Compositions and methods for preparing factor XA and derivatives

Abstract

To provide compositions and methods for preparing factor XA and derivatives.SOLUTION: The present disclosure relates to protein sequences which can be used to generate factor Xa proteins and derivatives thereof. The protein sequences include a factor Xa light chain portion, a heavy chain catalytic domain portion, and an activation peptide at a C-terminal with respect to the heavy chain catalytic domain portion. It is discovered that, when an activation peptide (AP) is fused to the C-terminal end of the heavy chain of the factor Xa protein or derivative, the resulting protein can be more efficiently expressed and attachment of the activation peptide (AP) to the heavy chain does not affect activity of the protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 884,652, filed August 8, 2019, and U.S. Provisional Patent Application No. 62 / 990,885, filed March 17, 2020, the contents of each of which are incorporated by reference in their entirety into this disclosure. [Background technology]

[0002] Recombinant factor Xa (fXa) and its derivatives, such as andexanet alpha, can be produced from host mammalian cell lines. Andexanet alpha (or simply andexanet) is a drug approved in the United States and Europe for patients treated with rivaroxaban or apixaban when life-threatening or uncontrollable bleeding necessitates reversal of anticoagulation. Rivaroxaban and apixaban are factor Xa inhibitors, a group of anticoagulant (blood-thinning) drugs that also includes betrixaban and edoxaban, as well as low molecular weight heparins (LMWHs). Andexanet is a modified recombinant derivative of factor Xa (fXa). Andexanet acts as a decoy molecule, binding to the inhibitor and recapitulating its inhibition of fXa, thus restoring the normal clotting activity of fXa.

[0003] Factor Xa and andexanet have two chains linked by a disulfide bond between the two chains. Recombinant native fXa is usually produced by first expressing an inactive precursor factor X (fX), followed by a second step of activating the expressed fX to fXa by physiological enzymes (e.g., FVIIa / TF, FIXa / FVIIIa) or non-physiological activators (e.g., RVV-X). The difference between fX and fXa lies in the removal of the 52 amino acid residues of the activation peptide (AP) at the N-terminus of the fX heavy chain. Since typical production cell lines, e.g., CHO cells, are unable to process and remove AP, an activation step is necessary to convert inactive fX to native fXa.

[0004] In contrast, andexanet is directly expressed as a fully processed and functional molecule that can be purified directly from harvested cell culture fluid. This is achieved by replacing the AP sequence in native fX with a -RKR-tripeptide to form a -RKRRKR- linker between the heavy and light chains that can be processed by CHO cells. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides compositions and methods for preparing two-chain activated factor Xa proteins or derivatives thereof. It has been discovered that when an activation peptide (AP) is fused to the C-terminal end of the heavy chain of a factor Xa protein or derivative, the resulting protein can be expressed more efficiently, and the addition of the activation peptide (AP) to the heavy chain does not affect the activity of the protein. In contrast, adding an activation peptide to other parts of the protein is not useful for enhancing expression (e.g., when added to the C-terminus of the light chain) or even poses manufacturing challenges (e.g., when added to the N-terminus of the heavy chain). Furthermore, contrary to the general knowledge that removal of the C-terminal 20 amino acid residues (beta peptide) of the FX heavy chain does not adversely affect protein expression of factor X (see, e.g., Branchini et al, J Thromb Haemost 2015;13:1468-74 and Ferrarese et al., Thrombosis Res. 2019,173:4-11), it is herein discovered that such a large truncation is not desirable for efficient expression of fXa and derivatives. Even more surprisingly, by fusing AP to the C-terminus of FXa and its derivatives, the effect of C-terminal truncation on protein expression can be abolished.

[0006] In one embodiment, the present disclosure provides a protein comprising: LC-L1-HC-L2-AP (I) (In the formula, the LC comprises the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; L1 is a peptide linker containing a protease recognition site, HC comprises the amino acid sequence of SEQ ID NO:11 or a peptide having at least 85% sequence identity to SEQ ID NO:11; L2 is absent or is a peptide linker that cannot be processed by proteases, and AP contains the activation peptide) wherein when L1 is processed by a protease, it is capable of producing a two-chain polypeptide comprising an LC and an HC on separate chains connected by a disulfide bridge, and the two-chain polypeptide is capable of binding to a factor Xa inhibitor.

[0007] In another embodiment, the protein comprises: HSA-L2-LC-L1-HC (II) (In the formula, HSA is human serum albumin (HSA) or a variant having at least 85% sequence identity to HSA; L1 is a peptide linker containing a protease recognition site, L2 is absent or is a peptide linker that cannot be processed by proteases, The LC comprises the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; and HC comprises the amino acid sequence of SEQ ID NO:11 or a peptide having at least 85% sequence identity to SEQ ID NO:11. wherein when L1 is processed by a protease, it is capable of producing a two-chain polypeptide comprising an LC and an HC on separate chains, and the two-chain polypeptide is capable of binding to a factor Xa inhibitor.

[0008] In another embodiment, the protein comprises: LC-L1-HC-L2-HSA (III) (In the formula, HSA is human serum albumin (HSA) or a variant having at least 85% sequence identity to HSA; L1 is a peptide linker containing a protease recognition site, L2 is absent or is a peptide linker that cannot be processed by proteases, The LC comprises the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; and HC comprises the amino acid sequence of SEQ ID NO:11 or a peptide having at least 85% sequence identity to SEQ ID NO:11. wherein when L1 is processed by a protease, it is capable of producing a two-chain polypeptide comprising an LC and an HC on separate chains, and the two-chain polypeptide is capable of binding to a factor Xa inhibitor.

[0009] In some embodiments, LC does not include amino acid residues 1-45 of SEQ ID NO: 2. In some embodiments, L2 is absent.

[0010] In some embodiments, there is also provided a two-chain polypeptide capable of binding to a factor Xa inhibitor, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 13, or a peptide having at least 85% sequence identity to SEQ ID NO: 13, and a heavy chain comprising a first fragment comprising the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 85% sequence identity to SEQ ID NO: 11, and a second fragment C-terminal to the first fragment and comprising an activation peptide.

[0011] Also provided is a two-chain polypeptide comprising a light chain comprising a first fragment comprising human serum albumin (HSA) or a variant having at least 85% sequence identity to HSA and a second fragment comprising the amino acid sequence of SEQ ID NO:13 or a peptide having at least 85% sequence identity to SEQ ID NO:13, and a heavy chain comprising the amino acid sequence of SEQ ID NO:11 or a peptide having at least 85% sequence identity to SEQ ID NO:11, wherein the two-chain polypeptide is capable of binding to a factor Xa inhibitor, and the light chain does not comprise amino acid residues 1 to 45 of SEQ ID NO:2.

[0012] Polynucleotides, cells transfected with the polynucleotides, and methods are also provided, in some embodiments, for preparing the double-chain polypeptide. In certain embodiments, for example, the following items are provided: (Item 1) A protein, LC-L1-HC-L2-AP (I) (In the formula, the LC comprises the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; L1 is a peptide linker containing a protease recognition site, HC comprises the amino acid sequence of SEQ ID NO:11 or a peptide having at least 85% sequence identity to SEQ ID NO:11; L2 is absent or is a peptide linker that cannot be processed by proteases, and AP contains the activation peptide) wherein said L1 comprises an amino acid sequence of formula (I), and wherein when said L1 is processed by said protease, it is capable of producing a two-chain polypeptide comprising said LC and said HC on separate chains, and said two-chain polypeptide is capable of binding to a factor Xa inhibitor. (Item 2) 2. The protein according to item 1, wherein the protease is furin. (Item 3) 3. The protein according to item 2, wherein the L1 comprises the amino acid sequence of RKR or RKRRKR (SEQ ID NO: 7). (Item 4) 4. The protein according to any one of items 1 to 3, wherein L2 has a length of 0 to 50 amino acid residues. (Item 5) 5. The protein according to item 4, wherein at least 50% of the amino acid residues of L2 are Gly or Ser. (Item 6) 6. The protein according to any one of items 1 to 5, wherein the activation peptide comprises a glycosylation site. (Item 7) 6. The protein according to any one of items 1 to 5, wherein the activation peptide is an activation peptide of factor IX, factor X, factor XIII, factor II, or protein C, or has at least 85% sequence identity to an activation peptide of factor IX, factor X, factor XIII, factor II, or protein C. (Item 8) 6. The protein according to any one of items 1 to 5, wherein the activation peptide comprises an amino acid sequence of SEQ ID NO: 12, 31, 32, 33, 39 or 40, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12, 31, 32, 33, 39 or 40. (Item 9) 9. The protein according to any one of items 1 to 8, further comprising human serum albumin (HSA) on the N-terminal side of the LC. (Item 10) 10. The protein according to item 9, wherein the HSA comprises the amino acid sequence of SEQ ID NO: 15. (Item 11) 10. The protein according to item 9, wherein the LC does not contain amino acid residues 1 to 45 of SEQ ID NO:2. (Item 12) 12. The protein according to any one of items 1 to 11, wherein the produced two-chain polypeptide has a reduced ability to assemble into the prothrombin complex compared to wild-type fXa. (Item 13) 13. The protein according to any one of items 1 to 12, which does not contain amino acid residues 6 to 39 of SEQ ID NO:2. (Item 14) 14. The protein according to item 13, wherein the LC comprises the amino acid sequence of SEQ ID NO:6 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:6. (Item 15) 15. The protein according to any one of items 1 to 14, wherein the produced two-chain polypeptide has reduced catalytic activity compared to wild-type human factor Xa. (Item 16) 16. The protein according to any one of items 1 to 15, wherein the HC comprises an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. (Item 17) 17. The protein according to any one of items 1 to 16, wherein the HC comprises the amino acid sequence of SEQ ID NO:11. (Item 18) 18. The protein according to any one of items 1 to 17, which does not contain amino acid residues 436 to 448 of SEQ ID NO:2. (Item 19) 19. The protein according to any one of items 1 to 18, which does not contain amino acid residues 434 to 448 of SEQ ID NO:2. (Item 20) A protein, HSA-L2-LC-L1-HC (II) (In the formula, HSA is human serum albumin (HSA) or a variant having at least 85% sequence identity to said HSA, L1 is a peptide linker containing a protease recognition site, L2 is absent or is a peptide linker that cannot be processed by proteases, The LC comprises the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; and HC comprises the amino acid sequence of SEQ ID NO:11 or a peptide having at least 85% sequence identity to SEQ ID NO:11. wherein said L1 comprises an amino acid sequence of formula (II) and wherein said L1 is capable of producing a two-chain polypeptide comprising said LC and said HC on separate chains when said L1 is processed by said protease, said two-chain polypeptide being capable of binding to a factor Xa inhibitor. (Item 21) 21. The protein according to item 20, wherein the HSA comprises the amino acid sequence of SEQ ID NO: 15. (Item 22) 21. The protein according to item 20, wherein the LC does not contain amino acid residues 1 to 45 of SEQ ID NO:2. (Item 23) 23. The protein according to item 22, wherein L2 is absent. (Item 24) 24. The protein according to item 22 or 23, wherein the protease is furin. (Item 25) 25. The protein according to item 24, wherein the L1 comprises the amino acid sequence of RKR or RKRRKR (SEQ ID NO: 7). (Item 26) 26. The protein according to any one of items 20 to 25, further comprising an activation peptide (AP) fused to the C-terminus of the HC. (Item 27) 27. The protein according to item 26, wherein the activation peptide comprises a glycosylation site. (Item 28) 28. The protein according to item 26 or 27, wherein the activation peptide is an activation peptide of factor IX, factor X, factor XIII, factor II or protein C or has at least 85% sequence identity to an activation peptide of factor IX, factor X, factor XIII, factor II or protein C. (Item 29) 29. The protein according to item 28, wherein the activation peptide comprises the amino acid sequence of SEQ ID NO: 12, 31, 32, 33, 39 or 40 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12, 31, 32, 33, 39 or 40. (Item 30) 30. The protein according to any one of Items 20 to 29, wherein the LC comprises an amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. (Item 31) 31. The protein according to any one of items 20 to 30, wherein the produced two-chain polypeptide has reduced catalytic activity compared to wild-type human factor Xa. (Item 32) 32. The protein according to item 31, wherein the HC comprises the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:10. (Item 33) 33. The protein according to item 32, wherein the HC comprises the amino acid sequence of SEQ ID NO:11. (Item 34) 34. The protein according to any one of items 1 to 33, further comprising a signal or signal / propeptide. (Item 35) 35. The protein according to item 34, wherein the signal or signal / propeptide is selected from the group consisting of SEQ ID NOs: 34 to 37. (Item 36) A protein comprising the amino acid sequence of SEQ ID NO:25. (Item 37) A protein comprising the amino acid sequence of SEQ ID NO:26. (Item 38) A protein comprising the amino acid sequence of SEQ ID NO:42. (Item 39) A protein comprising the amino acid sequence of SEQ ID NO:43. (Item 40) A polynucleotide encoding the protein according to any one of items 1 to 39. (Item 41) A cell comprising the polynucleotide of item 40. (Item 42) 42. The cell of item 41, further comprising a polynucleotide encoding the protease. (Item 43) 43. A method for preparing a protein, comprising culturing the cell according to item 41 or 42 and recovering the two-chain protein from the culture. (Item 44) 1. A two-chain polypeptide comprising: a light chain comprising the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; and a heavy chain comprising a first fragment comprising the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 85% sequence identity to SEQ ID NO: 11 and a second fragment located on the C-terminal side of the first fragment and comprising an activation peptide, wherein the two-chain polypeptide is capable of binding to a factor Xa inhibitor. (Item 45) 1. A double-chain polypeptide comprising: a light chain comprising a first fragment comprising human serum albumin (HSA) or a variant thereof having at least 85% sequence identity to said HSA, and a second fragment comprising the amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 85% sequence identity to SEQ ID NO: 11, wherein the double-chain polypeptide is capable of binding to a factor Xa inhibitor, and the light chain does not comprise amino acid residues 1 to 45 of SEQ ID NO: 2. (Item 46) A double-chain polypeptide obtainable by treating the protein according to any one of items 1 to 39 with the protease. [Brief description of the drawings]

[0013] [Figure 1] The structures of constructs C01 to C14 are shown. [Figure 2A] The results of expression and activity tests for C05, C007, C08 and C10 are shown. [Figure 2B]Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Diagram 3] Shown are the results of expression and activity studies for C12-C14, using the andexanet precursor (AnXa), C08 and C10 as standards. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] I. Definition All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 0.1. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0015] The terms "protein" and "polypeptide" are used interchangeably and in the broadest sense refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and no limit is set for the maximum number of amino acids that may comprise a protein or peptide sequence. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics. The one-letter and three-letter abbreviations of naturally occurring amino acids are listed below. Peptides of three or more amino acids are commonly referred to as oligopeptides if the peptide chain is short. If the peptide chain is long, the peptide is commonly referred to as a polypeptide or protein.

[0016] "Factor Xa", or "fXa", or "fXa protein" refers to a serine protease in the blood coagulation pathway that is generated from inactive factor X (fX). Factor Xa is activated either by its cofactor, factor IXa, together with factor VIIIa in a complex known as intrinsic Xase, or by its cofactor, factor VIIa, together with tissue factor in a complex known as extrinsic Xase. fXa forms a membrane-bound prothrombinase complex with factor Va, the active component in the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. Thrombin is the enzyme that catalyzes the conversion of fibrinogen to fibrin, which ultimately leads to blood clot formation. Thus, the biological activity of fXa is sometimes referred to herein as "procoagulant activity".

[0017] Factor Xa is a two-chain molecule linked by one disulfide bond between the two chains. The light chain (LC) has 139 amino acid residues (amino acids 1-139 of SEQ ID NO:2) and contains a gamma-carboxyglutamic acid (Gla)-rich domain (amino acids 1-45 of SEQ ID NO:2) with a short aromatic stack (AS) (amino acids 40-45 of SEQ ID NO:2) followed by two epidermal growth factor (EGF)-like domains (EGF1: amino acids 46-84 of SEQ ID NO:2, EGF2: amino acids 85-128 of SEQ ID NO:2).

[0018] The heavy chain (HC) before activation has 306 amino acids and contains a 52 amino acid activation peptide (AP: amino acids 143-194 of SEQ ID NO:2) followed by a catalytic domain (amino acids 195-448 of SEQ ID NO:2). The catalytic triad equivalent to H57-D102-S195 in the chymotrypsin numbering is located at His236, Asp282 and Ser379 in the fX sequence (SEQ ID NO:2) (amino acids 236, 282 and 379 of SEQ ID NO:2). The heavy chain contains a serine protease, a trypsin-like active site and an N-terminal activation peptide which is glycosylated. The heavy chain has at least three forms, α, β and γ, which differ by cleavage of the C-terminal peptide in the heavy chain.

[0019] The nucleotide sequence encoding human factor X ("fX") can be found in GenBank, "NM_000504". The corresponding amino acid sequence and domain structure of fX are described in Leytus et al, Biochemistry, 1986, 25:5098-5102. The domain structure of mature fX is also described in Venkateswarlu, D. et al, Biophysical Journal, 2002, 82:1190-1206. Upon catalytic cleavage of the first 52 residues of the heavy chain, fX is activated to fXa (SEQ ID NO:3). FXa contains a light chain and a heavy chain in which the glutamic acid residues become gamma-carboxylglutamic acid after translation. The first 45 amino acid residues of the light chain contain 11 post-translationally modified gamma-carboxyglutamic acid residues (Gla), and are therefore referred to as the Gla domain. The Gla domain also contains a short (6 amino acid residue) aromatic stack sequence. Chymotrypsin digestion selectively removes residues 1-44, resulting in fXa lacking the Gla domain. The serine protease catalytic domain of fXa is located in the C-terminal heavy chain. The heavy chain of fXa is highly homologous to other serine proteases such as thrombin, trypsin and activated protein C.

[0020] [Table 1]

[0021] [Table 2]

[0022] [Table 3]

[0023] "Native fXa" or "wild-type fXa" refers to fXa that is naturally present in plasma or isolated in its original, unmodified form, which possesses the biological activity of activating prothrombin, thus promoting the formation of blood clots. This term includes naturally occurring polypeptides isolated from tissue samples and recombinantly produced fXa. "Activated fXa" refers to fXa that has the biological activity of activating prothrombin. "Activated fXa" can be native fXa or modified fXa that retains procoagulant activity.

[0024] "fXa derivative" or "modified fXa" or "derivative of factor Xa protein" refers to a fXa protein that has been modified but is still capable of binding directly or indirectly to a factor Xa inhibitor.

[0025] Derivatives may have modified active sites and / or modified Gla domains. Additional modifications are also contemplated. It is contemplated that such modifications may be made by one or more of the following means: deletion of one or more amino acids from the sequence, replacement of one or more amino acid residues with one or more different amino acid residues, and / or manipulation of one or more amino acid side chains or their "C" or "N" termini.

[0026] The term "active site" refers to the portion of an enzyme or antibody where a chemical reaction occurs. A "modified active site" is an active site that has been structurally modified to provide an active site with increased or decreased chemical reactivity or specificity. It is contemplated that an active site includes not only the actual site, but also the domain that contains the active site. Examples of active sites include, but are not limited to, the catalytic domain of human factor X, which contains amino acid residues 235-488, and the catalytic domain of human factor Xa, which contains amino acid residues 195-488. The catalytic triad equivalent to H57-D102-S195 in the chymotrypsin numbering is located at His236, Asp282, and Ser379. Examples of modified active sites include, but are not limited to, the catalytic triad, either individually or in combination. One modification relates to a fXa derivative with a modified active site Ser379Ala. Further examples include modifications to the catalytic domain of human factor Xa comprising amino acid residues 195-448 by at least one amino acid substitution at positions Arg306, Glu310, Arg347, Lys351, Lys414 or Arg424.

[0027] The terms "factor Xa inhibitors" or "inhibitors of factor Xa" refer to compounds capable of directly or indirectly inhibiting the activity of coagulation factor Xa, which catalyzes the conversion of prothrombin to thrombin in vitro and / or in vivo. Examples of known fXa inhibitors include, without limitation, edoxaban, fondaparinux, idraparinux, biotinylated idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, DX-9065a (described, for example, in Herbert, JM, et al, J Pharmacol Exp Ther. 1996 276(3):1030-8), YM-60828 (described, for example, in Taniuchi, Y., et al, Thromb Haemost. 1998 79(3):543-8), YM-150 (described, for example, in Eriksson, BI et. al, Blood 2005;106(11), Abstract 1865), apixaban, rivaroxaban, PD-348292 (described, for example, in Pipeline Insight: Antithrombotics-Reaching the Untreated Prophylaxis Market, 2007), otamixaban, razaxaban (DPC906), BAY59-7939 (e.g., as described in Turpie, AG, et al, J. Thromb. Haemost. 2005, 3(11):2479-86), edoxaban (e.g., as described in Hylek EM, Curr Opin Invest Drugs 2007 8(9):778-783), LY517717 (e.g., as described in Agnelli, G., et al, J. Thromb. Haemost. 2007 5(4):746-53), GSK913893, betrixaban and its derivatives. Low molecular weight heparins ("LMWH") are also considered to be factor Xa inhibitors.

[0028] In one embodiment, the derivatives of the invention bind directly or indirectly to factor Xa inhibitors. The terms "binding", "binding", "recognizing" or "recognizing" as used herein are meant to include interactions between molecules, which may be detected, for example, using a hybridization assay. The term is also meant to include "binding" interactions between molecules. The interactions may be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature. Binding may be "direct" or "indirect". "Direct" binding includes direct physical contact between molecules. "Indirect" binding between molecules includes molecules that simultaneously have direct physical contact with one or more intermediate molecules. For example, it is envisioned that the derivatives of the invention indirectly bind and substantially neutralize low molecular weight heparin and other indirect inhibitors of factor Xa. This binding may result in the formation of a "complex" that includes the interacting molecules. A "complex" refers to the binding of two or more molecules that are held together by covalent or non-covalent bonds, interactions or forces.

[0029] "Neutralizing," "counteracting," or "counteracting" the activity of an inhibitor of fXa, or similar phrases, refers to inhibiting or blocking the factor Xa inhibitory or anticoagulant function of the fXa inhibitor. Such phrases refer to partial inhibition or blocking or most or all of the function of fXa inhibitor activity in vitro and / or in vivo. These terms also refer to at least about 20% neutralization of pharmacological or surrogate markers of fXa inhibitor dependence. Examples of markers include, but are not limited to, INR, PR, aPTT, ACT, anti-fXa units, thrombin generation (Technothrombin TGA), thromboelastography, CAT (autocalibrated thrombogram), and others.

[0030] A "composition" is intended to mean a combination of an active agent and another compound or composition, inert (eg, a detectable agent or label) or active, such as an adjuvant.

[0031] A "pharmaceutical composition" is intended to comprise a combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0032] As used herein, the term "equivalent thereof" refers to a reference protein, polypeptide or nucleic acid that has only minimal homology but still maintains the desired functionality. Any modified protein referred to herein is also intended to include its equivalent. For example, the homology may be at least 75% homology, alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% homology percentage and can exhibit substantially equivalent biological activity to the reference polypeptide or protein. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. It should be noted that if only the heavy chain of fXa (or a related serine protease) is used, the overall homology can be lower than 75%, for example 65% or 50%, while still retaining the desired functionality.

[0033] When a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) "sequence identity" to another sequence, it means that, when aligned, that percentage of bases (or amino acids) are the same when the two sequences are compared.

[0034] II. Preparation of Factor Xa and Derivatives Andexanet alfa, or simply andexanet, is a modified factor Xa polypeptide approved in the United States and Europe for patients treated with rivaroxaban or apixaban when life-threatening or uncontrolled bleeding necessitates reversal of anticoagulation. Also referred to as r-Antidote, the structure and activity of andexanet are described in U.S. Patent No. 8,153,590.

[0035] Andexanet is a processed two-chain polypeptide, the processed product of SEQ ID NO: 4 after cleavage of the -RKRRKR- (SEQ ID NO: 7) linker. Andexanet is represented by SEQ ID NO: 5, which comprises a light chain (SEQ ID NO: 6) and a heavy chain (SEQ ID NO: 8). The light and heavy chains are connected by a single disulfide bond between cysteine ​​98 (Cys98) of the light chain and cysteine ​​108 (Cys108) of the heavy chain. Like wild-type fXa, in a given production unit, andexanet undergoes post-translational modification resulting in glycosylation at certain amino acid residues, such as Ser56, Ser72, Ser76, and Thr82 of the light chain and Thr249 of the heavy chain, and a modified residue (3R)-3-hydroxyAsp at Asp29 of the light chain. In addition to the interchain disulfide bonds, intrachain disulfide bonds may form between cysteines 16 and 27, 21 and 36, 38 and 47, 55 and 66, 62 and 75, and 77 and 90 in the light chain, and between cysteines 7 and 12, 27 and 43, 156 and 170, and 181 and 209 in the heavy chain.

[0036] [Table 4]

[0037] [Table 5]

[0038] The precursor of andexanet (SEQ ID NO: 4) contains three mutations compared to wild-type fXa. The first mutation is a deletion of aa 6-39 in the Gla domain of fX. The second mutation is a replacement of the activation peptide sequence 143-194 aa by -RKR-. This produces a -RKRRKR- (SEQ ID NO: 7) linker that connects the light and heavy chains. Upon secretion, this linker is cleaved, resulting in a two-chain polypeptide (andexanet). The third mutation is a mutation of the active site residue S379 to an Ala residue.

[0039] Due to these structural changes, andexanet does not compete with fXa for assembly into the prothrombinase complex and has reduced or no catalytic activity. Thus, andexanet is able to scavenge circulating fXa inhibitors without interfering with the natural clotting mechanism. Similar antidotes have also been disclosed, including those with additional deletions in the EGF-1 or EGF-2 domains.

[0040] SEQ ID NO:4 is the precursor protein expressed in host cells to produce andexanet, which can be digested by endogenous or supertransfected furin proteins that target the -RKRRKR- linker (SEQ ID NO:7). The activation peptide (AP) from wild-type fX was not thought to be necessary, as it was replaced with the artificial -RKRRKR- linker.

[0041] However, it has been discovered herein that inclusion of AP can increase the expression of processed two-chain products. However, inclusion of AP in precursor proteins, such as at the N-terminus of the heavy chain, presents challenges to production. Protein production is typically performed by CHO cells, which do not have the enzyme activity to naturally process factor X at the cleavage site -LTR- between AP and the N-terminus of the heavy chain (SEQ ID NO: 2). Therefore, fXa derivatives contain a specific linker that can be cleaved by endogenous furin or co-transfected furin protease in CHO cells, which recognizes the -RKR- and -RKRRKR- (SEQ ID NO: 7) linkers.

[0042] In this regard, it was discovered that when AP is added to a protein through a furin-recognizable linker (e.g., C04-C06), furin may process the linker too early, defeating the purpose of increasing expression. In addition, if the furin-recognizable linker is not properly processed, the processed two-chain molecule may be inactive. On the other hand, AP had no ability to increase protein expression when fused to the light chain so that it is not cleaved (e.g., C07). AP had the greatest effect in increasing protein expression and functional activity only when fused to the C-terminal end of the heavy chain so that it is not cleaved.

[0043] It was further discovered that deletion of 13 or 15 C-terminal amino acid residues of the heavy chain (Delta HC-13 and Delta HC-15, respectively) had no significant effect on protein expression or activity. In contrast, deletion of 20 C-terminal amino acid residues from the C-terminus of the HC (Delta HC-20) significantly reduced protein expression. This was unexpected in view of the general knowledge that truncation of native FX to 20 amino acid residues did not significantly affect factor X expression, as demonstrated, for example, in Branchini et al., Journal of Thrombosis and Haemostasis, 13:1468-1474 (2015). Even more surprisingly, fusing AP to the C-terminus of FXa and derivatives could abolish the effect of C-terminal truncation on protein expression.

[0044] According to one embodiment of the present disclosure, therefore, a compound of formula (I): LC-L1-HC-L2-AP (I) A precursor protein comprising the sequence of

[0045] Here, LC refers to a protein fragment comprising the amino acid sequence of SEQ ID NO: 13 or a biological equivalent such as a peptide having at least 85% sequence identity to SEQ ID NO: 13. HC refers to a protein fragment comprising the amino acid sequence of SEQ ID NO: 11 or a biological equivalent such as a peptide having at least 85% sequence identity to SEQ ID NO: 11. In some embodiments, L2 comprises at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the beta peptide (RGLPKAKSHAPEVITSSPLK, SEQ ID NO: 38).

[0046] L1 denotes a protein linker that includes a protease recognition site. The protease may be furin in some embodiments. L2 may be null (in other words, optional) or a peptide linker on the other hand. However, when L2 is a linker, it cannot be processed by the protease. Therefore, when the protein is incubated with the protease, the protease can digest the protein and generate a two-chain polypeptide, one containing LC and the other containing HC-L2-AP. In some embodiments, the two-chain polypeptide produced is capable of binding to a factor Xa inhibitor.

[0047] Interestingly, in the first set of experiments, the expression-enhancing effect of AP was not observed with human serum albumin (HSA), a protein commonly used to enhance the expression or stability of expressed proteins. See C10 in Example 2. However, in further experiments (Example 3), two precursor constructs, C13 and C14, in which HSA was fused to the N-terminus of the light chain, exhibited greatly improved expression and activity. Therefore, it is assumed that the total deletion of the Gla domain (C10 had a partial deletion of the Gla domain) enhanced the effect of HSA. In other words, when HSA was directly fused to the EGF1 domain, the expression-enhancing effect of HSA was more prominent.

[0048] According to another embodiment of the present disclosure, therefore, a compound of formula (II): HSA-L2-LC-L1-HC (II) A precursor protein comprising the sequence of

[0049] In another embodiment, the compound of formula (III): LC-L1-HC-L2-HSA (III) Also provided is a precursor protein comprising the sequence:

[0050] Here, HSA refers to human serum albumin (HSA) or a variant having at least 85% sequence identity to HSA, L1 is a peptide linker containing a protease recognition site, L2 is absent or a peptide linker that cannot be processed by a protease, LC comprises an amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13, and HC comprises an amino acid sequence of SEQ ID NO: 11 or a peptide having at least 85% sequence identity to SEQ ID NO: 11. The protein of formula (II), when L1 is processed by a protease, produces a two-chain polypeptide comprising LC and HC on separate chains, and the two-chain polypeptide is capable of binding to a factor Xa inhibitor. In some embodiments, LC does not include amino acid residues 1-45 of SEQ ID NO: 2. In some embodiments, L2 is absent.

[0051] In some embodiments, the protein of formula (I) can further comprise HSA fused to the N-terminal portion of the light chain or the C-terminal portion of the heavy chain. In some embodiments, the protein of formula (II) can further comprise AP fused to the C-terminal portion of the heavy chain. In some embodiments, the protein of formula (III) can further comprise AP fused to the N-terminal portion of the light chain.

[0052] In some embodiments, any protein of the present disclosure may be fused to an Fc fragment of an immunoglobulin. The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and with some proteins of the complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody.

[0053] In some embodiments, the Fc fragment used is an Fc fragment of an IgG, such as IgG1, IgG2 or IgG4. In some embodiments, the Fc fragment further comprises a CH1 domain in addition to the CH2 and CH3 domains. An exemplary Fc fragment sequence is provided in Table 24, SEQ ID NO: 46. In some embodiments, the fusion protein comprises a signal peptide, such as SEQ ID NO: 34-36 or 45.

[0054] In some embodiments, only one chain of the Fc fragment is fused to a fXa derivative, hi some embodiments, both chains of the Fc fragment are fused to a fXa derivative.

[0055] The term "furin" or "paired basic amino acid cleaving enzyme" as used herein refers to a protein having an amino acid sequence substantially identical to any of the representative furin sequences in GenBank Accession Nos. NP_002560 (human), NP_001074923 (mouse) or NP_062204 (rat). Suitable cDNAs encoding furin are provided in GenBank Accession Nos. NM_002569 (human), NM_001081454 (mouse) or NM_019331 (rat). In certain embodiments, furin refers to human furin.

[0056] Human serum albumin (HSA) is the serum albumin found in human blood. HSA constitutes about half of serum proteins. HSA is produced in the liver and is soluble in water. Albumin transports hormones, fatty acids and other compounds, regulates pH and maintains oncotic pressure, among other functions. Albumin is synthesized in the liver as preproalbumin, which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. This product, proalbumin, is then cleaved in Golgi vesicles to produce secretory albumin. HSA can have a native sequence or, as tested herein, a single Cys34Ser mutation (SEQ ID NO: 15) to remove the free cysteine ​​in native HSA.

[0057] AP refers to a protein fragment that contains an activation peptide. An "activation peptide" is a peptide that is covalently or non-covalently added to a protein and keeps the protein in an inactive state until the activation peptide is removed. In some embodiments, the activation peptide contains four or more glycosylation sites. Amino acid residues such as Asp, Ser, Tyr and Thr are known to be suitable glycosylation sites. In some embodiments, the AP is 10-100 amino acid residues in length (or alternatively 10-80, 15-70, 20-60 or 10-50 amino acid residues in length) and contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 glycosylation sites.

[0058] Activation peptides are present in wild-type proteins such as factor IX (fIX), factor X (fX), factor XIII (fXIIII), factor II (prothrombin) and the precursor of protein C. Their respective sequences are shown in Table 6.

[0059] [Table 6]

[0060] In some embodiments, the activation peptide comprises an amino acid sequence of SEQ ID NO: 12, 31, 32, 33, 39, or 40, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12, 31, 32, 33, 39, or 40. In some embodiments, the activation peptide comprises an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the activation peptide comprises an amino acid sequence derived from SEQ ID NO: 12 with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the activation peptide comprises an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 31. In some embodiments, the activation peptide comprises an amino acid sequence derived from SEQ ID NO: 31 with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the activation peptide comprises an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 32. In some embodiments, the activation peptide comprises an amino acid sequence derived from SEQ ID NO: 32 with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the activation peptide comprises an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the activation peptide comprises an amino acid sequence derived from SEQ ID NO: 33 with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the activation peptide comprises an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 39. In some embodiments, the activation peptide comprises an amino acid sequence derived from SEQ ID NO: 39, with one, two or three amino acid additions, deletions and / or substitutions.In some embodiments, the activation peptide comprises an amino acid sequence having at least 85%, or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 40. In some embodiments, the activation peptide comprises an amino acid sequence derived from SEQ ID NO: 40 with 1, 2 or 3 amino acid additions, deletions and / or substitutions. In some embodiments, the activation peptide is 10-100 amino acid residues in length (or alternatively 10-80, 15-70, 20-60 or 10-50 amino acid residues in length) and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 glycosylation sites.

[0061] In some embodiments, the protease is furin, although other proteases are within the scope of the disclosure, such as other proprotein convertases, such as PC5, or coagulation enzymes, such as FXa and thrombin. Examples of suitable peptide linkers for furin include -RKR- and -RKRRKR- (SEQ ID NO: 7).

[0062] As stated, in some embodiments, L2 is absent (null). In some embodiments, L2 is a peptide linker of 1-50 (or 1-40, 1-30, 1-25, 1-20, 5-40, 10-30, 15-35) amino acid residues in length. The peptide linker is preferably flexible, such as having at least 30%, 40%, 50%, 60%, 70%, 80% or 90% Gly and / or Ser.

[0063] The two-chain polypeptide produced is suitable, in some embodiments, for use as an antidote to fXa inhibitor-based anticoagulant therapy. In some embodiments, the two-chain polypeptide is unable to compete with fXa for assembly into the prothrombinase complex, has a reduced ability to assemble into the prothrombin complex, or is unable to assemble into the prothrombin complex. Assembly into the prothrombin complex requires a functional Gla domain. In some embodiments, the LC does not include a substantial portion of a Gla domain, such as not including amino acids 6-39 of SEQ ID NO:2. In some embodiments, the LC does not include amino acids 1-45 of SEQ ID NO:2.

[0064] In some embodiments, the LC comprises the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 85%, or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:6.

[0065] In some embodiments, the LC comprises the entire light chain sequence of wild-type fXa (i.e., amino acid residues 1-139 of SEQ ID NO: 3) or an amino acid sequence having at least 85%, or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to the entire light chain sequence of wild-type fXa (i.e., amino acid residues 1-139 of SEQ ID NO: 3) or the full-length light chain sequence of wild-type fXa.

[0066] The two-chain polypeptide produced, in some embodiments, has reduced catalytic activity compared to wild-type human factor Xa, which can be achieved, for example, by mutation of one or more active site residues in the heavy chain, such as His236, Asp282, and Ser379 (amino acids 236, 282, and 379 of SEQ ID NO:2).

[0067] In some embodiments, the HC comprises at least the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the HC comprises at least the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the HC comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85% or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 7.

[0068] In some embodiments, the HC comprises the entire heavy chain sequence of wild-type fXa (i.e., amino acid residues 140-393 of SEQ ID NO: 3) or an amino acid sequence having at least 85%, or alternatively at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to the full-length heavy chain sequence of wild-type fXa.

[0069] In some embodiments, the protein (or HC) does not include the 13 C-terminal amino acid residues of the heavy chain (436-448 of SEQ ID NO:2). In some embodiments, the protein (or HC) does not include the 15 C-terminal amino acid residues of the heavy chain (434-448 of SEQ ID NO:2).

[0070] Non-limiting examples of protein sequences include SEQ ID NOs:25 and 26.

[0071] In some embodiments, a signal peptide (or combined with a propeptide) is included at the N-terminal end of the protein. Exemplary signal / propeptides are shown in Table 7.

[0072] [Table 7]

[0073] The disclosed precursor proteins can have the same amino acid residues as wild-type fX, except for the deletions shown. In a preferred embodiment, specific amino acid substitutions are introduced, such as the Ser379Ala substitution shown in SEQ ID NO: 4, leading to the production of an effective antidote to fXa inhibitors. In some embodiments, the precursor protein is capable of producing a two-chain polypeptide capable of binding to factor Xa inhibitors when L1 and L2 are digested. When amino acid substitutions are introduced, in some embodiments, the digested two-chain polypeptide is unable to compete with fXa for assembly into the prothrombinase complex, has a reduced ability to assemble into the prothrombin complex, or is unable to assemble into the prothrombinase complex, and / or has reduced catalytic activity compared to wild-type human factor Xa.

[0074] In some embodiments, a two-chain polypeptide obtainable by processing a precursor protein of the present disclosure is also provided. In one embodiment, a two-chain polypeptide is provided that comprises a light chain comprising an amino acid sequence of SEQ ID NO: 13 or a peptide having at least 85% sequence identity to SEQ ID NO: 13, and a heavy chain comprising a first fragment comprising an amino acid sequence of SEQ ID NO: 11 or a peptide having at least 85% sequence identity to SEQ ID NO: 11 and a second fragment C-terminal to the first fragment and comprising an activation peptide, the two-chain polypeptide being capable of binding to a factor Xa inhibitor. Examples of such two-chain polypeptides are provided in Tables 8 and 9 below.

[0075] [Table 8]

[0076] [Table 9]

[0077] Polynucleotides and host cells The present disclosure also provides host cells containing the polynucleotides encoding the disclosed proteins and one or more polypeptides. In one embodiment, the polypeptides are expressed and present on the cell surface (extracellularly). Suitable cells containing the polypeptides of the present invention include prokaryotic and eukaryotic cells, including but not limited to bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, mouse cells, rat cells, sheep cells, monkey cells and human cells. Examples of bacterial cells include Escherichia coli, Salmonella enterica and Streptococcus gordonii. Cells can be purchased from commercial vendors such as the American Type Culture Collection (ATCC, Rockville Maryland, USA) or can be cultured from isolates using methods known in the art. Examples of suitable eukaryotic cells include, but are not limited to, 293T HEK cells and the hamster cell lines CHO, BHK-21; mouse cell lines designated NIH3T3, NS0, C127, monkey cell lines COS, Vero; and human cell lines HeLa, PER.C6 (commercially available from Crucell), U-937, and Hep G2. Non-limiting examples of insect cells include Spodoptera frugiperda. Examples of yeast useful for expression include, but are not limited to, the genera Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or Pichia. See U.S. Patent Nos. 4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and 6,258,559.

[0078] In some embodiments, the host cell is further transfected with a polynucleotide encoding a furin protein.

[0079] In addition to being species specific, the cells may be of any particular tissue type, such as neurons, or alternatively somatic or embryonic stem cells, such as stem cells that may or may not differentiate into neuronal cells, e.g., embryonic stem cells, adipose stem cells, neuronal stem cells, and blood stem cells. The stem cells may be of human or animal origin, such as mammalian. EXAMPLES

[0080] The present invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the present invention. The present invention is not limited in scope by the exemplified embodiments, which are intended to merely illustrate a single aspect of the invention. Any methods that are functionally equivalent are within the scope of the present invention. Various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications fall within the scope of the appended claims.

[0081] Example 1. Preparation of expression vector In accordance with this example, polynucleotide constructs were generated that encode precursor proteins that can be used to produce factor Xa derivatives, including andexanet, that can be used to neutralize factor Xa inhibitors. The precursor protein sequences are shown in Tables 10-20, shown in Figure 1, and designated C01-C11.

[0082] Compared to the precursor of andexanet (SEQ ID NO: 4), C01 (SEQ ID NO: 18) contained a deletion of 20 C-terminal amino acid residues of the heavy chain. Similarly, C02 (SEQ ID NO: 19) and C03 (SEQ ID NO: 20) contained deletions of 13 and 15 C-terminal amino acid residues, respectively.

[0083] Using C03 as a reference, the factor Xa activation peptide (AP) was added back into precursor C04 (SEQ ID NO:21). Unlike wild-type fX, a -RKRRKR- linker was placed between the AP and the heavy chain to facilitate processing by furin (as with wild-type fX, a -RKR- linker was also between the light chain and AP). Another version of this precursor, C05 (SEQ ID NO:22), was also prepared, which did not have a C-terminal truncation, but in which the N-terminal 11 amino acids of the light chain were removed. Yet another precursor, C06 (SEQ ID NO:23), was also prepared, which contained both the N-terminal 11 amino acid truncation and the C-terminal 15 amino acid truncation. In C07 (SEQ ID NO:24), the native -RKR- linker between the light chain and AP was removed.

[0084] In precursors C08 (SEQ ID NO: 25) and C09 (SEQ ID NO: 26), AP was placed at the C-terminus of the heavy chain. AP was fused directly to the heavy chain in C08 and fused via an artificial linker (-KSS(GSS) 9 GSS-, SEQ ID NO:14).

[0085] In precursors C10 (SEQ ID NO:27) and C11 (SEQ ID NO:28), human serum albumin (HSA, SEQ ID NO:15) was fused to either the N- or C-terminus, where HSA could have either the native sequence or a sequence with a single Cys34Ser mutation (SEQ ID NO:15) to remove the free cysteine ​​in native HSA. In C10, the heavy chain was intact, and in C11, the heavy chain had a 15 amino acid truncation at the C-terminus.

[0086] [Table 10]

[0087] [Table 11]

[0088] [Table 12]

[0089]

Table 13

[0090]

Table 14

[0091]

Table 15

[0092]

Table 16

[0093]

Table 17

[0094]

Table 18

[0095]

Table 19

[0096]

Table 20

[0097] Polynucleotides encoding these precursor proteins (with appropriate signal peptides) were synthesized de novo. After verification of these polynucleotide sequences, they were ligated into expression vectors suitable for transfection into mammalian host cells. An exemplary expression vector was the AB1 vector. Another expression vector used was the pcDNA3.3 vector (Thermo Fisher Scientific).

[0098] The expression vectors were transfected into host cells CHO-DUXB11, CHO-S (Thermo Fisher Scientific), ExpiCHO-S (Thermo Fisher Scientific), DG44 (Thermo Fisher Scientific) or CHO-M. Transfections were performed using the ExpiFectamine™ CHO Transfection Kit (Thermo Fisher Scientific). Electroporation was performed using either ELISA kits (Microchip Scientific) or reagents and an electroporator (Maxcyte) according to the manufacturer's recommendations.

[0099] The transfected CHO cells were cultured in shake flasks and protein expression was monitored over time. Protein expression was monitored transiently without prior use of antibiotics for stable cell selection. Protein expression was further monitored using stable CHO cell pools. The stable pools were expanded and cell banks were prepared and stored in liquid nitrogen. Protein expression with stable pools proceeded similarly to transient expression by thawing a vial of the cell bank.

[0100] To improve the processing of the linker connecting the heavy and light chains of the fXa derivatives, a second polynucleotide sequence encoding the human furin protein was co-transfected into certain samples. The furin construct was introduced using a separate vector. For stable cell production, a stable cell pool was first selected using pcDNA3.3 with the fXa derivative and G-418 (0-1500 μg / mL) to generate a stable pool (parent pool). The parent pool was further transfected with a second vector carrying furin (supertransfection). The supertransfected pool was further selected using antibiotics and prepared for cell banking.

[0101] Protein expression levels in cell cultures were measured by ELISA (FX-EIA, Enzyme Research Laboratories) and characterized by Western blot using monoclonal antibodies against FX / FXa heavy and light chains. Andexanet was used to generate a standard curve. Proteins in the harvested cell culture fluids were purified by an affinity-based method using STI-resin to capture functional proteins.

[0102] Example 2. Testing expression levels and activity In this example, the expression level and activity of the fXa derivative prepared in Example 1 were examined.

[0103] The expression and functional activity of andexanet (AnXa) precursor (SEQ ID NO: 4) and C01-C03 were tested in transient transfections (co-transfection with furin). Conditions were chemical transfection with Expifectamine followed 24 hours later by incubation at 37°C, 8% CO 2 , 135 rpm including enhancer and feed addition. The results are shown in the table below.

[0104] [Table A]

[0105] Stable pools of these constructs were generated. Total cell culture volume was 30 mL. Cells were passaged every 3 days until cells fully recovered to >97% viability. Conditions for titering were as follows: 32°C, 5% CO 2 , 135 rpm; feed addition on days 1, 4, 7 and 10. The results are shown in the table below.

[0106] [Table B]

[0107] The table below shows the results of tests where the cells were additionally transfected with furin.

[0108] [Table C]

[0109] The results showed that in transient and stable expression, C01 had low levels of expression, while C02 and C03 functioned similarly to the andexanet precursor. Thus, truncation of 13 or 15 C-terminal residues from the heavy chain did not affect protein expression or activity, whereas deletion of the 20 terminal residues had a significant adverse effect.

[0110] Compared to C01-C03, C04-C11 further contained a factor X activation peptide (AP) or human serum albumin (HSA). When wild-type factor X is activated, the AP is removed. In the production of andexanet, the AP was not part of the construct. As shown in the results below, the inclusion of the AP significantly increased protein expression. Furthermore, when the AP was fused to the C-terminal portion of the heavy chain (as opposed to the light chain), the constructs yielded the most functionally active product (C08 and C09). Fusion to HSA was also useful in increasing expression and activity, although the impact was less pronounced compared to AP.

[0111] [Table D]

[0112] [Table E]

[0113] Based on the above results, C05, C07, C08 and C10 were further examined for their expression and functional activity. As shown in Figures 2A-2D, co-transfection of the constructs with furin promoted increasing the titer of functional protein in most cases. However, among C05, C07, C08 and C10, only C08 retained both high expression level and functional activity.

[0114] Furthermore, as shown in the table below, co-transfection of C08 with 5% furin resulted in the highest picograms / cell / day (PCD) of approximately 1.9 calculated from both ELISA and functional quantitative analysis.

[0115] [Table F]

[0116] Example 3. Preparation and testing of additional constructs Based on the test results in Example 2, this example prepared and tested a few additional constructs whose sequences are provided in the table below.

[0117] [Table 21]

[0118] [Table 22]

[0119] [Table 23]

[0120] The domain structures of these constructs are also shown in Figure 1. C12, compared to C08, has an intact heavy chain and an additional AP inserted between the EGF2 and heavy chain. C13 is similar to C10, but has an N-terminally truncated light chain. Finally, C14 contained both an N-terminal HSA (as in C14) and a C-terminal AP (as in C08).

[0121] The expression levels and activities of these constructs were measured along with the andexanet precursor (AnXa), C10 and C12 as controls, and the results are shown in the table below and plotted in Figure 3.

[0122] [Table G]

[0123] C13, which has HSA directly fused to the EGF1 domain of the light chain, and C14, which further contains an AP domain at the C-terminus of the heavy chain, exhibited the highest expression and activity. Interestingly, the only difference between C10 and C13 was that C10 further contains certain amino acids (A1-K11) from the Gla-domain, but the expression of C10 was significantly lower, suggesting that the direct fusion between HSA and the EGF1 domain is beneficial. Furthermore, the results of C12 suggest that adding an additional AP between the light and heavy chains is not necessary.

[0124] This example further demonstrates the benefit of fusing HSA to the N-terminus of the light chain and the AP domain to the C-terminus of the heavy chain in the construct.

[0125] Example 4. Preparation and testing of Fc fusions This example tested Fc fragments and fusions between Fc fragments and factor Xa derivatives such as andexanet.

[0126] Two fusion constructs were prepared, as shown in Tables 25 and 26, containing the Fc fragment of Table 24 and two different signal peptides.

[0127] [Table 24]

[0128] [Table 25]

[0129] [Table 26]

[0130] Expression of these constructs was tested in transient transfections. Conditions included chemical transfection with Expifectamine followed by incubation for 24 hours at 37°C, 8% CO 2 , including the addition of enhancer and feed at 135 rpm. As shown in the results below, the addition of the signal peptide of fXa (construct F01) promoted increased expression of the fusion protein.

[0131] [Table H]

[0132] While the present invention has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art.

Claims

[Claim 1] The invention described in the specification.

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