280hm. Column calibration for all assays was performed using a size exclusion calibration standard from BioRad; markers contain thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobulin (17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographic profiles of glucagon-Y288, glucagon-Y144, glucagon-Y72, glucagon-Y36 are illustrated as an overlay in FIG. 21. The data demonstrate that the apparent molecular weight of each compound is proportional to the length of the linked XTEN sequence. Despite this, the data also demonstrate that the apparent molecular weight of each construct is significantly greater than that expected for a globular protein (as shown in comparison to using standard proteins in the same assay). Based on the SEC analyzes for all constructs evaluated that contain the CFXTEN composition, average molecular weights, average molecular weight ratio (expressed as ratio of apparent molecular

- 229 041874 lar mass to the calculated molecular weight) and hydrodynamic radius (RH in nm) are shown in Table. 35. The results indicate that the introduction of various XTENs of 576 or more amino acids confers an apparent molecular weight, in the case of a fusion protein, of about 339 to 760 kDa, and that XTENs of 864 or more amino acids confers an apparent molecular weight above about 800 kDa. The results of a proportional increase in apparent molecular weight to actual molecular weight are consistent with fusion proteins generated with XTEN from several different families of motifs; those. AD, AE, AF, AG, and AM, with an increase of at least four times and ratios up to about 17. Additionally, the inclusion of XTEN fusion partners with 576 or more amino acids in fusion proteins with different payloads (and 288 residues in the case of glucagon fused to Y288) results in a hydrodynamic radius of 7 nmol or greater, far from a glomerular pore size of approximately 3-5 nm. Accordingly, fusion proteins that contain growth and XTEN are expected to reduce renal clearance, resulting in increased terminal half-life and improved therapeutic or biological effect relative to the corresponding non-fused bioburden protein.

Table 35

SEC analysis of various polypeptides

AI construct name XTEN or merged partner Therapeutic protein Current MM (kDA) Apparent MM (kDA) Apparent molecular weight coefficient Rh (nm) AC 14 Y288 glucagon 28.7 370 12.9 7.0 AC28 Y144 glucagon 16.1 117 7.3 5.0 AC34 Y72 glucagon 9.9 58.6 5.9 3.8 ASZZ Y36 Glucagon 6.8 29.4 4.3 2.6 AC89 AF120 glucagon 14.1 76.4 5.4 4.3 AC88 AF108 glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.3 95.2 5.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.1 7.7 AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6 AC398 AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7 Yu,1 AC85 AE864 Exendin-4 83.6 938 AND 2 8.9 AC114 AM875 Exendin-4 82.4 1344 16.3 9.4 AC 143 AM875 hGH 100.6 846 8.4 8.7 AC227 AM875 IL-1ha 95.4 1103 I,6 9.2 AC228 AM1318 IL-1ha 134.8 2286 17.0 10.5

Example 41 Pharmacokinetic Properties of Expanded GFP Fusion Polypeptides in Cynomolgus Monkeys.

The pharmacokinetic properties of GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-XTEN_Y576, and XTEN_AD836-GFP were tested in cynomolgus monkeys to determine the effect of formulations and length of unstructured polypeptides on PK parameters. Blood samples are analyzed at various time points after injection and plasma GFP concentration is measured by ELISA using a polyclonal anti-GFP antibody for capture and biotinylated production of the same polyclonal antibody for detection. The results are illustrated in FIG. 19. They show an unexpected increase in half-life with increasing length of the XTEN sequence. For example, a half-life of 10 hours is determined for GFP-XTEN_L288 (with 288 amino acid residues in XTEN). Doubling the length of the unstructured polypeptide fusion partner to 576 amino acids increases the half-life to 20-22 hours for multiple fusion protein constructs; those. GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTENY576. Further lengthening of the unstructured fusion partner polypeptide to 836 residues results in a half-life of 72-75 hours for XTEN_AD836-GFP. Thus, increasing the length of the polymer by 288 residues, from 288 to 576 residues, increases the in vivo half-life by about 10 hours. 50 hours. These results demonstrate that there is an unexpected threshold for the length of the unstructured polypeptide, resulting in a more than proportional increase in in vivo half-life. Thus, fusion proteins that contain extended, unstructured polypeptides are expected to have improved pharmacokinetic properties compared to shorter length polypeptides.

Example 42 Serum Stability of XTEN.

The fusion protein, which contains XTEN_AE864 fused to the N-terminus of GFP, is incubated in monkey plasma and rat kidney lysate for up to 7 days at 37°C. Samples were taken at time 0, Day 1 and Day 7 and analyzed by SDS PAGE followed by detection using Western blot and anti-GFP antibody detection as illustrated in FIG. 20. The XTEN_AE864 sequence shows little evidence of degradation after 7 days in plasma. Despite this, XTEN_AE864 is rapidly degraded in rat kidney lysate within 3 days. In vivo stability of the fusion protein is tested in plasma samples while GFPAE864 is immunoprecipitated and analyzed by SDS PAGE as described above. Samples taken up to 7 days after injection show very little evidence of degradation. The results demonstrate the resistance of CFXTEN to degradation due to serum proteases; factor for increasing the pharmacokinetic properties of the hybrid protein CFXTEN.

Example 43 Increasing Peptide Solubility and Payload Stability by Binding to XTEN.

In order to evaluate the ability of XTEN to enhance the physicochemical properties of solubility and stability, shorter length glucagon plus XTEN fusion proteins were prepared and evaluated. Test substances were prepared in TRIS-buffered saline at neutral pH and characterization of the Gcg-XTEN solution was performed by reverse phase HPLC and gel permeation chromatography to confirm that the protein was homogeneous and non-aggregated in solution. The data are presented in table. 36. For the purpose of comparison, the solubility limit of unmodified glucagon in the same buffer is measured at 60 µmol (0.2 mg/mL) and the result shows that for all lengths of XTEN added, a significant increase in solubility is achieved. It is important to note that, in most cases, XTEN glucagon fusion proteins are prepared to achieve target concentrations and are not evaluated to determine the maximum solubility limits for this construct. Despite this, in the case of glucagon associated with AF-144 XTEN, the limit of solubility is determined, which ultimately achieves a 60-fold increase in solubility compared to glucagon not associated with XTEN. In addition, CFXTEN glucagon-AF144 was evaluated for stability and found to be stable in liquid formulation for at least 6 months under refrigeration and for approximately one month at 37° C. (data not shown).

The data provide the conclusion that binding of short-length XTEN polypeptides to a biologically active protein, such as glucagon, can markedly enhance the protein's solubility properties of the resulting fusion protein, as well as confer stability at high protein concentrations.

Table 36 _____Solubility of Glucagon-XTEN Constructs

Investigational drug Solubility Glucagon 60 µM Glucagon-Y3 6 >370 µM Glucagon-Y72 >293 µM G lucagon-AF108 >145 µM Glucagon-AP120 >160 µM Glucagon-A144 >497 µM Glucagon-AE144 >467 µM G lucagon-AF144 >3600 µM Glucagon-A288 >163 µM

Example 44 Analysis of sequences for secondary structures using prediction algorithms.

Amino acid sequences can be assessed for secondary structure by certain computer programs or algorithms such as the widely known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and Garnier-Osguthorpe-Robson, or the method GOR (Gamier J, Gibrat JF, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Enzymol Methods 266:540-553). In the case of a given sequence, the algorithms can predict whether or not some secondary structure exists at all, expressed as a whole and/or as a percentage of sequence residues that form, for example, alpha helices or beta sheets, or a percentage of sequence residues that, as is assumed to lead to the formation of a random

- 231 041874 spirals.

Some representative sequences from the XTEN family can be assessed using two algorithm based methods in the case of the Chou-Fasman and GOR methods to assess the degree of secondary structure in these sequences. The Chou-Fasman method is provided by William R. Pearson and the University of Virginia, on the Biosupport website, URL located on the Internet at fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 accessed June 19, 2009. The GOR method is provided by Pole Informatique Lyonnais on the Network Protein Sequence Analysis website, URL located on the Internet at npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl, accessed June 19, 2008.

As a first step in the assays, the unique XTEN sequence is analyzed using two algorithms. Composition AE864 is an XTEN with 864 amino acid residues constructed from multiple copies of four 12-amino acid sequence motifs consisting of the amino acids G, S, T, E, P, and A. Sequence motifs are characterized in that there is limited repeatability within motifs, and within a sequence in general, that the sequence of any two consecutive amino acids does not repeat more than twice in any 12-amino acid motif, and that the three adjacent amino acids of full-length XTEN are not identical. Gradually longer stretches of the AF 864 sequence from the N-terminus are analyzed using the Chou-Fasman and GOR algorithms (the latter requires a minimum length of 17 amino acids). The sequences are analyzed by entering the FASTA format sequences into prediction tools and performing the analysis. The results of the analysis are presented in table. 37.

The results indicate that, using Chou-Fasman calculations, the short XTEN of the AE and AG families, up to at least 288 amino acid residues, has no alpha helices or beta sheets, but the predicted percentage of random helix by the GOR algorithm can vary from 78 up to 99%. As XTEN lengths increase from 504 to over 1300 residues, XTEN analyzed with the Chou-Fasman algorithm has predicted percentages of alpha helices or beta sheets from 0 to about 2%, while the calculated percentages of random helix are increased to from 94 to 99%. XTENs with alpha helices or beta sheets are sequences with one or more occurrences of three contiguous serine residues, resulting in the predicted formation of a beta sheet. Despite this, even these sequences still have approximately 99% random helix formation.

The data provided herein suggests that 1) an XTEN constructed from several G, S, T, E, P, and A sequence motifs that have limited repeatability, as in the case of contiguous amino acids that are predicted to have very low numbers of alpha helices and beta sheets; 2) an increase in the length of XTEN does not significantly increase the likelihood of alpha helix or beta sheet formation; and 3) gradual increase in the length of the XTEN sequence by the addition of non-repeating 12-mers consisting of the amino acids G, S, T, E, P and A leads to an increased percentage of random helix formation. The results further indicate that the XTEN sequences defined herein (which contain, for example, an XTEN constructed from sequence motifs G, S, T, E, P, and A) have limited repeatability (including no more than two identical contiguous amino acids in one motif) are expected to have a very limited secondary structure. Any order or combination of sequence motifs from Table. 3 can be used to generate an XTEN polypeptide, resulting in an XTEN sequence that is essentially devoid of secondary structure, with three contiguous serines not being preferred. The unfavorable property of three adjacent in a row, however, can be mitigated by increasing the length of XTEN. Such sequences are expected to have the characteristics described in the CFXTEN embodiments disclosed herein.

- 232 041874

Table 37

Predictive Calculations of CHOU-FASMAN and GOR Polypeptide Sequences

NAME ate. SEQ ID NO: No. balance OB ChouFasman calculation GOR Calculation AE36: LCW0402_002 1489 36 Total residues: Η: 0 E: 0 percent: Η: 0.0 E: 0.0 94.44% AE36: LCW0402_003 1490 36 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 94.44% AG36: LCW0404_001 1491 36 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 77.78% AG36: LCW0404_003 1492 36 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 83.33% AE42_1 1493 42 Total residues: N: 0 U. 0 percent: N: 0.0 E: 0.0 90.48% AE42_1 1494 42 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 90.48% AG42_1 1495 42 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 88.10% AG42_2 1496 42 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 88.10% AE144 1497 144 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 98.61% AG144_1 1498 144 Total residues: H: 0 E: 0 percent: H: 0.0 E: o.o 91.67% AE288 1499 288 Total residues: H: 0 E: 0 percent: H: 0.0 E: o.o 99.31% AG288_2 1500 288 Total residues: H: 0 E: 0 percent: H: 0.0 E: o.o 92.71

- 233 041874

AF504 1501 504 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 94.44% AD 576 1502 576 Total residues: H: 7 E: 0 percent: H: 1.2 E: 0.0 99.65% AE576 1503 576 Total residues: H: 2 E: 0 percent: H: 0.4 E: 0.0 99.65% AG576 1504 576 Total residues: H: 0 E: 3 percent: H: 0.4 E: 0.5 99.31% AF540 1505 540 Total residues: H: 2 E: 0 percent: H: 0.4 E: 0.0 99.65 AD836 1506 836 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 98.44% AE864 1507 864 Total residues: H: 2 E: 3 percent: H: 0.2 E: 0.4 99.77% AF864 1508 875 Total residues: H: 2 E: 0 percent: H: 0.2 E: 0.0 95.20% AG864 1509 864 Total residues: H: 0 E: 0 percent: H: 0.0 E: 0.0 94.91% AM875 1510 875 Total residues: H: 7 E: 3 percent: H: 0.8 E: 0.3 98.63% AM1318 1511 1318 Total residues: H: 7 E: 0 percent: H: 0.7 E: 0.0 99.17% AM923 1512 924 Total residues: H: 4 E: 3 percent: H: 0.4 E: 0.3 98.70% AE912 1513 913 Total residues: H: 8 E: 3 percent: H: 0.9 E: 0.3 99.45% VS 864 1514 Total residues: N: 0 E: 0 percent: N: 0 E: 0 99.77%

H: alpha helix E: beta sheet.

Example 45 Analysis of polypeptide sequences for repeatability.

In this example, various polypeptides that contain some of the XTEN sequences are evaluated for repeatability in amino acid sequence. The amino acid sequences of a polypeptide can be assessed for repeatability by quantifying the number of times a shorter subsequence occurs within the entire polypeptide. For example, a polypeptide with a length of 200 amino acid residues has a total of 165 overlapping 36 amino acid blocks (or 36-mers) and 198 3-mer subsequences, but the number of unique 3-mer subsequences will depend on the number of repeats within the sequence. In the case of assays, different polypeptide sequences are evaluated for repeatability by determining the number of subsequences obtained using the following equation:

Number of BOi

Number of subsequences = -Gzl------- I

771 where m = (length of the polypeptide in amino acids) - (length of the subsequence in amino acids) + 1; and Num _i = the cumulative number of matches for each unique subsequence within the sequence.

In the analyzes of this example, the number of subsequences in the case of polypeptides from table. 38 is determined using the previous equation in a computer program using the algorithm illustrated in FIG. 27, with the subsequences set to 3 amino acids in length. The resulting number of subsequences is a reflection

- 234 041874 degree of repeatability within a polypeptide.

The results presented in table. 38 indicate that unstructured polypeptides composed of 2 or 3 amino acid types have high subsequence results, while those composed of 12-amino acid motifs of the six amino acids G, S, T, E, P, and A have low intrinsic repeatability have subsequence results of less than 10, and in some cases less than 5. For example, the sequence L288 has two types of amino acids and has short, frequently repeated sequences, resulting in a subsequence count of 50.0. The J288 polypeptide has three types of amino acids, but also has short, repetitive sequences, resulting in a subsequence count of 33.3. Y576 also has three types of amino acids, but not created from internal repeats, reflected in the number of subsequences equal to 15.7 for the first 200 amino acids. W576 consists of four types of amino acids, but has a high degree of internal repeatability, such as GGSG (SEQ ID NO: 1694), resulting in a subsequence count of 23.4. AD576 is composed of four types of 12-amino acid motifs, each consisting of four types of amino acids. Due to the low degree of internal repetition of individual motifs, the total number of subsequences for the first 200 amino acids is 13.6. In contrast, XTEN is composed of four motifs containing six types of amino acids, each with a low degree of internal repeatability, with lower subsequence results; those. AE864 (6.1), AF864 (7.5), and AM875 (4.5), while XTEN, consisting of four motifs that contain five types of amino acids, is intermediate; those. AE864, with a score of 7.2.

Conclusions: The results indicate that combining 12-amino acid sequence submotifs, each consisting of four to six amino acid types that are non-repetitive, in the longer XTEN polypeptide, as a whole, results in a sequence that is essentially non-repetitive, as indicated an overall subsequence score of less than 10 and, in many cases, less than 5. This is despite the fact that each subsequence motif may be used multiple times throughout the sequence. In contrast, polymers made from smaller numbers of amino acid types lead to larger subsequence outcomes, with polypeptides that consist of two amino acid types have higher outcomes than those that consist of three amino acid types.

Table 38

Subsequence Calculations of Polypeptide Sequences

Ate name. SEQID NO: Result J288 1515 33.3 K288 1516 46.9 L288 1517 50.0 Y288 1518 26.8 Q576 1519 18.5 U576 1520 18.1 W576 1521 23.4 Y576 1522 15.7 AE288 1523 6.0 AG288_1 1524 6.9 AD576 1525 13.6 AE576 1526 6.1 AF540 1527 8.8 AF504 1528 7.0 AE864 1529 6.1 AF864 1530 7.5 AG864 1531 7.2 AG868 1532 7.5 AM875 1533 4.5 AE912 1534 4.5 AM923 1535 4.5 AM1296 1536 4.5

Example 46 Calculation of TEPITOPE results.

TEPITOPE results for a 9-mer peptide sequence can be calculated by adding pocket potentials as described by Sturniolo [Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555]. In this example, individual TEPITOPE results are calculated for individual alleles.

- 235 041874

HLA. Tab. 39 illustrates as an example of pocket potentials in the case of HLA0101B, which is often observed in the population of the Caucasus. To calculate the result of the TEPITOPE peptide with the sequence

P1-P2-P3-P4-P5-P6-P7-P8-P9, add the corresponding individual pocket potentials from the table. 39.

The HLA0101B result for the 9-mer peptide with the sequence FDKLPRTSG (SEQ ID NO: 1695) is the sum of 0, -1.3, 0, 0.9, 0, -1.8, 0.09, 0, 0.

In order to evaluate the results of TEPITOPE in the case of long peptides, the method can be repeated for all 9-mer subsequences of the sequences. This method can be repeated for proteins encoded by other HLA alleles. Tab. 40-43 give pocket potentials in the case of protein products of HLA alleles, which are often observed in Caucasian populations.

TEPITOPE results calculated using this method range from about -10 to +10. Despite this, 9-mer peptides that do not have a hydrophobic amino acid (FKLMVWY (SEQ ID NO: 1696)) at position P1 score TEPITOPE results ranging from -1009 to -989. This value is biologically meaningless and reflects the fact that a hydrophobic amino acid serves as an anchor residue for HLA binding and peptides lacking a hydrophobic residue in P1 are considered non-HLA binding. Since most XTEN sequences do not have hydrophobic residues, all combinations of 9-mer subsequences will have TEPITOPE results ranging from -1009 to -989. This method confirms that XTEN polypeptides may have little or no predictive T-cell epitopes.

Table 39

Pocket potential in the case of the HLA0101B allele

Amino acid P1 P2 RZ R4 P5 R6 R7 R8 P9 A -999 0 0 0 - 0 0 - 0 WITH -999 0 0 0 - 0 0 - 0 D -999 -1.3 -1.3 -2.4 - -2.7 -2 - -1.9 E -999 0.1 -1.2 -0.4 - -2.4 -0.6 - -1.9 F 0 0.8 0.8 0.08 - -2.1 0.3 - -0.4 G -999 0.5 0.2 -0.7 - -o,z -1.1 - -0.8 H -999 0.8 0.2 -0.7 - -2.2 0.1 - -1.1 I -1 1.1 1.5 0.5 - -1.9 0.6 - 0.7 TO -999 1.1 0 -2.1 - -2 -0.2 - -1.7 L -1 1 1 0.9 - -2 0.3 - 0.5 M -1 1.1 1.4 0.8 - -1.8 0.09 - 0.08 N -999 0.8 0.5 0.04 - -1.1 0.1 - -1.2 R -999 -0.5 oh, s -1.9 - -0.2 0.07 - -1.1 Q -999 1.2 0 0.1 - -1.8 0.2 - -1.6 R -999 2.2 0.7 -2.1 - -1.8 0.09 - -1 S -999 -o,z 0.2 -0.7 - -0.6 -0.2 - -o,z T -999 0 0 -1 - -1.2 0.09 - -0.2 V -1 2.1 0.5 -0.1 - -1.1 0.7 - oh, s W 0 -0.1 0 -1.8 - -2.4 -0.1 - -1.4 Y 0 0.9 0.8 -1.1 - -2 0.5 - -0.9

- 236 041874

Table 40

Pocket potential, in the case of the HLA0301B allele

Amino acid P1 R2 RZ P4 P5 R6 R7 R8 R9 A -999 0 0 0 - 0 0 - 0 WITH -999 0 0 0 - 0 0 - 0 D -999 -1.3 -1.3 2.3 - -2.4 -0.6 - -0.6 E -999 0.1 -1.2 -1 - -1.4 -0.2 - -0.3 F -1 0.8 0.8 -1 - -1.4 0.5 - 0.9 G -999 0.5 0.2 0.5 - -0.7 0.1 - 0.4 H -999 0.8 0.2 0 - -0.1 -0.8 - -0.5 I 0 1.1 1.5 0.5 - 0.7 0.4 - 0.6 TO -999 1.1 0 -1 - 1.3 -0.9 - -0.2 L 0 1 1 0 - 0.2 0.2 - -0 M 0 1.1 1.4 0 - -0.9 1.1 - 1.1 N -999 0.8 0.5 0.2 - -0.6 -0.1 - -0.6 R -999 -0.5 oh, s -1 - 0.5 0.7 - -o,z Q -999 1.2 0 0 - -o,z -0.1 - -0.2 R -999 2.2 0.7 -1 - 1 -0.9 - 0.5 S -999 -o,z 0.2 0.7 - -0.1 0.07 - 1.1 T -999 0 0 -1 - 0.8 -0.1 - -0.5 V 0 2.1 0.5 0 - 1.2 0.2 - oh, s W -1 -0.1 0 -1 - -1.4 -0.6 - -1 Y -1 0.9 0.8 -1 - -1.4 -0.1 - oh, s

Table 41

Pocket potential in the case of the HLA0401B allele

Amino acid P1 R2 RZ P4 P5 R6 R7 R8 P9 A -999 0 0 0 - 0 0 - 0 WITH -999 0 0 0 - 0 0 - 0 D -999 -1.3 -1.3 1.4 - -1.1 -0.3 - -1.7 E -999 0.1 -1.2 1.5 - -2.4 0.2 - -1.7 F 0 0.8 0.8 -0.9 - -1.1 -1 - -1 G -999 0.5 0.2 -1.6 - -1.5 -1.3 - -1 H -999 0.8 0.2 1.1 - -1.4 0 - 0.08 I -1 1.1 1.5 0.8 - -0.1 0.08 - -o,z TO -999 1.1 0 -1.7 - -2.4 -o,z - -o,z L -1 1 1 0.8 - -1.1 0.7 - -1 M -1 1.1 1.4 0.9 - -1.1 0.8 - -0.4 N -999 0.8 0.5 0.9 - 1.3 0.6 - -1.4 R -999 -0.5 oh, s -1.6 - 0 -0.7 - -1.3 Q -999 1.2 0 0.8 - -1.5 0 - 0.5 R -999 2.2 0.7 -1.9 - -2.4 -1.2 - -1 S -999 -o,z 0.2 0.8 - 1 -0.2 - 0.7 T -999 0 0 0.7 - 1.9 -0.1 - -1.2 V -1 2.1 0.5 -0.9 - 0.9 0.08 - -0.7 W 0 -0.1 0 -1.2 - -1 -1.4 - -1 Y 0 0.9 0.8 -1.6 - -1.5 -1.2 - -1

- 237 041874

Table 42

Pocket potential in the case of the HLA0701B allele Amino acid P1 R2 RZ R4 P5 R6 R7 R8 P9 A -999 0 0 0 - 0 0 - 0 WITH -999 0 0 0 - 0 0 - 0 D -999 -1.3 -1.3 -1.6 - -2.5 -1.3 - -1.2 E -999 0.1 -1.2 -1.4 - -2.5 0.9 - -o,z F 0 0.8 0.8 0.2 - -0.8 2.1 - 2.1 G -999 0.5 0.2 -1.1 - -0.6 0 - -0.6 H -999 0.8 0.2 0.1 - -0.8 0.9 - -0.2 I -1 1.1 1.5 1.1 - -0.5 2.4 - 3.4 TO -999 1.1 0 -1.3 - -1.1 0.5 - -1.1 L -1 1 1 -0.8 - -0.9 2.2 - 3.4 M -1 1.1 1.4 -0.4 - -0.8 1.8 - 2 N -999 0.8 0.5 -1.1 - -0.6 1.4 - -0.5 R -999 -0.5 oh, s -1.2 - -0.5 -0.2 - -0.6 Q -999 1.2 0 -1.5 - -1.1 1.1 - -0.9 R -999 2.2 0.7 -1.1 - -1.1 0.7 - -0.8 S -999 -o,z 0.2 1.5 - 0.6 0.4 - -o,z T -999 0 0 1.4 - -0.1 0.9 - 0.4 V -1 2.1 0.5 0.9 - 0.1 1.6 - 2 W 0 -0.1 0 -1.1 - -0.9 1.4 - 0.8 Y 0 0.9 0.8 -0.9 - -1 1.7 - 1.1

Table 43

Pocket potential in the case of the HLA1501B allele Amino acid P1 R2 RZ P4 P5 R6 R7 R8 P9 A -999 0 0 0 - 0 0 - 0 WITH -999 0 0 0 - 0 0 - 0 D -999 -1.3 -1.3 -0.4 - -0.4 -0.7 - -1.9 E -999 0.1 -1.2 -0.6 - -1 -0.7 - -1.9 F -1 0.8 0.8 2.4 - -0.3 1.4 - -0.4 G -999 0.5 0.2 0 - 0.5 0 - -0.8 H -999 0.8 0.2 1.1 - -0.5 0.6 - -1.1 I 0 1.1 1.5 0.6 - 0.05 1.5 - 0.7 TO -999 1.1 0 -0.7 - -o,z -o,z - -1.7 L 0 1 1 0.5 - 0.2 1.9 - 0.5 M 0 1.1 1.4 1 - 0.1 1.7 - 0.08 N -999 0.8 0.5 -0.2 - 0.7 0.7 - -1.2 R -999 -0.5 oh, s -o,z - -0.2 oh, s - -1.1 Q -999 1.2 0 -0.8 - -0.8 -o,z - -1.6 R -999 2.2 0.7 0.2 - 1 -0.5 - -1 S -999 -o,z 0.2 -o,z - 0.6 oh, s - -o,z T -999 0 0 -o,z - -0 0.2 - -0.2 V 0 2.1 0.5 0.2 - -o,z oh, s - oh, s W -1 -0.1 0 0.4 - -0.4 0.6 - -1.4 Y -1 0.9 0.8 2.5 - 0.4 0.7 - -0.9

Example 46. Evaluation of insertion of XTEN into a loop available for embedding.

Insert AE42-4 ΧΤΕΝ.

The construction and expression of FVIII with AE42 XTEN inserts is described in Examples 17 and 24. Thus, when the X residue is the site of insertion and the Z residue is the next residue in the native FVIII polypeptide sequence, the polypeptide derived from the AE42 XTEN insert may contain the sequence:

X-GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS-Z (SEQ W NO: 1697)

In the case of AE42 XTEN insertion, 16 different sites in the FVIII sequences are selected and they form Series 1. An additional 21 sites selected for AE42 XTEN insertion form Series 2. Collectively, Series 1 and Series 2 sites represent 12 sites in the A1 domain, 7 sites in the A2 domain, 10 sites in the AZ domain, 4 sites in the C1 domain, and 3 sites in the C2 domain. The localizations of the Series 1 and 2 sites in the 3-D structure of FVIII are illustrated in FIG. 32.

The arrangement of these series 1 and series 2 insertion sites results in 37 constructs, designated pSDOOOl-pSD0004, pSD0009-pSD0012, pSD0023-pSD0032, pSD0034-pSD0063 [the above ranges also include all sub-numbers], the sequences of which are shown in Table 1. 21 and insertion sites, which are shown in table. 23.

-238 041874

In vitro assays.

To assess the tolerance of FVIII to the AE42-4 XTEN insert, FVIII activity in culture media samples from FVIII-XTEN cell cultures was analyzed using FVIII chromogenic assay.

Antigen expression levels are analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.

Measurement of FVIII activity by chromogenic assay.

FVIII activity was measured using the COATEST® SP FVIII kit from DiaPharma (lot# N089019) and all incubations were performed at 37° C. in a plate heater with shaking. Cell culture harvests from transient transfection medium of FVIII-XTEN variants AE42-4 from 6-well plates are diluted to the desired range of FVIII activity using 1x FVIII COATEST® buffer. FVIII standards are prepared in 1x FVIII COATEST® buffer which contains a sham transfection medium with the appropriate concentration of culture medium as test sample. The range of the recombinant factor VIII (rFVIII) standard is from 100 mIU/mL to 0.78 mIU/mL. Standards, diluted cell culture samples and pooled normal human plasma assay control are added to Immulon® 2HB 96-well plates in duplicate (25 µl/well).

Freshly prepared mixture of IXa/FX/phospholipid (50 μl), 25 μl of 25 mm CaCl ₂ and 50 μl of FXa substrate are added sequentially to each well, with 5 minutes of incubation between each addition. After incubation with the substrate, 25 µl of 20% acetic acid is added to stop the color reaction and absorbance at 405 nm is measured with a SpectraMAX® plus instrument (Molecular Devices).

Data analysis is carried out using SoftMax Pro software (option 5.2). The Lowest Level of Quantification (LLOQ) is 39 mIU/mL. The results are presented in table. 22.

Measurement of expression using FVIII-HC and FVIII-LC ELISA.

Variant expression is quantified using ELISA. The expression levels of the FVIII antigen of the DNA constructs corresponding to the XTEN inserts in the A1 and A2 domains of FVIII were analyzed by FVIII-LC ELISA. The expression levels of the FVIII antigen of the DNA constructs corresponding to the XTEN inserts in the A3, C1 and C2 domains of FVIII were analyzed by FVIII-HC ELISA. The results are presented in table. 22.

FVIII-XTEN antigens in the cell culture medium after harvest are captured with GMA011 antibodies (Green Mountain Antibodies) in the case of FVIII-LC ELISA) or with GMA016 antibodies (Green Mountain Antibodies) in the case of FVIII-HC ELISA. Immulon® 2HB 96-well plates are coated with 100 μl/well of anti-FVIII antibody (2 μg/ml) by incubating at 4° C. overnight. The plates are then washed four times with Phosphate Buffer with Tween-20 (PBST) and blocked with blocking buffer (PBST with 10% heat-inactivated horse serum) for 1 hour at room temperature.

Cell culture harvests from transient transfection medium of FVIII-XTEN variants from a 6-well plate are diluted to the desired FVIII antigen range using 1x blocking buffer. FVIII standards are prepared in 1x FVIII blocking buffer which contains a sham transfection medium with the appropriate concentration of medium as test samples. The range of the rFVIII standard is from 50 ng/mL to 0.39 ng/mL.

Standards diluted with cell culture samples and a pooled control for normal human plasma analysis are added to Immulon® 2HB 96-well plates in duplicate (100 µl/well) and incubated at 37°C for 2 hours. Washed a further four times with PBST , 100 μl of anti-hFVIII ovine HRP antibody (Affinity Biologicals, F8C-EIC-D) was added to each well and plates, incubated for 1 hour at 37°C. After four more washes with PBST, 100 µl of TMB Super Sensitive Substrate (BioFX) is added to each well, followed by 5-10 min of color development. To stop the color reaction, 50 μl of H2SO4 was added to each well and the absorbance at 450 nmol was measured with a SpectraMAX plus (Molecular Devices).

Data analysis is carried out using SoftMax Pro software (option 5.4). The Lowest Level of Quantification(LLOQ) is 0.0039 µg/mL. The results are presented in table. 22.

Permissive sites into which XTEN sequences are introduced without abolishing the procoagulant activity of the recombinant protein or the ability of the recombinant protein to be expressed in the host cell cluster within a loop within each of the three FVIII domains. Fig. 36 illustrates the location of insertion sites in recombinant FVIII proteins that exhibit FVIII activity in the A1, A2 and A3 domains. Fig. 33 illustrates a structural representation depicting the location of insertion sites in recombinant FVIII proteins that exhibit FVIII activity.

The permissive sites are concentrated in highly conformationally labile solvent-exposed outer loops (the XTEN loop available for embedding). The loops of the A1 domain are located in a region approximately corresponding to amino acid positions 15 to 45 and 201

- 239 041874 to 232, respectively, in mature human FVIII sequences (FIG. 30). The A2 domain loops are located in a region approximately corresponding to amino acid positions 395 to 421 and 577 to 635, respectively, in mature human FVIII sequences (FIG. 30). The A3 domain loops are located in a region approximately corresponding to amino acid positions 1705 to 1732 and 1884 to 1917, respectively, in mature human FVIII sequences (FIG. 30). Fig. 37A and 37B illustrate the location in the 3D FVIII structure of the XTEN loops available for embedding in relation to secondary structure elements.

Example 47 CFXTEN with XTEN inserts that have 144 amino acids.

An analysis of the preliminary data presented above (Example 46) suggested the existence of certain regions within the linear polypeptide sequences and 3-D structures of FVIII A domains where the XTEN sequence insert could be located. To test this hypothesis and further define the boundaries of putative regions where an insert of XTEN sequences can be accommodated without loss of FVIII activity, 23 additional insertion sites not represented in either Series 1 or 2 are selected and designated Series 3.

Series 3 constructs are generated by insertion of residue 144 of the AE XTEN polypeptide, which contains the amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), Thr (T), and Glu (E), or residue 144 of the polypeptide AG XTEN, which contains the amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), and Thr (T). Five different variants of residue 144 of the AE polypeptide are obtained and are designated XTEN-AE144-2A, XTEN-AE144-3B, XTEN-AE144-4A, XTEN-AE144-5A, XTEN-AE144-6B. Amino acid sequences are as indicated in table. 4. Five different variants of residue 144 of the polypeptide are prepared and are designated XTEN-AG144-1, XTEN-AG144-A, XTEN-AG144-B, XTEN-AG144-C, and XTENAG144-F. Amino acid sequences are as indicated in table. 4.

Residue 144 of XTEN encoding the DNA sequence was introduced by chemical synthesis of DNA segments (DNA 2.0, Redwood City, CA) spanning the nearest unique restriction sites within the main vector on either side of the insertion site.

The DNA sequences corresponding to the 144 XTEN peptides are introduced such that the resulting DNA construct will encode the FVIII protein, in which the XTEN sequence of 144 proteins is inserted immediately after the residue specified in the site selection and flanked by the AscI and XhoI sites.

In addition to these sites, sites from Series 1 and 2, while insertion of the AE42 XTEN polypeptide does not abolish the procoagulant activity of FVIII, are altered by excising the AE42 polypeptide encoding the DNA segment with restriction enzymes AscI and XhoI and introducing AE144 XTEN and AG144XTEN encoding sequences at the same sites. The location of these Series 1, Series 2 and Series of insertion sites are summarized in Table 1. III. Fig. 34 is a structural representation of FVIII which shows the location of XTEN 144 insertion sites.

A total of 48 designs are created with 144 XTEN inserts. The constructs are the sequences pSD0001-pSD0004, pSD0009-pSD0012, pSD0023-63 [the above ranges also contain all intermediate numbers], which are given in Table 1. 21 and the insertion sites of which are described in detail in Table. 22.

Expression of 144 FVIII-XTEN variants.

FVIII variants with 144 XTEN inserts are transfected into HEK293F cells (Invitrogen, Carlsbad, CA) using polyethyleneimine (PEI, Polysciences Inc. Warrington, PA) or Lipofectamine transfection agent (Invitrogen, Carlsbad, CA). Transiently transfected cells are grown in 293 Free Style media or a mixture of 293 Free Style media and CD Opti CHO (Invitrogen, Carlsbad, CA). Cell culture medium is harvested 3-5 days post-transfection and assayed for FVIII expression by FVIII activity chromogenic assay and FVIII ELISA as described herein.

Cell culture medium from the transient transfection is concentrated 10-fold in a Centricon® centrifuge column (cut off 100 kDa). The concentrated material is then flash frozen and stored at -80°C for further in vitro analysis and in vivo PK studies.

In vitro assays.

To assess the tolerability of FVIII to inserts, FVIII activity in culture media samples from cell cultures is analyzed using FVIII chromogenic assay. Antigen expression levels are analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.

Measurement of FVIII activity by chromogenic assay and expression measurement by FVIII-HC and FVIII-LC ELISA.

Chromogenic assay methods and ELISA assays are performed as described. The results are shown in table. 23.

Permissive sites into which XTEN sequences are introduced without abolishing the procoagulant activity of the recombinant protein or the ability of the recombinant proteins to be expressed in the host cell are located within the loop in each of the three FVIII domains. The same regions of the XTEN loop available for embedding allow shorter XTEN sequences,

- 240 041874 when introduced show an insertion tolerance for longer XTEN sequences. Fig.

illustrates the location of 144 XTEN insertion sites in recombinant FVIII proteins that exhibit FVIII activity in the A1, A2, and A3 domains. Fig. 35 illustrates a structural representation depicting the location of insertion sites in recombinant FVIII proteins that exhibit FVIII activity.

These observations indicate that two regions within each of the FVIII domains are able to accommodate an insert of XTEN sequences without loss of FVIII cofactor activity. Structural representation of these so-called insertable XTEN loops (FIGS. 40 and 41) demonstrates that they occupy structurally similar positions in each of the domains and are shown on one side of the FVIII molecule. Certain XTEN loops available for insertion correspond to highly conformationally labile loops located between the beta strands in the three-dimensional structures of the A1, A2, and A3 domains, as shown in FIG. 37A and 37B.

In vivo evaluation of the 144 XTEN insert for increased FVIII half-life, as determined by pharmacokinetic properties, is described in Example 32.

Example 48 Facilitate or increase FVIII expression by inserting XTEN sequences within the a3 acidic peptide region of FVIII.

Adherent HEK293 cells are transfected (as described in Example 24) with FVIIIXTEN DNA constructs in which the Factor VIII coding sequence without B domains contains 2 to 4 XTEN inserts of 144 amino acid residues each, the composition and arrangement of the inserts as shown in Table 1. 44 below. 5 days after transfection, cell culture supernatants are analyzed for FVIII activity by chromogenic assay (as described in Example 25). The results are shown in table. 44.

Table 44

Expression levels of FVIII activity by CFXTEN variants that contain XTEN at position 1720 and _______one, two, or three additional XTEN inserts_______

Design name Domain, position, and XTEN insert type Activity (mIU/ml) Al A2 a3 AZ-1 AZ-2 LSD0040.002 26AG144 1720 AG144 175 LSD0041.008 403 AE144 1720AG144 279 LSD0045.002 1656 AG144 1720AG144 2598 PSD080.002 26AG144 1656 AG144 1720AG144 1081 PSD083.001 403 AE144 1656 AG144 1720 AG144 789 PSD082.001 26AG144 1720AG144 1900 AE144 <LLOQ PSD090.003 26AG144 1656 AG144 1720AG144 1900 AE144 316

For comparison purposes, all FVIII-XTEN constructs have an AG144 XTEN insert at amino acid position 1720 (numbering relative to full-length factor VIII) in the A3 domain. Expression levels of FVIII-XTEN variants are determined by chromogenic assay and expressed in units of mIU/mL. Constructs with a unique additional XTEN insert at either position 26 in the A1 domain (LSD0040.002) or position 403 in the A2 domain (LSD0041.008) resulted in expression levels of 175 and 279 mIU/mL, respectively. In contrast, the construct with a unique additional XTEN insert at position 1656 within the a3 acidic peptide gave an expression level of 2598 mIU/mL, showing an increase in expression level for the a3 XTEN insert construct relative to the A1 and A2 insert constructs. Furthermore, compared to the FVIII-XTEN construct with XTEN inserts at positions 26 in the A1 domain and 1720 in the A3 domain (LSD0040.002), the construct with an additional XTEN insert at position 1656 within the a3 acidic peptide region (PSD080.002) gives significantly greater expression (175 and 1081 mIU/ml, respectively). Consistent with these results, the construct with XTEN inserts at positions 403 in the A2 domain and 1720 in the A3 domain (LSD0041.008) resulted in an expression level of 279 mIU/mL, while an additional XTEN insert at position 1656 within the a3 acidic peptide region (PSD083.001) leads to an increase in the expression level to 789 mIU/ml. Finally, the FVIII-XTEN construct with an XTEN insert at position 26 in the A1 domain and two XTEN inserts at positions 1720 and 1900 in the A3 domain (PSD082.001) did not give activity above the lower limit of quantitation. Regardless, the FVIII-XTEN construct with an additional XTEN insert within the a3 acidic peptide region (PSD090.003) results in detectable activity, demonstrating that incorporation of XTEN sequences in the A3 domain can result in recovery of expression (as measured by activity) in FVIII-XTEN designs, which

- 241 041874 are, in other cases, expressed at levels below the lower limit of quantitation. Under experimental conditions, the results provide the conclusion that the insertion of XTEN at position 1656 and, by magnification, within the a3 region, results in improved expression of FVIII-XTEN procoagulant formulations.

Example 49 Effect of XTEN insert on FVIII activity as measured by aPTT.

The one-step activated partial prothrombin (aPTT) coagulation activity assay was used in addition to the chromogenic assay (as described in Example 25) to determine the FVIII activity of various FVIII-XTEN fusion proteins.

Method: FVIII-XTEN aPTT activity was measured using a Sysmex CA-1500 instrument (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). To create a standard curve for analysis, the WHO factor VIII standard is diluted with 2% sham transfection medium to 100 mU/mL and then a 2-fold serial dilution series is performed, with the final standard being 0.78 mU/mL. FVIII-XTEN cell culture samples are first diluted to 1:50 with aPTT assay buffer, additional dilutions are made with 2% sham transfection medium as needed.

After dilution, the aPTT assay is performed using the Sysmex instrument as follows: 50 µl of the diluted standards and samples are mixed with 50 µl of FVIII deficient human plasma and then with 50 µl of the aPTT substance. The mixture is incubated at 37° C. for 4 minutes, and with further incubation, 50 μl CaCl ₂ is added to the mixture and the clotting time is measured immediately.

To determine samples for testing FVIII activity, the clotting time of the standards is plotted using a semi-logarithmic scale (clotting time: linear; standard concentration: Log) to extrapolate the alignment between clotting time and FVIII activity, and FVIII-XTEN activity is then calculated against the standard curve. The sensitivity of the assay is 40 mU/ml factor VIII.

Results. The results are illustrated in FIG. 44-46. If a unique XTEN of 144 or 288 amino acids is introduced into FVIII, all FVIII-XTEN fusion proteins that show activity in the chromogenic assay are also active in the aPTT assay. aPTT activity followed by a chromogenic assay trend, for example, molecules that show low FVIII activity in the chromogenic assay also have low aPTT values. Typically, aPTT results for fusion proteins are lower than those obtained by chromogenic assay, with a ratio of chromogenic assay to aPTT as 1.1 to 2.2, as illustrated in FIG. 44 for unique XTEN inserts. FVIII-XTEN fusion proteins with multiple XTEN inserts generally show additional reductions in aPTT activity compared to chromogenic assay. FVIIIXTEN assays with two XTEN inserts show activity with all constructs, but with chromogenic assay/aPTT ratios as high as 4 in some cases (FIG. 45). FVIII-XTEN assays with three XTEN inserts also show activity in both assays, with chromogenic assay/aPTT ratios reaching up to 5 in some cases (Figure 46), while the ratios for the BDD-FVIII control are more similar (right FIG 46). Additionally, the XTEN insertion site that has appeared results in a visible difference between the activities in aPTT and chromogenic assays. For example, while some molecules with 2 XTEN inserts result in 4-fold lower activity than values in the chromogenic assay, the aPTT activities of other FVIII molecules with 2 XTEN are quite similar to those in the chromogenic assay (Fig. 45). Some molecules with 3 XTEN inserts show up to 5-fold lower activities than in the case of the chromogenic assay, other FVIII molecules with 3 XTEN have aPTT activity less than 2-fold lower than that in the case of the chromogenic assay (Fig. 45). Under experimental conditions, the results provide the conclusion that the FVIII-XTEN fusion protein constructs retain procoagulant activity, but that the chromogenic assay generally provides greater levels of activity than those of the aPTT assay system used in the study.

Example 50 Evaluations of the influence of the XTEN insertion site on the increase in FVIII half-life.

Ways. Six FVIII-XTEN fusion proteins with unique AG-144 XTEN inserts at specific locations were tested in FVIII/VWF DKO mice (as described in Example 32) to evaluate the effect of the XTEN insertion site on FVIII half-life. Six representative XTEN variants (shown in Table 1) with an XTEN insert either within A1, A2, a3, A3-region 1 (A3-R1), A3-region 2 (A3-R2), or at the C-terminus, are selected for this studies, and BDD-FVIII derived from the main vector is used as a control. FVIII/VWF DKO mice are treated with a single intravenous injection of cell culture medium concentrate for transient transfection of six FVIII-XTEN constructs (or positive control medium) at 100-200 IU/kg, and plasma samples are subsequently collected at 5 min, 7 h and 16 hours after dosing. Plasma FVIII activity was tested using the FVIII chromogenic assay and the half-life of FVIII-XTEN was assessed using the WinNonlin program. Data from the study are given in table. 45 and FIG. 47.

Results. The significantly longer half-life observed with all FVIII-XTEN variants is tested compared to the BDD-FVIII control, but the degree of time increase

- 242 041874 half life is variable, with the XTEN variant in insertion site 403 providing a measure of a 10-fold increase in half-life (compared to control), while the 1900 insert variant provides the largest 18-fold increase in half-life. Differences in the XTEN insertion site in increasing FVIII half-life may reflect the roles of different FVIII domains in FVIII clearance in vivo.

Table 45

RK of AG-144 FVIII-XTEN Single Insert Variants in FVIII/VWF DKO Mice

Treatment BDDFVIII PSD-050 PSD0003 PSD0039 PSD0010 PSD063 PSD014 Insertion site No 26 403 1656 1720 1900 CT Recovery 21.3 33.8 34.8 36.0 33.6 39.6 32.4 tl/2 (h) 0.25 3.15 2.4 3.3 4.28 4.54 3.91 Increase tl/2 (times) 13 10 13 17 18 16

Example 51 Evaluations of the Additive Effect of XTEN Inserts on Increasing FVIII Half-Life.

Ways. In order to assess the effects of multiple XTEN inserts on the half-life of the FVIII-XTEN fusion protein, the half-lives of FVIII-XTEN variants with 1-3 XTEN inserts were determined in FVIII-XTEN DKO mice using a cell culture concentrate of five constructs (as described in Example 32). The study tested five variants of FVIII-XTEN: pSD-062, with an AE144 insert at position 1900 (numbering relative to full-length factor VIII); pSD-0005 with AE144 in the B domain of FVIII (amino acid position 745 in the B domain); PSD-0019 with AE288 at the C-terminus of FVIII (CT); LSD0003.006 with AE144 inserted at the B domain and AE288 inserted at the C-terminus, and LSD0055.021 with three XTEN AE144, AE144 and AE288 inserted at position 1900, with a B domain and at the C-terminus. FVIII-XTEN half-life values assume the use of the WinNonlin program.

Results. The results of the study are given in table. 46 and the PK curves are illustrated in FIG. 48. The results of the study clearly demonstrate the additive effect of multiple XTEN inserts in increasing the half-life of FVIII. With the unique XTEN inserts, the half-life of FVIII was extended from 0.25 h to 3.2-4.0 h, a 13 to 16-fold increase. When inserts B and CT XTEN are combined together, the half-life of FVIII is further increased to 10.6 hours, a 42-fold prolongation. Finally, with a third XTEN insert added at position 1900 to the B/CT construct, the half-life reaches 16 hours in FVIII-VWF DKO mice, a 64-fold increase.

Table 46

Additive effect of XTEN inserts on ti/ ₂ FVIII in FVIII/VWF DKO mice

Treatment BDDFVIII PSD -062 PSD0005 PSD0019 LSD0003,006 LSD0055,021 XTEN Insertion site No 1900 IN CT B/CT 1900/B/CT Recovery 21.3 35.3 44.9 33.3 39.0 37.2 tl/2 (h) 0.25 3.8 3.2 4.0 10.6 16.0 Increasing tl/2 (times) 15 13 16 42 64

Example 52 Evaluation of FVIII-XTEN disruption associated with anti-FVIII antibody binding using the Bethesda assay.

The ability of XTEN inserts in the FVIII molecule to interfere with the binding of preexisting anti-FVIII antibodies to the FVIII-XTEN fusion protein is evaluated to determine their utility in the treatment of patients with anti-FVIII inhibitory antibodies.

Ways. To evaluate anti-FVIII antibody binding, two variants of FVIII-XTEN (PSD088, with 144 XTEN administered at locations 26/403/1656/1900; and PSD-090, with 144 XTEN administered at locations 26/1656/1720/1900 ) is tested against Refacto (marketed rFVIII) against plasma samples from three hemophilia A patients with factor VIII inhibitors (designated 04-483, 05-505 and GK18382079) and an anti-FVIII sheep polyclonal antibody from Affinity Biologicals Inc (F8C -EIA-C). The Bethesda titer of four anti-FVIII antibodies against two FVIII-XTEN variants (pSD-088 and pSD-090) and the Refacto control were determined using modified Bethesda assay methods described as follows. Heat-inactivated anti-FVIII antibody samples at various dilutions are incubated with 1 IU/ml of each FVIII variant (diluted in IX in FVIII chromogenic assay buffer) at a 1:1 ratio. The FVIII/antibody mixtures are then incubated for 2 hours in a 37°C incubator. After incubation, samples are diluted 10-fold with 1x FVIII chromogenic assay buffer and 25 µl of the diluted mixture is then used for FVIII chromogenic assay, the percentage of FVIII activity remaining is calculated against the activity after incubation of a known non-neutralized sample. Bethesda units are calculated using the following formula: FU = dilution factor X

-243 041874 (LN(% activity remaining) + 6.6438).

Results. The results are shown in table. 47. Reduced Bethesda Unit (BU) titers are observed for all four antibodies when tested against two FVIII-XTEN variants, compared to Refacto. A 5-8 fold increase against PSD-088 and a 3-5 fold increase against PSD-090 are observed, respectively. Inhibition curves were plotted against FVIII variants for each antibody (FIG. 49) and compared to Refacto, and showed a clear leftward shift in the inhibition curve for two FVIII-XTEN molecules, with FVIII-XTEN variant pSD-088 resulting in an additional shift to the left, compared to PSD-090. These results clearly demonstrate that: 1) both FVIII-XTEN variant fusion proteins are more resistant compared to pre-existing anti-FVIII inhibitory antibodies than Refacto; and 2) PSD-088 is more resistant to anti-FVIII antibodies than PSD-090 may provide information useful in determining differences in XTEN insertion sites while preventing anti-FVIII antibodies from binding. Under experimental conditions, the results provide some support for the potential use of the FVIII-XTEN formulation in the treatment of haemophilia A patients with factor VIII inhibitors.

Table 47

Bethesda anti-FVIII antibody titer against FVIII-XTEN variants

^^^ngi-FVIII antibody FVin'-\^^ 04-483 05-505 GK1838- 2079 F8C-EIA- C PSD-088 (16/403/1656/1900) 2.5 8 47 57 PSD-090 (26/1656/1720/1900) 3.4 12 55 96 Refacto 12 66 268 337

Example 53 Half-life estimates of FVIII XTEN fusion molecules that contain four XTEN inserts in mice with hemophilia A.

Ways. Eight FVIII-XTEN fusion proteins with four XTEN inserts, each at specific locations, were tested in FVIII/VWF DKO mice to evaluate the effect of XTEN inserts on increasing FVIII half-life: LSD0071.001, contains XTEN inserts 403-AG144, 1900- AE144, 745(B)-AE144, 2332(CT)-AE288 (designated as FVIII amino acid number and inserted XTEN); LSD0071.002 which contains inserts XTEN 403-AE144, 1900-AE144, 745(B)-AE144, 2332(CT)-AE288; LSD0072.001 which contains inserts XTEN 403-AG144, 1900-AG144, 745(B)AE144, 2332(CT)-AE288; LSD0072.002 which contains inserts XTEN 403-AE144, 1900-AG144, 745(B)-AE144, 2332(CT)-AE288; pBC0247.004, which contains XTEN inserts 18-AG144, 403-AE144, 1656-AG144, 2332(CT)-AE288; pBC0251.002, which contains XTEN inserts 18-AG144, 1656-AG144, 1900-AE144, 2332(CT)-AE288; pSD088 which contains XTEN inserts 26-AG144, 403-AE144, 1656-AG144, 1900-AE144 and pSD090 which contains inserts XTEN 26-AG144, 1656-Ag144, 1720-AG144, 1900-AE144. FVIII/VWF DKO mice are treated as described in Example 32 with a single intravenous injection of FVIII-XTEN transfection cell medium concentrate of eight constructs at 100200 IU/kg and plasma samples are subsequently collected at 5 min, 8 h, 24 h, 48 h, 72 h and 96 h after dosing. Plasma FVIII activity was tested using the FVIII chromogenic assay and the half-life of FVIII-XTEN was assessed using the WinNonlin program.

Results. all of the eight FVIII XTEN fusion molecules that contain four XTEN inserts exhibit longer half-life than unmodified FVIII (results in Table 48). Three molecules with XTEN inserts at positions 403, 1900, the B domain, and at the C-terminus achieve a half-life of up to 16.3 hours, representing a 65-fold improvement over unmodified BDD FVIII. Despite this, tested molecules with XTEN inserts at 26/403/1656/1900 (pSD088), or at 26/1656/1720/1900 (pSD090) show a half-life of 9.1 h and 9.5 h, respectively, which, compared to BDD FVIII, represents an increase of 36-fold and 38-fold, respectively. pBC247.004 (XTEN inserts in 18/403/1656/CT) and pBC251.002 (XTEN inserts in 18/1900/1656/CT) achieve half-lives of 14.1 h and 13 h, respectively. The results demonstrate that multiple XTEN inserts (in this case, four XTEN inserts for each FVIII molecule) can significantly improve the half-life of FVIII. This further illustrates that the effect of XTEN on FVIII half-life is insertion site dependent, even in the case of multiple XTEN inserts.

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Table 48

PK FVIII-XTEN variants with four XTEN inserts in FVIII/VWF DKO mice Treatment XTEN inserts 11/2 (h) Increasing tl/2 (once) BDD-FVIII No 0.25 n/a LSD0071.001 403AG/1900AE/B/CT 16.2 64.8 LSD0071.002 403AE/1900AE/B/ST 16.3 65.2 LSD0072.001 403AG/1900AG/B/CT 11.8 47.2 LSD0072.002 403AE/1900AG/B/CT 16.1 64.4 pBC247.004 18/403/1656/ST 14.1 56.4 рВС251.002 18/1900/1656/ST 13.0 52 PSD088 26/403/1656/1900 9.1 36.4 PSD090 26/1656/1720/1900 9.5 38

Table 49

Illustrative Biological Activity, Illustrative Assays and Preferred Indications

Bioactive Protein Biological activity Illustrative Activity Assays Preferred indication: Factor VIII (factor VIII; octocog alfa; moroctocog alfa; recombinant antihemophilic factor; nordiate; re facto; Kogenate; Kogenate SF; helixate; recombinate) Coagulation factor VIII is a factor essential for hemostasis. This gene, which codes for coagulation factor VIII, is involved in the intrinsic pathway of blood clotting; factor VIII is a cofactor in the case of factor 1Xa which, in the presence of Ca+2 and phospholipids, converts factor X to the activated form Xa. This gene produces two alternatively spliced templates. Option 1 of the template encodes a large glycoprotein, isoform a, which Chromogenix analysis (Rosen S, Scand J Haematol (1984) 33 (Suppl 40): 139-45); Chromogenix Coatatic® factor VIII assay; one-step coagulation activity assay (Lethagen, S., et al., Scandinavian J Haematology (1986) 37:448-453. One-step coagulation activity assay and two-step coagulation activity assay (Barrowcliffe TW, Hemophilia A; bleeding; factor VIII deficiency; cases of bleeding in patients with a factor VIII inhibitor; surgery associated with cases of hemorrhage

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circulates in plasma and is associated with von Willebrand factor in a non-covalent complex. This protein undergoes many cleavage events. Template variant 2 encodes a putative small protein, isoform b, which consists primarily of the factor VI Hc phospholipid binding domain. This binding domain is necessary in case of coagulant activity. Defects in this gene result in hemophilia A, a common recessive X-linked coagulation disorder. Semin Thromb Hemost. (2002) 28(3):247-256); Development of a simple factor VIII chromogenic assay for clinical use. (Wagenvoord RJ, Hendrix HH, Hemker HC. Haemostasis 1989; 19(4): 196204) Bethesda analysis (Verbruggen B, et al.. Improvements in factor VIII inhibitory detection: From Bethesda to Nijmegen. Semin Thromb Hemost. 2009 Nov; 35(8):752759)

Table 50 Exemplary CFXTEN which contains FVIII and XTEN internal/external sequences (SEQ ID NOS 1537-1554, respectively, in order of appearance)

Name CFXTEN Amino acid sequence FVIII BDD2 (A1-K127 -AE144- V128- N745AE288P1640- Y2332) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKGGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPAT SGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSTEPSEGSAPGVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNS LMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTT PEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQ HDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDN SPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKN QASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVED

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FVIII BDD2 (AlA375AE576K376N745AE144P1640Y2332)

GPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVF DSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSY EDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREIT RTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAV ERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELN EHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKN FVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGP LLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSI HFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHL HAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYS GSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY SLDG KKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHL QGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLY_____________________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAGGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTE EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQ RIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQA SRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGP TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRN VILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDI SAYLLSKNNAIEPRSFSQNGGSEPATSGSETPGTSESATPESGPGSEPATSGS

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ETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSE PATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEG SAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQS PRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQE FTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSL ISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYF SDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSW YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQK FSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQIT AS S YFT NMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTT QGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2 (AlY1792- AF144- E1793Y2332AE864) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQS DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWD YGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLL GPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYGGTSTPESGSASPGTSPSGE SSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPG TSPSGESST APGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGES STAPGTSPSGESSTAPGEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAP TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIM DTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALY NLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLA PMIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMES KAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQV DFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKV FQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYG GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS

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FVIII BDD2 (AlY2043AG144G2044Q2222AG864V2223Y2332)

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQS DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWD YGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLL GPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHT NTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPT FKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMST LFLVYSNKCQTPLGMASGHIRDFQITASGQYGPGSSPSASTGTGPGSSPSAS TGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GTPGSGT AS S SPGS STP SGATGSPGTPGSGT A S S SPGASPGT S STGSPGASPG TSSTGSPGTPGSGTASSSGGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFF GNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGM ESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQGGASPGTSS TGSPGSSPS ASTGTGPGS SPS ASTGTGPGTPGSGT AS S SPGS STPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT AS S SPGASPGTS STGSPGASPGTS STGSPGTPGSGT AS S SPGSSTP SGATGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS TGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSP GASPGTS STGSPGTPGSGTAS S SPGASPGTS STGSPGASPGTS STGSPGASPG TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGS

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GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS S SPGS STP SGATGSPGS SPS ASTGTGPGS SP S ASTGTGPGASPGT S STGSPGAS PGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGV NNPKEWLQ VDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQDGHQWT LFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEV LGCEAQDLY FVIII BDD2 (AlG1799AE144A1800- F2093AE42S2094- V2223AE42N2224AE42N2225G2278- AE42K2279Y2332) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMA SDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIG CHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDD DLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPL VLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLL ICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSG YTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTA LLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIA AVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGGG SEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGSEPATSGSETPGSE PATSGSETPGSEPATSGSETPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGAEPRKNFVKPNETKT YFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFK ENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTL FLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWS TKEPFSWIKVDLLAPMIIHGIKTQGARQKFGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGSPAGSPGSSLYISQFIIMYSLDGKKWQTYRGNSTGTL MVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCS MPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVGP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGGNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLIS S SQDGHQWTLFFQNGGTEP S EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSKVKVFQGNQDSFTP VVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2 (A1-R28AG144F29- ATRRYYLGAVELSWDYMQSDLGELPVDARGPGSSPSASTGTGPGSSPSAST GTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG TPGSGTAS S SPGS STPSGATGSPGTPGSGT AS S SPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSGFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPR PPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEY

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G244AG288L245R2090AG576Q2091Y2332AG864)

DDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVD LVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKN SLMQDRDAASARAWPKMHTVNGYVNRSLPGGPGASPGTSSTGSPGASPGT SSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGS SPS ASTGTGPGS SPS A STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTA QTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDY DDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYA PLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHES GILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPL LICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQ LEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFS GYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTA LLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREI TRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSF QKKTRHYFIAA VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGEL NEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCN IQMEDPTFKENYRFHAGINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENI HSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGE HLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLH YSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGQKFS SLYISQFIIMYSLDGKKWQTYRGNSTGT LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCS MPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFF QNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS

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FVIII (AlT1651AG576R1652K1808AG144P1809F2093AG288S2094Y2332)

TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAP_______________________________________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKG VKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPE NDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYE TFSDDPSPGAIDSNNSLSEMTIIFRPQLHHSGDMVFTPESGLQLRLNEKLGTT AATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLD TTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESG RLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLI ENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSK NMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQ GPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSR NLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFM KNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEE ENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETE LEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSH SIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESS HFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKK VENTVLPK PDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIK WNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQE KSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLC SQNPPVLKRHQREITGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTAS S SPGS STPSGATGSPGSSTPSGATGSPGS STPSGATGSPGS SP SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST GSPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGTPGSGT ASS SPGS S TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT

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GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSSGRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYR GELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAE PRKNFVKGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGPNETKTY FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLN PAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNI<CQTPLGMASGHIRDFQITASGQYGQWAPI<LARLHYSGSINAWSTI<E PFSWIKVDLLAPMIIHGIKTQGARQKFGPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGT GPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG SSTPSGATGSGSSL YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGV KSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDP PLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2 (A1-A28AG42F29-E124AG42D125E124AG42D125РЗЗЗAG42Q334Y2332) ATRRYYLGAVELSWDYMQSDLGELPVDAGGAPSPSASTGTGPGTPGSGTA SSSPGSSTPSGATGSPGPSGPGRFPPRVPKSFPFNTSVVYKKTLFVEFTVHLF NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEGGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGASPGDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGGSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGAGKSWHSETKNSLMQDRDAASA RAWPKMHTVNGYVNSSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHT FLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPGSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGGQL RMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVH YIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTD ETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFV NMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLT ENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYIL SIGAQTDFLS VFF SGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSF SQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSP RSFQKKTR HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF TDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ

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FVIII (AlD345AE144Y346D403AE144R405R1797AE288Q1798Y2322)

YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWTPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY_______________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHIS SHQHDGMEAYVKVD SCPEEPQLRMKNNEEAEDGGSEP AT SGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP GSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSTEPSEGSAPGYDDDLTDSEMDVVRFDDDNSP SFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDGGTSTPESGSASP GTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSST AESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTA P GTSPSGESSTAPGTSPSGESSTAPGRSYKSQYLNNGPQRIGRKYKKVRFMA YTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDV RPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSS FVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWY LTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYW YILSIGAQTDFLS VFF SGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWI LGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SF SQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVS S SDLLM LLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHS GDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAA GTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLL ESGLMNSQES SWGKNVS STESGRLFKGKRAHGPALLTKDNALFKVSISLLK TNKTSNNSATNRKTHroGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRML MDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKML FLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVV VGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKE TLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQ NFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPST LTQID YNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQ DNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVG SLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSN GSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLD PLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAIN EGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYD DTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPH VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEV EDNIMVTFRNQASRPYSFYSSLISYEEDQRGGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE

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FVIII(AlN745)AE864(P1640Y2332)

SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQG AEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERN CRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMG SNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGSINAWSTKEPF SWIKVDLLAPMIIHGIKTQGARQKF S SLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTH YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR YLRIHPQSWVHQIALRMEVLGCEAQDLY______________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQ HDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE GTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTL QSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVK PNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLV CHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQME DPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFS

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GHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAG MSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSIN AWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKW QTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRS NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSS QDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY FVIII BDD9 (AlN745)AE288(P1640Y2332) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD9 (AlS743)AE288(Q1638Y2332) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD

-256041874

FVIII BDD9 (AlN745)AG288_2(P1640Y2332)AG288_2

SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG QNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQ RIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY_______________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIM SDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGD YYED SYEDIS AYLLSKNNAIEPRSF SQNGPGASPGTS STGSPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSGPPVLKRHQREITRTTLQ SDQEEID YDDTISVEMKKEDFDIYDEDENQ SP RSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF TDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLI

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FVIII BDD9 (AlS743)AG288_2 (Q1638Y2332)AG288_2

SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGS STPSGATGSPGTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA SSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG___________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMAS HPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGD YYED SYEDIS AYLLSKNNAIEPRSF SGPGASPGTS STGSPGASPGT SSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS GQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMK KEDFDIYDEDENQSP RSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF TDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS

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FVIII BDD 10 (AlN745)AE288(P1640Y2332)AE288

FVIII BDD 10 (AlLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGS STPSGATGSPGTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA SSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG___________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYL TENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLN SC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAP__________________________________________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS

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S743)AE288(Q1638Y2332)AE288 DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG QNPPVLKRHQ AEITRTTLQ SDQEEID YDDTISVEMKKEDFDIYDEDENQ SPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFK KVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAP FVIII BDD10 (AlN745)AG288_2(P1640Y2332)AG288_2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK

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MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSP SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISDAQITASS YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGS STPSGATGSPGTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA S S SPGS STPSGATGSGAETAEQKLISEEDLSP ATG FVIII BDD 10 (AlS743)AG288_2(Q1638Y2332)AG288_2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAV FDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAP DDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLL YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPG EIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSC DKNTGD YYED SYEDIS AYLLSKNNAIEPRSF SGPGASPGT S STGSPGASPGT SSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGS SPS ASTGTGPGS SPS A STGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPS GATGSPGSSPSA STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS QNPP VLKRHQ AEITRTTLQ SDQEEID YDDTISVEMKKEDFDIYDEDENQ SPR YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGS STPSGATGSPGTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGSSP SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA S S SPGS STPSGATGSGAETAEQKLISEEDLSP ATG

The sequence name reflects the N- to C-terminal conformation of the FVIII segments (amino acid spanning the number relative to the mature sequence) and XTEN components.

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Exemplary CFXTENs that contain FVIII, cleavage sequences, and XTEN sequences (SEQ ID NOS 1555-1590, respectively, in order of appearance)

Table 51

Name CFXTEN

SP-AE288CS-L(FVIII1745)AE288(FVIII168 6-2332)-LCS-AE288

Amino acid sequence

MQIELSTCFFLCLLRFCFSGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQ GPEGPSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTS VVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMA SHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQ VLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEK TQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNG YVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS LEISPITFLT AQTLLMDLGQFLLFCHIS SHQHDGMEAYVKVD SCPEEPQLR MKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVH YIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYT DETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYS SFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRS

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WYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMEN PGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSK NNAIEPRSFSQNGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRH YFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQP LYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQ RQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE NMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGA RQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNI FNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQI TASSYFTNMFATWSPSKARLHLQGRSN AWRPQVNNPKEWLQVDFQKT MKVTGVTTQGVKSLLTSMYVKEFLIS S SQDGHQWTLFFQNGKVKVFQG NQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGP EGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP SP-AE576CS-L(FVIIIl745)AE576(FVIII168 6-2332)-LCS-AE288 MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPQSPRSFQGPSGPATRRYYLGAVELSWDYMQ SDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPW MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETK NSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGM GTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHI SSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVR FDDDNSPSFIQIRSV AKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYK SQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEV GDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIF

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KYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEF QASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTF KHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALL KVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQK KTRHYFIAAVERLWD YGMS S SPHVLRNRAQ SGS VPQFKKVVFQEFTDGS FTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYE EDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD VDLEKDVHS GLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEM LPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQIT ASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKH NIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDA QITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQK TMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYG PEGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP SP(FVIIIl745)AE576(FVIII168 6-2332)-LCS-AE576 MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARF PPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDK VFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGA LLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAAS ARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLE GHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEA YVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRI GRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQA SRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVED GPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSD KRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSING YVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL TLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMT ALLKVS SCDKNTGD

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SP-AE576CS-L(FVIIIl745)AE576(FVIII168 6-2332)

YYEDSYEDISAYLLSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVER LWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGL IGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP KLARLHYSGSINAW STKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR LHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQG SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPQSPRSFQGPEGPSATRRYYLGAVELSWDYM QSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPP WMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYD

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SP-AE576CS-L(FVIIIl743)AE288(FVIII168 6-2332)-LCS-AE576

DQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVD LVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETK NSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGM GTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHI SSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVR FDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYK SQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEV GDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIF KYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEF QASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTF KHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALL KVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQK KTRHYFIAAVERLWD YGMS S SPHVLRNRAQ SGS VPQFKKVVFQEFTDGS FTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYE EDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY FTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEM LPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQIT ASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKH NIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDA QITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQK TMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG S EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPIEPRSPSGSPGATRRYYLGAVELSWDYMQS DLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWM

-266041874

GLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNS LMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGT TPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQ YLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGD TLLUFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKY KWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKV SSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPQSPRSFQKKTRHYFIAAVER LWDYGMSSSPHVLRNRA QSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIM VTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGR QVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTK EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYR GNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRS NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISS SQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSW VHQIALRMEVLGCEAQDLYGSPGIEPRSPSGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEP TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP

SP-AG288CS-L(FVIIIl743)AG576(FVIII 168

MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTS STGSPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGATGSIEPRSP

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6-2332)-LCS-AG288

SP-AG576CS-L(FVIIIl745)SGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMAS HPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQV LKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKT QTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNG YVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS LEISPITFLT AQTLLMDLGQFLLFCHIS SHQHDGMEAYVKVD SCPEEPQLR MKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVH YIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYT DETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYS SFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRS WYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMEN PGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSK NNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA STGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGT SSTGSPGA SPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGT SSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGS STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGT SSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTS STGSQSPRSFQKKTRHYFIAAVERLWD YGMS S SPHVLRNR AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNI MVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHG RQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNI<CQTPLGMASGHIRDFQITASGQYGQWAPI<LARLHYSGSINAWSTI< EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYR GNSTGTLMVFFGNVD S SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRS NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISS SQDGHQWTLFFQNGKVKVFQGNQD SFTPVVNSLDPPLLTRYLRIHPQSW VHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS PGTPGSGTASS SPGS STPSGATGSPGS STPSGATGSPGS SPS ASTGTGPGS S PSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS PGS SPS ASTGTGPGASPGTS STGSPGS SPS ASTGTGPGTPGSGT AS S SPGS S TPSGATGS____________________________________________ MQIELSTCFFLCLLRFCFSPGTPGSGTAS S SPGS STPSGATGSPGS SPS AST GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GASPGT S STGSPGASPGT S STGSPGTPGSGT AS S SPGASPGT S STGSPGASP GTS STGSPGASPGT S STGSPGS SP S ASTGTGPGTPGSGT AS S SPGASPGT S S

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AG288(FVIII168 6-2332)-LCS-AE576

TGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSP GASPGTS STGSPGTPGSGT AS S SPGS STPSGATGSPGS STPSGATGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSQSPRSFQGSPGATRRYYLGAVELSWDY MQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRP PWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEY DDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSET KNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIG MGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFC HISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGE VGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI FKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKES VDQRGNQIMSDKRNVILFS VFDENRSWYLTENIQRFLPNPAGVQLEDPEF QASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTF KHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALL KVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPGASPGTSSTGS PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS PGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGT ASSSPGSSTPSGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVED NIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWK VQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPA HGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMST LFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA WSTKEPF S W IK VDLLAPMIIHGIKTQG ARQKF S SLYISQFIIMYSLDGKKW QTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRM ELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWS PSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT SESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT

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SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP

SP(FVIIIl743)AG576(FVIII168 6-2332)-LCS-AG576

MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARF PPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDK VFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGA LLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAAS ARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLE GHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEA YVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRI GRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQA SRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVED GPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSD KRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSING YVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL TLFPF SGET VFMSMENPGLWILGCHNSDFRNRGMT ALLK VS SCDKNTGD YYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPG SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSPGSSTPSGATGSPGSSTP SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPS ASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEH LGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKN FVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLI GPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN ENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFETVEMLPSI<AGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP KLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR LHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGT PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASS SPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGS SPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT

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SSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGS STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSG TASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG s

SP-AG288CS-L(FVIIIl743)AG288(FVIII168 6-2332)-LCS-AE288

MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTS STGSPGS SPS ASTGTGPGTPGSGTAS S SPGS STPSGATGSQSPRS FQGPSGPATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT SVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVW QVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKE KTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQA SLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQL RMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWV HYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDV RPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYY SSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHE VAYW YILSIGAQTDFLS VFF SGYTFKHKMVYEDTLTLFPF SGETVFMSME NPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSF SPGASPGT S STGSPGASPGT S STGSPGTPGSGT AS S SPGS S T PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA TGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTS STGSPGASPGTS STGSPGS STPSGATGSPGS SPS ASTGTGPGASPGTS S TGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQKKTRH YFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQP LYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQ RQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE NMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGA RQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNI FNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQI TASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKT MKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQW TLFFQNGKVKVFQG NQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGP SGPQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA

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PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

SP-AE576CS-L(FVIIIl743)AG576(FVIII168 6-2332)

MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPQSPRSFQGSPGATRRYYLGAVELSWDYMQS DLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWM GLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNS LMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGT TPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVA KKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQ YLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGD TLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKY KWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKV SSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG ATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGS STPSGATGSPGASPGTS STGSPGTPGSGTAS S SPGS STPSGATGS PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS PGTS STGSPGTPGSGTAS S SPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS PGS STPSGATGSPGTPGSGTAS S SPGS STPSGATGSPGS STPSGATGSPGS S PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTR HYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQ PLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQ RQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE NMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIR WYLLSMGSNENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFETVEMLP SKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGA RQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNI FNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQI TASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKT

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FVIII BDD2 S367-FXIaAE42F368Y2332FXIaAE864

FVIII

MKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQG NQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNSSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSKLTRAETGEPSEGSAPGSPAGSPT STEEGTSESATPESGPGSEPATSGSGFIQIRSVAKKHPKTWVHYIAAEEED WDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SF SQNPPVLKRHQREITRTTLQ SDQEEID YDDTIS VEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRA QSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQT YRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWTPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYKLTRAETGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEG SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP______ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT

-273 041874

BDD2 N745-FIXaAG288FlXaP1640Y2332FlXaAG864

LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPLGRIVGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS GATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGT SSTGSPGSSPSASTGTGPGTPGSGTA SSSPGSSTPSGATGSGPLGRIVGGPP VLI<RHQREITRTTLQSDQEEIDYDDTISVEMI<I<EDFDIYDEDENQSPRSF QKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAY FSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETK SWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMA QDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFE TVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIR DFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHG IKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQD LYPLGRIVGGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS GTAS S SPGS STPSGATGSPGS SPS ASTGTGPGASPGTS STGSPGTPGSGT AS SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GAT GSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS

- 274 041874

GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

FVIII

BDD2

V128FVIIaAG42FVIIaG2044FVIIaAG144Y2332FVIIaAG576

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVLQVRIVGGGAPSPSASTGT GPGTPGSGTASSSPGSSTPSGATGSPGPSGPGLQVRIVGG

FPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGH TFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYV KVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRS VAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPT KSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRN VILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYED SYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDD TIS VEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWD YGMS S SP HVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIR AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKT YFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTN TLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDP TFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSG HVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHA GMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGLQVRIVGGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGLQVRIVGGQWAPKLA RLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP THYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFAT WSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLD PPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYLQVRIVGGPGTPGSGT ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGS PGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS PGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGT AS S SPGSSTPSGATGSPGTPGSGT AS S SPGS STPSGATGSPGS STPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS

AE864FVIIIThrombinAE144

EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST

- 275 041874

EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGATRRYYLGAVELSWDYMQS DLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWM GLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNS LMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGT TPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYV KVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQ YLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGD TLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKY KWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKV SSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNA TTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQ EAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRL NEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMP VHYD SQLDTTLFGKKS SPLTESGGPLSLSEENNDSKLLESGLMNSQES SW GKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSAT NRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATAL RLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESAR WIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGE FTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQ ENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTS QQ NFVTQRSKRALKQFRLPLEETELEKRUVDDTSTQWSKNMKHLTPSTLTQ IDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRV LFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQ REVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDL FPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSA KTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNAC ESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTT LQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVE RLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELN EHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPR I<NFVI<PNETI<TYFWI<VQHHMAPTI<DEFDCI<AWAYFSDVDLEI<DVHSG

-276041874

LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRV ECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWA PKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS SYFTNM FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGLTPRSLLVGGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGSEPATSGSETPGSEPATSGSETPGSEP ATSGSETPGTSTEPSEGSAP GTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAP FVIII BDD3- FXIIa- AE144 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMT ALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQ VTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGTMTRIVGGGGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGS EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAP FVIII BDD3ElastaseAE144 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI

-277041874

FVIII

BDD3FXIaAE144

LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKE PFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQT YRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSET PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGN QIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY

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PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEP ATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAP FVIII BDD3 ThrombinAE144 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV TVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGS EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAP AE144FVIII BDD2- MMP-17- AE864 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGATRRYYLGAV ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLF NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWK ASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLT

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AE144FVIII

BDD2FXIIaAE864

YSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEG KSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV LAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA GVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFL SVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPV LKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ KKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYF SDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQ DQRIRWYLLSMGSNENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFET VEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGM ASGHIRD FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGI KTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQD LYGAPLGLRLRGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE S ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP_________ GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGATRRYYLGAV ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLF NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWK ASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLT YSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEG

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KSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV LAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA GVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFL SVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPV LKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ KKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYF SDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQ DQRIRWYLLSMGSNENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFET VEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLA PMIIHGI KTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQD LYGTMTRIVGGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

AG144FVIII

BDD2FXIaAG576

SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA TGSPGS SPS ASTGTGPGS SPS ASTGTGPGASPGTS STGSPGTPGSGT AS S SP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGATRRYYLGAV ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLF NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWK ASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLT YSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEG KSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS

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VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV LAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA GVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFL SVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPV LKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ KKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYF SDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQ DQRIRWYLLSMGSNENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFET VEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGI KTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM VFFGNVDSS GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQD LYGKLTRAETGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS PSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTS STGSPGS SP S ASTGTGPGTPGSGT AS S SPGASPGT S STGSPGAS PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGS PGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS TGTGPGASPGTS STGS AE144FXIa-FVIII BDD2- AE864 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGKLTRAETGAT RRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF VEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPM ASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFIL LFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLT AQTLLMDLGQFLLFCHIS SHQHDGMEAYVKVD SCPEEPQLRMKNNEEA EDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEED WDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER

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DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMT ALLKVS SCDKNTGD YYED S YEDIS A YLLS KNN AIEPR SF SQNPPVLKRHQREITRTTLQ SDQEEID YDDTIS VEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQT YRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

AE144FVIII

BDD2Y2332 ThrombinAE864

GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGATRRYYLGAV ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLF NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWK ASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLT YSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEG KSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV LAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG

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PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA GVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFL SVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPV LKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ KKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYF SDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQ DQRIRWYLLSMGSNENIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFET VEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGI KTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQD LYGLTPRSLLVGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT PESGPGSEPATSG SETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE864FVIIIMMP-17- AE144 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS

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EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGATRRYYLGAVELSWDYMQS DLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWM GLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNS LMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGT TPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQ YLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGD TLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKY KWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKV SSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNA TTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQ EAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRL NEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGT DNTSSLGPPSMP VHYD SQLDTTLFGKKS SPLTESGGPLSLSEENNDSKLLESGLMNSQES SW GKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSAT NRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATAL RLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESAR WIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGE FTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQ ENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQ NFVTQRSKRALKQFRLPLEETELEKRUVDDTSTQWSKNMKHLTPSTLTQ IDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRV LFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQ REVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDL FPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSA KTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNAC ESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTT LQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVE RLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELN EHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPR KNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG LIGPLLVCHTNTLNPAHGRQVTVQEF ALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRV ECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWA PKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTNM FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAPLGLRLRGGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS

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EGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP

AF144FXIIaFVIIIFXIIaAF864

GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSE SPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESP GPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTMTRIVGGATRR YYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVE FTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVG VSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLI GCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTA QTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAE DYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEED WDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SF SQNSRHPSTRQKQFNATTIPENDIEKTDPWF AHRTPMPKIQNVS S SDLL MLLRQSPTPHGLSLSDLQEAKYETFSDD PSPGAIDSNNSLSEMTHFRPQL HHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSD NLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEEN NDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALF KVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKV TPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQ NPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVE GQNFLSEKNKWVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNT HNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVE GSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTK QIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDT STQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSP LPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGA KKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPK TSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNE ANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKS PEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLC SQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVF QEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSF YSSLISYEEDQRQGA EPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE

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WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGTMTRIVGGGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTA PGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTS ESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESS TAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPES GSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAP GSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSS TAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGT APGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGS TSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESP SGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGP GTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPS GESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGT APGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTS PSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGES STAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPG TSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSP AE864FVIIIFXIa- AE144 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGATRRYYLGAVELSWDYMQS DLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWM GLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNS LMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGT TPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQ YLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGD TLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKY KWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKV

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SSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNA TTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQ EAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRL NEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMP VHYD SQLDTTLFGKKS SPLTESGGPLSLSEENNDSKLLESGLMNSQES SW GKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSAT NRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATAL RLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESAR WIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGE FTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQ ENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQ NFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQ IDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRV LFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQ REVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDL FPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSA KTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNAC ESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTT LQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ SPRSFQKKTRHYFIAAVE RLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELN EHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPR KNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRV ECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWA PKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTNM FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGKLTRAETGGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE144FXIa-FVIII BDD9- AE864 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGKLTRAETGAT RRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF VEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPM ASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFIL LFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLT AQTLLMDLGQFLLFCHIS SHQHDGMEAYVKVD SCPEEPQLRMKNNEEA EDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEED WDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER

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DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SF SQNPPVLKRHQREITRTTLQ SDQEEID YDDTIS VEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE48FXIa-FVIII BDD9- AE864 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGK LTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVW QVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKE KTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQA SLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQL RMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWV HYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDV RPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYY SSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHE VAYW YILSIGAQTDFLS VFF SGYTFKHKMVYEDTLTLFPF SGETVFMSME

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NPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEroYDDTISVEMKKEDF DIYDEDENQSPRSFQKKTRHYFIAAVERLWD YGMS S SPHVLRNRAQ SGS VPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFR NQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAP TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTV QEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAING YIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRI<I<EEYI< MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW IKVDLLAPMHHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTG TLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLN SCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRP QVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA LRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII BDD9FXIaAG288 2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMT ALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SF SQNPPVLKRHQREITRTTLQ SDQEEID YDDTIS VEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS

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FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYKLTRAETGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS PGS STPSGATGSPGTPGSGTAS S SPGS STPSGATGSPGTPGSGTAS S SPGS S TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS STGSPGTPGSGT AS S SPGS STP SGATGSPGS SP S ASTGTGPGS SP S ASTGTG PGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGAS PGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS FVIII BDD9- FXIa- AG864 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED SYEDIS AYLLSKNNAIEPR SF SQNPPVLKRHQREITRTTLQ SDQEEID YDDTIS VEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHG RQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG SGTASS SPGS STPSGATGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSS TGSPGTPGSGTAS S SPGS STPSGATGSPGASPGTS STGSPGTPGSGT AS S SP

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GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSST PSGATGSPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGTPGSGT A SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSST PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GASPGT S STGSPGTPGSGT AS S SPGASPGT S STGSPGASPGTS STGSPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GS SPS ASTGTGPGS SPSASTGTGPGASPGTS STGSPGTPGSGT AS S SPGS ST PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSS TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGP GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

FVIII BDD9(1745)AG288 2(1640Y2332)FXIaAG864

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSPG SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQREITRTTLQS DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLW DYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNF VKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAP CNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVEC LIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQ FIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFA TWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGV KSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSL DPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGT SSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG SPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT

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PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT SSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTG SPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGT PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGS SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSP FVIII BDD9(1743)AG288_2(1638Y2332)FXIaAG864 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SF SGPGASPGTSSTGSPGASPGTS STGSPGTPGSGTAS S SPGS STPSGATGS PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS PGASPGTS STGSPGS STPSGAT GSPGS SPS ASTGTGPGASPGTS STGSPGS S PSASTGTGPGTPGSGTASSSPGSSTPSGATGSQNPPVLKRHQREITRTTLQ SDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEH LGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKN FVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLI GPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP KLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR LHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISD AQITAS S YFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNS LDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPG

-293 041874

BDD10(l745)AG288_2(1640Y2332)FXIaAG864

TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGT ASS SPGASPGTS STGSPGASPGTS STGSPGTPGSGTAS S SPGS STP SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTG TGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG ASPGTS STGSPGASPGTS STGSPGTPGSGTAS S SPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST GSPGASPGTS STGSPGASPGTS STGSPGS STPSGATGSPGS STPSGATGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTP SGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSP_________________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDT WITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMT ALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQS DQEEID YDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLW DYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNF VKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAP CNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE NIHSIHFSGHVFTVRI<I<EEYI<MALYNLYPGVFETVEMLPSI<AGIWRVEC LIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQ FIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFA TWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGV

-294041874

KSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSL DPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGT SSTGSPGSSPS ASTGTGPGS SP S ASTGTGPGTPGSGTAS S SPGS STPSGATG SPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT SSTGSPGASPGTS STGSPGS SPS ASTGTGPGTPGSGT AS S SPGASPGTS STG SPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGASPGTS STGSPGT PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGS SPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA STGTGPGASPGTS STGSPGS SPSASTGTGPGTPGSGTASS SPGS STPSGATG SPGSSTPSGATGSPGASPGTSSTGSP FVIII BDD10FXIaAG288_2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV TVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYKLTRAETGAGSPGAETAPGASPGTS STGSPGASPGTS STGSPG TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS

- 295 041874

FVIII

BDD 10FXIaAG864

GTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSGAETAEQKLISEEDLSPATG_____________________________ ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSS TGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSST PSGA TGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGP GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSST PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GASPGT S STGSPGTPGSGT AS S SPGASPGT S STGSPGASPGTS STGSPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GSSPS ASTGTGPGS SPS ASTGTGPGASPGTS STGSPGTPGSGT AS S SPGS ST PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSS TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGP

-296041874

GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

FVIII

BDD10FXIaAE864

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGP MASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLP GLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFL TAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEE AEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEE DWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILG CHNSDFRNRGMTALLKVS SCDKNTGD YYED S YEDIS AYLLSKNNAIEPR SFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDEN QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVV FQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV TVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLY PGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKF S SLYISQFIIMYSLDGKKWQT YRGNSTGTLMVFFG NVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKE WLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLYKLTRAETGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

The name of the sequence reflects the N- to C-terminal conformation of the FVIII variant and XTEN components: signal sequence (SP); linker (L); a cleavage sequence (CS) can be designated by naming the proteases active in the sequence and XTEN components by family name and length, with insertion points in the case of components designated by the amino acid FVIII and numbered positions adjacent to the inserted sequence or A1, which represents is the N-terminus and Y2332, which is the C-terminus of FVIII.

Claims (31)

1. A recombinant factor VIII fusion protein containing a factor VIII polypeptide fused to a first extended recombinant polypeptide (XTEN), wherein the factor VIII fusion protein has procoagulant activity when cleaved by thrombin, where said factor VIII polypeptide contains amino acid residues 1-745 and 1649-2332 mature human factor VIII (SEQ ID NO: 2), and XTEN inserted directly 5'-3' from the amino acid at position 745 of mature native human factor VIII (SEQ ID NO: 2); where the factor VIII polypeptide contains a partial deletion in the B domain, and where the factor VIII polypeptide does not contain the amino acid residue R1648 corresponding to mature native human factor VIII (SEQ ID NO: 2). 2. The recombinant factor VIII fusion protein of claim 1, wherein the XTEN contains at least 36 amino acids. 3. The recombinant factor VIII fusion protein of claim 2, wherein the XTEN contains at least 144 amino acids. - 297 041874 4. The recombinant factor VIII fusion protein according to claim 2, wherein the XTEN contains at least 288 amino acids. 5. The recombinant factor VIII fusion protein according to any one of claims 1 to 4, wherein the XTEN comprises one or more XTEN sequence motifs, wherein one or more XTEN sequence motifs are selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48. 6. The recombinant factor VIII fusion protein of claim 5, wherein one or more XTEN sequence motifs are selected from SEQ ID NOs: 23, 24, 25 and 26. 7. The recombinant factor VIII fusion protein according to any one of claims 1 to 6, wherein XTEN contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 49, 50, 51, 52, 57, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 78 and 79. 8. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67. 9. The recombinant factor VIII fusion protein according to any one of claims 1 to 8, wherein XTEN contains the amino acid sequence of SEQ ID NO: 67. 10. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 50. 11. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains the amino acid sequence of SEQ ID NO: 50. 12. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 52. 13. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains the amino acid sequence of SEQ ID NO: 52. 14. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 78. 15. The recombinant factor VIII fusion protein according to any one of claims 1 to 7, wherein XTEN contains the amino acid sequence of SEQ ID NO: 78. 16. A recombinant factor VIII fusion protein according to any one of claims 1 to 6, wherein XTEN contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 279, 281, 283, 285, 287, 289 , 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339 , 341, 343, 345, 347, 349 and 351. 17. The recombinant factor VIII fusion protein of any one of claims 1 to 16, wherein the factor VIII polypeptide comprises a single chain factor VIII polypeptide. 18. The recombinant factor VIII fusion protein of any one of claims 1 to 17, wherein the factor VIII fusion protein further comprises an immunoglobulin Fc fragment conjugated at the C-terminus of the FVIII polypeptide. 19. An isolated nucleic acid encoding a recombinant factor VIII fusion protein according to any one of claims 1-18. 20. A vector containing the isolated nucleic acid according to claim 19. 21. The vector of claim 20, wherein said vector is a plasmid, cosmid, viral particle, or phage. 22. A vector according to claim 20 or 21, wherein said vector is an adenoviral vector, or a baculovirus vector, or an autonomously replicating vector. 23. A vector according to any one of claims 20-22, wherein the isolated nucleic acid is operably linked to a promoter, leader sequence, terminator, or enhancer. 24. A host cell containing the vector according to any one of claims 20-23, wherein said cell is a mammalian cell. 25. A pharmaceutical composition comprising a recombinant factor VIII fusion protein according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier. 26. A pharmaceutical composition comprising an isolated nucleic acid according to claim 19, a vector according to any one of claims 20-23, or a host cell according to claim 24, and a pharmaceutically acceptable carrier. 27. A method for producing a recombinant factor VIII fusion protein, comprising culturing a host cell according to claim 24 in a medium under suitable conditions. 28. The method of claim 27, further comprising isolating the recombinant factor VIII fusion protein from the medium. 29. The use of a pharmaceutical composition according to claim 25 or 26 for the manufacture of a medicament for the treatment of coagulopathy. - 298 041874 30. The use of a pharmaceutical composition according to claim 25 or 26 for the manufacture of a medicament for the treatment of bleeding. 31. The use of a pharmaceutical composition according to claim 25 or 26 for the manufacture of a medicament for the treatment of hemophilia A.