Patent Application
20110040073
The present invention relates to novel covalent complexes of a Factor VII polypeptide and a Tissue Factor polypeptide, in particular to such complexes which are functionally active and which have an enhanced proteolytic activity towards Factor X compared to the corresponding free Factor VII polypeptide. Such complexes are believed to particularly useful in the treatment of bleeding disorders.
Factor VIIa polypeptides with enhanced biological activity are desirable molecules for the treatment of uncontrolled bleedings that are partially or completely refractory to conventional recombinant Factor VIIa therapy due to sub-optimal potency of recombinant Factor VIIa. Current approaches to enhance potency reflect the mechanism of action of recombinant Factor VIIa which appears to be direct Factor X activation on the activated platelet independent of Tissue Factor.
Tissue Factor is a 263 amino acid integral membrane glycoprotein receptor residing on the cells of the vascular adventitia. It consists of an extracellular part folded into two compact fibronectin type III-like domains (1-219) each stabilized by a single disulfide bond, a transmembrane segment (220-242), and a short cytoplasmic tail (243-263). It serves as the key inhibitor of coagulation by forming a tight Ca2+-dependent complex with Factor VII which is captured from circulation upon vascular injury. Tissue Factor also greatly enhances the proteolytic activity of Factor VIIa towards its physiologic substrates Factor IX and Factor X by providing a scaffold for optimal macromolecular exosite interactions and by inducing conformational changes in the protease domain of Factor Vita resulting in correct definition of the active-site region. The conformational activation of FVIIa by TF which results from direct protein-protein interaction can be recapitulated in vitro by supplying Factor VIIa with the soluble ectodomain of Tissue Factor (sTF).
Miyata et al., Biochemistry, 36, 1997, 5120-5127, disclose a covalent complex of recombinant Factor VIIa and soluble Tissue Factor being chemically cross-linked by means of a homo-bifunctional amine-specific reagent. However the cross-linked complex did not exhibit proteolytic activity towards Factor X.
The present invention relates to novel covalent complexes of a Factor VII polypeptide and a Tissue Factor polypeptide. The complexes exhibit enhanced proteolytic activity towards the macromolecular substrate Factor X relative to the corresponding free Factor VII polypeptide.
More particular, the present invention relates to a covalent complex of a Factor VII polypeptide and a Tissue Factor polypeptide, wherein the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more designated links between amino acid side chains of sets of (i) an amino acid of the Factor VII polypeptide and (ii) an amino acid of the Tissue Factor polypeptide.
Moreover, the present invention also relates to a covalent complex of a Factor VIIa polypeptide and a Tissue Factor polypeptide, wherein the Factor VIIa polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more links between amino acid side chains of sets of (i) an amino acid of the Factor VIIa polypeptide and (ii) an amino acid of the Tissue Factor polypeptide, wherein the proteolytic activity of the Factor VIIa polypeptide is at least 2-fold, such as at least 50-fold, e.g. at least 100-fold, such as at least 200-fold, or at least 250-fold, of that of the free native (wild-type) factor VIIa, as determined by the in vitro proteolysis assay defined herein.
The present invention also relates to a method for production of a complex of a Factor VII polypeptide and a Tissue Factor polypeptide as defined herein, the method comprising a) transfecting a cell with (i) an expression vector comprising a nucleic acid molecule encoding the Factor VII polypeptide and expression control regions operatively linked to thereto and (ii) and an expression vector comprising a nucleic acid molecule encoding the Tissue Factor polypeptide and expression control regions operatively linked to thereto, b) culturing the transfected cell under conditions for expression of Factor VII polypeptide and Tissue Factor polypeptide, and c) isolating the expressed complex by suitable means.
The present invention, i.a., relates to a covalent complex of a Factor VII polypeptide and a Tissue Factor polypeptide, wherein the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more designated links between amino acid side chains of sets of (i) an amino acid of the Factor VII polypeptide and (ii) an amino acid of the Tissue Factor polypeptide.
In the present context, the term “covalent complex” refers to two molecules (here: a Factor VII polypeptide and a Tissue Factor polypeptide) associated by at least one covalent link or bond and a plurality of non-covalent interactions, e.g. hydrogen bonds, hydrophobic interactions, etc. Hence, the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked in this manner.
The two polypeptides are linked by means of one or more designated links between amino acid side chains of sets of (i) an amino acid of the Factor VII polypeptide and (ii) an amino acid of the Tissue Factor polypeptide.
When used herein, the term “Factor VII polypeptide” encompasses wild-type Factor VII (i.e. a polypeptide having the amino acid sequence disclosed in U.S. Pat. No. 4,784,950), as well as variants of Factor VII, Factor VII derivatives and Factor VII conjugates exhibiting substantially the same or improved biological activity relative to wild-type Factor VII. The term “Factor VII” is intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor VIIa. Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor VIIa. The term “Factor VII polypeptide” also encompasses polypeptides, including variants, in which the Factor VIIa biological activity has been substantially modified or somewhat reduced relative to the activity of wild-type Factor VIIa. These polypeptides include, without limitation, Factor VII or Factor VIIa into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.
The biological activity of Factor VIIa in blood clotting derives from its ability to (i) bind to Tissue Factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).
In the present context, the term “functionally active Factor VII polypeptide” refers to “Factor VII polypeptide” that have been proteolytically processed to yield their respective bioactive forms, and which frequently are referred to as “Factor VIIa polypeptides”.
In some important embodiments, the Factor VII polypeptide is functionally active.
As used herein, “wild type human FVIIa” is a polypeptide having the amino acid sequence disclosed in U.S. Pat. No. 4,784,950.
The term “Factor VII derivative” as used herein, is intended to designate a FVII polypeptide exhibiting substantially the same or improved biological activity relative to wild-type Factor VII, in which one or more of the amino acids of the parent peptide have been genetically and/or chemically and/or enzymatically modified, e.g. by alkylation, glycosylation, PEGylation, acylation, ester formation or amide formation or the like. This includes but is not limited to PEGylated human Factor VIIa, cysteine-PEGylated human Factor VIIa and variants thereof. Non-limiting examples of Factor VII derivatives includes GlycoPegylated FVII derivatives as disclosed in WO 03/31464 and US Patent applications US 20040043446, US 20040063911, US 20040142856, US 20040137557, and US 20040132640 (Neose Technologies, Inc.); FVII conjugates as disclosed in WO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, US patent application 20030211094 (University of Minnesota).
The term “improved biological activity” refers to FVII polypeptides with i) substantially the same or increased proteolytic activity compared to recombinant wild type human Factor VIIa or ii) to FVII polypeptides with substantially the same or increased TF binding activity compared to recombinant wild type human Factor VIIa or iii) to FVII polypeptides with substantially the same or increased half life in blood plasma compared to recombinant wild type human Factor VIIa. The term “PEGylated human Factor VIIa” means human Factor VIIa, having a PEG molecule conjugated to a human Factor VIIa polypeptide. It is to be understood, that the PEG molecule may be attached to any part of the Factor VIIa polypeptide including any amino acid residue or carbohydrate moiety of the Factor VIIa polypeptide. The term “cysteine-PEGylated human Factor VIIa” means Factor VIIa having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor VIIa.
Non-limiting examples of additional substitutions of amino acids in the Factor VII polypeptide providing Factor VII polypeptides with substantially the same or increased proteolytic activity compared to recombinant wild type human Factor VIIa include S52A, S60A (Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); substitutions providing FVIIa polypeptides exhibiting increased proteolytic stability as disclosed in U.S. Pat. No. 5,580,560; substitutions providing FVIIa polypeptides that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); oxidized forms of Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54, 1999); substitutions in FVIIa polypeptides as disclosed in WO 02/077218; and substitutions in FVIIa polypeptides, which exhibit increased pro-teolytic stability as disclosed in WO 02/38162 (Scripps Research Institute); substitutions in FVIIa polypeptides, which provides polypeptides having a modified Gla-domain and exhibiting an enhanced membrane binding as disclosed in WO 99/20767, U.S. Pat. No. 6,017,882 and U.S. Pat. No. 6,747,003, US patent application 20030100506 (University of Minnesota) and WO 00/66753, US patent applications US 20010018414, US 2004220106, and US 200131005, U.S. Pat. No. 6,762,286 and U.S. Pat. No. 6,693,075 (University of Minnesota); and substitutions in FVIIa polypeptides as disclosed in WO 01/58935, U.S. Pat. No. 6,806,063, US patent application 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (Maxygen ApS), WO 04/083361 (Maxygen ApS), and WO 04/111242 (Maxygen ApS), as well as in WO 04/108763 (Canadian Blood Services).
Non-limiting examples of further substitutions in FVIIa polypeptides pro-viding FVII polypeptides having increased biological activity compared to wild-type FVIIa include substitutions as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, WO 03/027147, WO 04/029090, WO 03/027147; WO 02/38162 (Scripps Research Institute); and FVIIa polypeptides with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).
Examples of further substitutions in the FVIIa polypeptides of the present invention include, without limitation, L305V, L305V/M306D/D309S, L3051, L305T, F374P, V158T/M298Q, V158D/E296V/M298Q, K337A, M298Q, V158D/M298Q, L305V/K337A, V158D/E296V/M298Q/L305V, V158D/E296V/M298Q/K337A, V158D/E296V/M298Q/L305V/K337A, K157A, E296V, E296V/M298Q, V158D/E296V, V158D/M298K, and S336G, L305V/K337A, L305V/V158D, L305V/E296V, L305V/M298Q, L305V/V158T, L305V/K337A/V158T, L305V/K337A/M298Q, L305V/K337A/E296V, L305V/K337A/V158D, L305V/V158D/M298Q, L305V/V158D/E296V, L305V/V158T/M298Q, L305V/V158T/E296V, L305V/E296V/M298Q, L305V/V158D/E296V/M298Q, L305V/V158T/E296V/M298Q, L305V/V158T/K337A/M298Q, L305V/V158T/E296V/K337A, L305V/V158D/K337A/M298Q, L305V/V158D/E296V/K337A, L305V/V158D/E296V/M298Q/K337A, L305V/V158T/E296V/M298Q/K337A, S314E/K316H, S314E/K316Q, S314E/L305V, S314E/K337A, S314E/V158D, S314E/E296V, S314E/M298Q, S314E/V158T, K316H/L305V, K316H/K337A, K316H/V158D, K316H/E296V, K316H/M298Q, K316H/V158T, K316Q/L305V, K316Q/K337A, K316Q/V158D, K316Q/E296V, K316Q/M298Q, K316Q/V158T, S314E/L305V/K337A, S314E/L305V/V158D, S314E/L305V/E296V, S314E/L305V/M298Q, S314E/L305V/V158T, S314E/L305V/K337A/V158T, S314E/L305V/K337A/M298Q, S314E/L305V/K337A/E296V, S314E/L305V/K337A/V158D, S314E/L305V/V158D/M298Q, S314E/L305V/V158D/E296V, S314E/L305V/V158T/M298Q, S314E/L305V/V158T/E296V, S314E/L305V/E296V/M298Q, S314E/L305V/V158D/E296V/M298Q, S314E/L305V/V158T/E296V/M298Q, S314E/L305V/V158T/K337A/M298Q, S314E/L305V/V158T/E296V/K337A, S314E/L305V/V158D/K337A/M298Q, S314E/L305V/V158D/E296V/K337A, S314E/L305V/V158D/E296V/M298Q/K337A, S314E/L305V/V158T/E296V/M298Q/K337A, K316H/L305V/K337A, K316H/L305V/V158D, K316H/L305V/E296V, K316H/L305V/M298Q, K316H/L305V/V158T, K316H/L305V/K337A/V158T, K316H/L305V/K337A/M298Q, K316H/L305V/K337A/E296V, K316H/L305V/K337A/V158D, K316H/L305V/V158D/M298Q, K316H/L305V/V158D/E296V, K316H/L305V/V158T/M298Q, K316H/L305V/V158T/E296V, K316H/L305V/E296V/M298Q, K316H/L305V/V158D/E296V/M298Q, K316H/L305V/V158T/E296V/M298Q, K316H/L305V/V158T/K337A/M298Q, K316H/L305V/V158T/E296V/K337A, K316H/L305V/V158D/K337A/M298Q, K316H/L305V/V158D/E296V/K337A, K316H/L305V/V158D/E296V/M298Q/K337A, K316H/L305V/V158T/E296V/M298Q/K337A, K316Q/L305V/K337A, K316Q/L305V/V158D, K316Q/L305V/E296V, K316Q/L305V/M298Q, K316Q/L305V/V158T, K316Q/L305V/K337A/V158T, K316Q/L305V/K337A/M298Q, K316Q/L305V/K337A/E296V, K316Q/L305V/K337A/V158D, K316Q/L305V/V158D/M298Q, K316Q/L305V/V158D/E296V, K316Q/L305V/V158T/M298Q, K316Q/L305V/V158T/E296V, K316Q/L305V/E296V/M298Q, K316Q/L305V/V158D/E296V/M298Q, K316Q/L305V/V158T/E296V/M298Q, K316Q/L305V/V158T/K337A/M298Q, K316Q/L305V/V158T/E296V/K337A, K316Q/L305V/V158D/K337A/M298Q, K316Q/L305V/V158D/E296V/K337A, K316Q/L305V/V158D/E296V/M298Q/K337A, K316Q/L305V/V158T/E296V/M298Q/K337A, F374Y/K337A, F374Y/V158D, F374Y/E296V, F374Y/M298Q, F374Y/V158T, F374Y/S314E, F374Y/L305V, F374Y/L305V/K337A, F374Y/L305V/V158D, F374Y/L305V/E296V, F374Y/L305V/M298Q, F374Y/L305V/K158T, F374Y/L305V/S314E, F374Y/K337A/S314E, F374Y/K337A/V158T, F374Y/K337A/M298Q, F374Y/K337A/E296V, F374Y/K337A/V158D, F374Y/V158D/S314E, F374Y/V158D/M298Q, F374Y/V158D/E296V, F374Y/V158T/S314E, F374Y/V158T/M298Q, F374Y/V158T/E296V, F374Y/E296V/S314E, F374Y/S314E/M298Q, F374Y/E296V/M298Q, F374Y/L305V/K337A/V158D, F374Y/L305V/K337A/E296V, F374Y/L305V/K337A/M298Q, F374Y/L305V/K337A/V158T, F374Y/L305V/K337A/S314E, F374Y/L305V/V158D/E296V, F374Y/L305V/V158D/M298Q, F374Y/L305V/V158D/S314E, F374Y/L305V/E296V/M298Q, F374Y/L305V/E296V/V158T, F374Y/L305V/E296V/S314E, F374Y/L305V/M298Q/V158T, F374Y/L305V/M298Q/S314E, F374Y/L305V/V158T/S314E, F374Y/K337A/S314E/V158T, F374Y/K337A/S314E/M298Q, F374Y/K337A/S314E/E296V, F374Y/K337A/S314E/V158D, F374Y/K337A/V158T/M298Q, F374Y/K337A/V158T/E296V, F374Y/K337A/M298Q/E296V, F374Y/K337A/M298Q/V158D, F374Y/K337A/E296V/V158D, F374Y/V158D/S314E/M298Q, F374Y/V158D/S314E/E296V, F374Y/V158D/M298Q/E296V, F374Y/V158T/S314E/E296V, F374Y/V158T/S314E/M298Q, F374Y/V158T/M298Q/E296V, F374Y/E296V/S314E/M298Q, F374Y/L305V/M298Q/K337A/S314E, F374Y/L305V/E296V/K337A/S314E, F374Y/E296V/M298Q/K337A/S314E, F374Y/L305V/E296V/M298Q/K337A, F374Y/L305V/E296V/M298Q/S314E, F374Y/V158D/E296V/M298Q/K337A, F374Y/V158D/E296V/M298Q/S314E, F374Y/L305V/V158D/K337A/S314E, F374Y/V158D/M298Q/K337A/S314E, F374Y/V158D/E296V/K337A/S314E, F374Y/L305V/V158D/E296V/M298Q, F374Y/L305V/V158D/M298Q/K337A, F374Y/L305V/V158D/E296V/K337A, F374Y/L305V/V158D/M298Q/S314E, F374Y/L305V/V158D/E296V/S314E, F374Y/V158T/E296V/M298Q/K337A, F374Y/V158T/E296V/M298Q/S314E, F374Y/L305V/V158T/K337A/S314E, F374Y/V158T/M298Q/K337A/S314E, F374Y/V158T/E296V/K337A/S314E, F374Y/L305V/V158T/E296V/M298Q, F374Y/L305V/V158T/M298Q/K337A, F374Y/L305V/V158T/E296V/K337A, F374Y/L305V/V158T/M298Q/S314E, F374Y/L305V/V158T/E296V/S314E, F374Y/E296V/M298Q/K337A/V158T/S314E, F374Y/V158D/E296V/M298Q/K337A/S314E, F374Y/L305V/V158D/E296V/M298Q/S314E, F374Y/L305V/E296V/M298Q/V158T/S314E, F374Y/L305V/E296V/M298Q/K337A/V158T, F374Y/L305V/E296V/K337A/V158T/S314E, F374Y/L305V/M298Q/K337A/V158T/S314E, F374Y/L305V/V158D/E296V/M298Q/K337A, F374Y/L305V/V158D/E296V/K337A/S314E, F374Y/L305V/V158D/M298Q/K337A/S314E, F374Y/L305V/E296V/M298Q/K337A/V158T/S314E, F374Y/L305V/V158D/E296V/M298Q/K337A/S314E, S52A, S60A; R152E, S344A, T106N, K143N/N145T, V253N, R290N/A292T, G291N, R315N/V317T, K143N/N145T/R315N/V317T; and substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn; substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys; and substitutions, additions or deletions in the amino acid sequence from 153Ile to 223Arg.
The terms “variant” or “variants”, as used herein, is intended to designate Factor VII having the amino acid sequence disclosed in U.S. Pat. No. 4,784,950, wherein one or more amino acids of the parent protein have been substituted by another amino acid and/or wherein one or more amino acids of the parent protein have been deleted and/or wherein one or more amino acids have been inserted in protein and/or wherein one or more amino acids have been added to the parent protein. Such addition can take place either at the N-terminal end or at the C-terminal end of the parent protein or both. The “variant” or “variants” within this definition still have FVII activity in its activated form. In one embodiment a variant is 70% identical with the amino acid sequence disclosed in U.S. Pat. No. 4,784,950. In one embodiment a variant is 80% identical with the amino acid sequence disclosed in U.S. Pat. No. 4,784,950. In another embodiment a variant is 90% identical with the amino acid sequence disclosed in U.S. Pat. No. 4,784,950. In a further embodiment a variant is 95% identical with the amino acid sequence disclosed in U.S. Pat. No. 4,784,950.
In the present context, the term “Tissue Factor polypeptide” refers to a polypeptide comprising the soluble ectodomain of Tissue Factor, i.e. amino acids 1-219 (in the following referred to as sTF or sTF(1-219)), or a functional variant or truncated form thereof. Preferably, the Tissue Factor polypeptide at least comprises a fragment corresponding to the amino acid sequence 6-209 of Tissue Factor. Examples hereof are, in particular, sTF(6-209), sTF(1-209) and sTF(1-219).
The amino acid sequence of mature human Tissue Factor (1-263) is given in SEQ ID NO:2 and disclosed in Spicer et al. (1987) Proc. Natl. Acad. Sci. USA, 84, 5148-5152.
In the present context, the terms “designated link” and “designated links” are intended to mean that the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more sets of covalently linked amino acid side chains (possibly via a linker moiety), where the set(s) for at least 90% of the complex species are identical with respect to the position and type of at least one of the two amino acids within each of the set(s) throughout a population of complex species. Such links may either be established as direct bonds between amino acid side chains, e.g. in the form of an S-S bridge between cysteine amino acid side chains, or by means of a linker moiety linking amino acid side chains.
Preferably, at least 95%, or even at least 98%, or even virtually all, of the complex species are identical with respect to the position and type of at least one of the two amino acids within each of the set(s) throughout a population of complex species.
The number of sets of covalently linked amino acid side chains is not particularly critical, but in view of the current experimental results, it is believed that only one set is necessary. Because the links are “designated”, i.e. position and type of at least one of the two amino acids within each of the set(s) is predetermined, it should be understood that the functionality and proteolytic activity of the involved polypeptides can be maintained and even enhanced, contrary to a “shotgun” approach using a common (homo)bifunctional cross-linking agents. This being said, it is believed that the number of sets of covalently linked amino acid side chains typically is in the range of 1-10, e.g. 1-5, such as 1-4, or 1-3, 1-2, or 2-4, or 2-3, or 1 or 2 link(s), in particular 1 or 2 links, preferably just one link.
Formation of one or more designated links can be accomplished either by incorporation of one or more amino acids in one or both polypeptide(s) which each are capable of reacting with an amino acid of the other polypeptide, or by addition of a cross-linking agent (most preferable a heterobifunctional cross-linking agent) which is capable of reacting with one or more specific amino acids (preferably only 1-10 amino acids).
In one important embodiment, each of the Factor VII polypeptide and the Tissue Factor polypeptide include one or more cysteine amino acids which is not designated for establishing a intramolecular disulfide bond, but which are capable of forming intermolecular disulfide bonds between the Factor VII polypeptide and the Tissue Factor polypeptide, i.e. covalent links.
In order to preserve the proteolytic activity of the Factor VII polypeptide, it is advantageous if such cysteine amino acids are located in the native interface between the Factor VII polypeptide and the Tissue Factor polypeptide in the corresponding non-covalently linked complex, e.g. as illustrated in
In another embodiment, the Factor VII polypeptide and/or the Tissue Factor polypeptide include one or more reactive amino acids, in particular photo-reactive amino acids which, upon photo activation, are capable of forming corresponding intermolecular bonds between the Factor VII polypeptide and the Tissue Factor polypeptide, i.e. covalent links. Examples of such photo-reactive amino acids are photo-isoleucine (photo-Ile), photo-methionine (photo-Met), and photo-leucine (photo-Leu) developed by Suchanek et al. (Suchanek et al. 2005; see Scheme 1), and p-benzoyl-L-phenylalanine.
Incorporation of photo-Ile, photo-Met, and photo-Leu into proteins and subsequent photo-induced cross-linking has been described by Suchanek et al. (2005) Nature methods, 2, 261-267.
Alternatively, the one or more designated links are established by means of a linker moiety between amino acid side chains of one or more sets of an amino acid of the Factor VII polypeptide and an amino acid of the Tissue Factor polypeptide. Examples of such linker moieties are those derived from a heterobifunctional reagent which is capable of reacting with a specific amino acid side chain, e.g. a cysteine side chains in one of the polypeptides, and subsequently with another amino acid side chain which is in close spatial proximity of that specific amino acid side chain when the Factor VII polypeptide and the Tissue Factor polypeptide form a non-covalently linked complex.
Examples of particularly suitable heterobifunctional reagents are those which are capable of first reacting with a cysteine amino acid side chain, and subsequently (preferably upon activation, e.g. photoactivation or rhodium catalysed) with another amino acid side chain which is in close spatial proximity. Particular examples hereof are those disclosed by Zhang et al. (Biochem. Biophys. Res. Comm., Vol. 217, 3, 1995, 1177-1184), e.g. p-azidoiodoacetanilide, p-azidophenacyl bromide and p-azidobromoacetanilide, and
In view of the above, it is clear that the embodiments where the covalent link is established by means of a reactive side chain or by a heterobifunctional reagent may give rise to a population of complex species where not all species are identical. This is due to the fact that a reactive side chain (e.g. a photo-reactive side chain) and a heterobifunctional reagent (even after specific reaction with an amino acid side chain) may experience more than one potential “reaction partner” upon further reaction, although the number of “reaction partners” may be dramatically reduced if the size (length) of the side chain/reagent is reduced.
However in some embodiments it is possible to select the involved amino acid side chains (or bifunctional reagent) in such a manner that virtually on one possible “reaction partner” exist. This is particularly true when the link is in the form of a disulfide bond, because the abundance of possible “reaction partners” (i.e. another cysteine) is very limited.
Hence in a preferred embodiment, the present invention provides the complex as defined hereinabove, wherein the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more specific links between amino acid side chains of sets of (i) an amino acid of the Factor VII polypeptide and (ii) an amino acid of the Tissue Factor polypeptide.
In the present context, the terms “specific link” and “specific links” are intended to mean that the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more sets of covalently linked amino acid side chains (possibly via a linker moiety), where the set(s) for at least 90% of the complex species are identical with respect to the position and type of each amino acid within each of the set(s) throughout a population of complex species.
Preferably, at least 95%, or even at least 98%, or even virtually all, of the complex species are identical with respect to the position and type of each amino acid within each of the set(s) throughout a population of complex species.
Even more preferable, the one or more specific links are established as one or more direct bonds between amino acids side chains of one or more sets of an amino acid of the Factor VII polypeptide and an amino acid of the Tissue Factor polypeptide.
Hence the present invention further provides the complex defined hereinabove, wherein the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more direct bonds between amino acid side chains of sets of (i) an amino acid of the Factor VII polypeptide and (ii) an amino acid of the Tissue Factor polypeptide.
In the present context, the terms “direct bond” and “direct bonds” are intended to mean that the at least one covalent link between an amino acid side chain of the Factor VII polypeptide and an amino acid side chain of the Tissue Factor polypeptide does not include residues derived from cross-linking agents, reagents, etc., but is formed by atoms derived from the respective amino acid side chains, e.g. in the form of an S-S bridge between cysteine amino acid side chains, however, taking into account that such amino acids need not to be natural amino acids, but may, e.g., be photo-reactive side chains. Consequently, formation of the direct bond does not include bifunctional reagents and the like.
In view of the above, it is preferred that the sets of amino acids comprise one or more cysteine amino acids.
In one variant, each of the sets comprises one cysteine amino acid, e.g. for use upon coupling to a heterobifunctional reagent.
In another variant, each of the sets comprises two cysteine amino acids. In this variant, disulfide link are established.
It has been found that the proteolytic activity is mainly preserved or even enhanced. Thus, preferred embodiments are those where the proteolytic activity of the Factor VIIa polypeptide is at least 2-fold, such as at least 50-fold, e.g. at least 100-fold, such as at least 200-fold, or at least 250-fold, of that of the free native (wild-type) factor VIIa, as determined by the in vitro proteolysis assay defined herein.
Hence, the invention also relates to a covalent complex of a Factor VIIa polypeptide and a Tissue Factor polypeptide, wherein the Factor VIIa polypeptide and the Tissue Factor polypeptide are covalently linked by means of one or more links between amino acid side chains of sets of (i) an amino acid of the Factor VIIa polypeptide and (ii) an amino acid of the Tissue Factor polypeptide, wherein the proteolytic activity of the Factor VIIa polypeptide is at least 2-fold, such as at least 50-fold, e.g. at least 100-fold, such as at least 200-fold, or at least 250-fold, of that of the free native (wild-type) factor VIIa, as determined by the in vitro proteolysis assay defined herein.
As mentioned above, the Tissue Factor polypeptide is the soluble ectodomain of Tissue Factor (1-219) or a functional variant or truncated form thereof.
In some important embodiments, at least one of the amino acids in the positions Thr17, Lys20, Ile22, Ser42, Gly43, Asp44, Trp45, Lys46, Ser47, Phe50, Cys57, Asp58, Asp61, Gly109, Gln110, Leu133, Ser205, Pro206 and Val207 in the soluble Tissue Factor polypeptide, in particular Trp45, Asp58, Gly109, Pro206 and Val207 in the soluble Tissue Factor polypeptide, has been replaced by an amino acid with a reactive side chain which gives rise to a designated link, such as a specific link, in particular a direct bond. In a preferred variant, the at least one amino acid is cysteine amino acid.
In other important embodiments, which preferably are combined with the foregoing, at least one of the amino acids in the positions Leu39, Phe40, Ile42, Ser43, Tyr44, Gln64, Leu65, Ile69, Glu77, Gly78, Arg79, Val92, Phe275, Val276, Arg277, Met306, Thr307, and Glu325 of the Factor VII polypeptide, in particular Phe40, Ser43, Gln64 Glu77 and Phe275 of the Factor VII polypeptide, has been replaced by an amino acid with a reactive side chain which gives rise to a designated link, such as a specific link, in particular a direct bond. In a preferred variant, the at least one amino acid is cysteine amino acid.
More particular, one or both of the amino adds of at least one of the amino acid sets
FVII-Ile69-sTF-Thr17,
FVII-Ile69-sTF-Lys20,
FVII-Arg79-sTF-Ile22,
FVII-Val92-sTF-Ser47,
FVII-Val92-sTF-Phe50,
FVII-Gly78-sTF-Cys57,
FVII-Glu77-sTF-Asp58,
FVII-Gly78-sTF-Asp58,
FVII-Arg79-sTF-Asp58,
FVII-Arg277-sTF-Ser42,
FVII-Val276-sTF-Gly43,
FVII-Arg277-sTF-Gly43,
FVII-Phe275-sTF-Asp44,
FVII-Val276-sTF-Asp44,
FVII-Arg277-sTF-Asp44,
FVII-Phe275-sTF-Trp45,
FVII-Val276-sTF-Trp45,
FVII-Arg134-sTF-Trp45,
FVII-Phe275-sTF-Lys46,
FVII-Gln64-sTF-Gly109,
FVII-Gln64-sTF-Gln110,
FVII-Leu65-sTF-Gln110,
FVII-Phe71-sTF-Leu133,
FVII-Ser43-sTF-Ser205,
FVII-Leu39-sTF-Pro206,
FVII-Phe40-sTF-Pro206,
FVII-Ile42-sTF-Pro206,
FVII-Ser43-sTF-Pro206,
FVII-Tyr44-sTF-Pro206,
FVII-Leu39-sTF-Val207,
FVII-Phe40-sTF-Val207, and
FVII-Ser43-sTF-Val207,
in particular at least one of the amino acids sets
FVII-Phe40-sTF-Val207,
FVII-Ser43-sTF-Pro206,
FVII-Gln64-sTF-Gly109,
FVII-Glu77-sTF-Asp58, and
FVII-Phe275-sTF-Trp45,
have been replaced by an amino acid with a reactive side chain which gives rise to a designated link, such as a specific link, in particular a direct bond.
In a preferred variant, at least one direct bond is a disulfide bond, i.e. a bond between two cysteine amino acids. More preferable, all covalent links are direct bonds in the form of disulfide bonds. In particular, the number of such bonds is 1-5, in particular 1 bond.
In another variant, at least one direct bond is formed via a photo-reactive amino acid, in particular selected from the group consisting of photo-Ile, photo-Leu and photo-Met.
In a still further embodiment, the Factor VII polypeptide and the Tissue Factor polypeptide are covalently linked by means of a linker moiety between amino acid side chains of one or more sets of (i) an amino acid of the Factor VII polypeptide and (i) an amino acid of the Tissue Factor polypeptide. In particular, the linker moiety is derived from a heterobifunctional reagent.
In one interesting variant, the method for the preparation of the complex involves production of a cysteine variant of soluble Tissue factor, subsequent labeling of the cysteine in soluble Tissue Factor with a heterobifunctional reagent in which one of the functionalities is cysteine reactive, and finally cross-linking to factor VIIa by virtue of the second functionality of the reagent. Methods for cloning and expression of cysteine variants of Tissue Factor in Escherichia coli as well as subsequent labeling with a cysteine-specific reagent have been described previously (Stone et al. (1995) Biochem. J., 310, 605-614; Freskgård et al. (1996) Protein Sci., 5, 1521-1540; Owenius et al. (1999) Biophys. J., 77, 2237-2250; Österlund et al. (2001) Biochemistry, 40, 9324-9328). Photo-crosslinking of proteins using heterobifunctional reagents containing one cysteine specific and one photo-activatable functionality have been described by Zhang et al. (1995) Biochem. Biophys. Res. Commun., 217, 1177-1184.
In one particularly interesting variant, the method for the preparation of the complex involves the coexpression of the Factor VII polypeptide and the Tissue Factor polypeptide, whereby the covalent link between the two polypeptides can be readily established.
More particular, the present invention also relates to a method for production of a complex of a Factor VII polypeptide and a Tissue Factor polypeptide as defined herein, the method comprising a) transfecting a cell with (i) an expression vector comprising a nucleic acid molecule encoding the Factor VII polypeptide and expression control regions operatively linked to thereto and (ii) and an expression vector comprising a nucleic acid molecule encoding the Tissue Factor polypeptide and expression control regions operatively linked to thereto, b) culturing the transfected cell under conditions for expression of Factor VII polypeptide and Tissue Factor polypeptide, and c) isolating the expressed complex by suitable means.
In one particularly interesting embodiment hereof, the two polypeptides are linked by means of a specific link, more particular by means of a direct link, such as one or more disulfide links between the Factor VII polypeptide and the Tissue Factor polypeptide.
Expression of protein in cells is well-known to the person skilled in the art of protein production. In practicing the method of the invention, the cells are typically eukaryote cells, more preferably an established eukaryote cell line, including, without limitation, CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), baby hamster kidney (BHK), and HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK cell line is the tk− ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982), hereinafter referred to as BHK 570 cells. The BHK 570 cell line is available from the American Type Culture Collection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCC accession number CRL 10314. A tk− ts13 BHK cell line is also available from the ATCC under accession number CRL 1632. A preferred CHO cell line is the CHO K1 cell line available from ATCC under accession number CCI61.
Other suitable cell lines include, without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1); DUKX cells (CHO cell line) (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) (DUKX cells also being referred to as DXB11 cells), and DG44 (CHO cell line) (Cell, 33: 405, 1983, and Somatic Cell and Molecular Genetics 12: 555, 1986). Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of myelomas with other cells. In some embodiments, the cells may be mutant or recombinant cells, such as, e.g., cells that express a qualitatively or quantitatively different spectrum of enzymes that catalyze post-translational modification of proteins (e.g., glycosylation enzymes such as glycosyl transferases and/or glycosidases, or processing enzymes such as propeptides) than the cell type from which they were derived. Suitable insect cell lines also include, without limitation, Lepidoptera cell lines, such as Spodoptera frugiperda cells or Trichoplusia ni cells (see, e.g., U.S. Pat. No. 5,077,214).
In some embodiments, the cells used in practicing the invention are capable of growing in suspension cultures. As used herein, suspension-competent cells are those that can grow in suspension without making large, firm aggregates, i.e., cells that are monodisperse or grow in loose aggregates with only a few cells per aggregate. Suspension-competent cells include, without limitation, cells that grow in suspension without adaptation or manipulation (such as, e.g., hematopoietic cells or lymphoid cells) and cells that have been made suspension-competent by gradual adaptation of attachment-dependent cells (such as, e.g., epithelial or fibroblast cells) to suspension growth.
The cells used in practicing the invention may be adhesion cells (also known as anchorage-dependent or attachment-dependent cells). As used herein, adhesion cells are those that need to adhere or anchor themselves to a suitable surface for propagation and growth. In one embodiment of the invention, the cells used are adhesion cells. In these embodiments, both the propagation phases and the production phase include the use of microcarriers. The used adhesion cells should be able to migrate onto the carriers (and into the interior structure of the carriers if a macroporous carrier is used) during the propagation phase(s) and to migrate to new carriers when being transferred to the production bioreactor. If the adhesion cells are not sufficiently able to migrate to new carriers by themselves, they may be liberated from the carriers by contacting the cell-containing microcarriers with proteolytic enzymes or EDTA. The medium used (particularly when free of animal-derived components) should furthermore contain components suitable for supporting adhesion cells; suitable media for cultivation of adhesion cells are available from commercial suppliers, such as, e.g., Sigma.
The cells may also be suspension-adapted or suspension-competent cells. If such cells are used, the propagation of cells may be done in suspension, thus microcarriers are only used in the final propagation phase in the production culture vessel itself and in the production phase. In case of suspension-adapted cells the microcarriers used are typically macroporous carriers wherein the cells are attached by means of physical entrapment inside the internal structure of the carriers. In such embodiments, the eukaryotic cell is typically selected from CHO, BHK, HEK293, myeloma cells, etc.
The Factor VII polypeptide and the Tissue Factor polypeptide may also be co-expressed in E. coli. or in bacteria or in transgenic animals, e.g. as discloses in the international patent application with publication number WO 05/075635.
The terminology for amino acid substitutions used in the following examples is as follows. The first letter represents the amino acid naturally present at a position of SEQ ID NO:1 or SEQ ID NO:2. The following number represents the position in SEQ ID NO:1 or SEQ ID NO:2. The second letter represents the different amino acid substituting for the natural amino acid. An example is factor VIIa F40C, where a phenylalanine at position 40 of SEQ ID No:1 is replaced by a cysteine. In another example, sTF(1-219) V207C, the valine in position 207 of SEQ ID NO:2 is replaced by a cysteine.
Materials—
Construction of DNA encoding factor VII and factor VII F40C mutant—The DNA coding sequence of factor VII including its pre (signal sequence) and pro-regions was amplified by PCR using Expand High Fidelity PCR system (Roche Diagnostics Corporation, Indianapolis, Ind.) according to manufacturer's recommendations and primers oHOJ1044 and oHOJ104-r, Introducing flanking NheI and NotI restriction sites (primer sequences are listed in Table 1). The DNA template for the PCR reaction was pLN174 as disclosed in international patent application with publication number WO 02/077218. The purified PCR product was cut with NheI and NotI and then ligated into the corresponding sites of pCI-neo (Promega, Madison, Wis.) to give pHOJ300. The amino acid of native (wild-type) factor VII is given in SEQ ID NO:1. The DNA sequence of native (wild-type) factor VII including its pre (signal sequence) and pro-regions is given in SEQ ID NO:3.
Plasmid pHOJ344 encoding factor VII F40C was constructed by QuickChange® Site-Directed Mutagenesis using primers oHOJ143-f and oHOJ143-r and pHOJ300 as template according to manufacturer's instructions (Stratagene, La Jolla, Calif.). The correct identity of all cloned sequences was verified by DNA sequencing.
Construction of DNA encoding sTF(1-219) and sTF(1-219) V207C mutant—The DNA coding sequence of sTF(1-219) including its signal sequence was amplified from a human whole brain cDNA library (Marathon-ready cDNA; Clontech Laboratories Inc., Mountain View, Calif.) by PCR using Expand High Fidelity PCR system (Roche Diagnostics Corporation, Indianapolis, Ind.) according to manufacturer's recommendations and primers oHO3152-f and oHO3152-r, introducing flanking NheI and XhoI restriction sites (primer sequences are listed in Table 1). The purified PCR product was cut with NheI and XhoI and then ligated into the corresponding sites of pCI-neo (Promega, Madison, Wis.) to give pHOJ356.
The amino acid of sTF(1-219) is given in SEQ ID NO:4. The DNA sequence of sTF(1-219) including its signal sequence is given in SEQ ID NO:5.
Plasmid pHOJ357 encoding sTF(1-219) V207C was constructed by QuickChange® Site-Directed Mutagenesis using primers oHOJ144-f and oHOJ144-r and pHOJ356 as template according to manufacturer's instructions (Stratagene, La Jolla, Calif.). The correct identity of all cloned sequences was verified by DNA sequencing.
Co-expression of factor VII F40C and sTF(1-219) V207C—Factor VIIa F40C and sTF(1-219) V207C were co-expressed by transient expression in the Freestyle™ 293 Expression System (Invitrogen, Carlsbad, Calif.) as instructed by the manufacturer. Briefly, HEK293F cells (Invitrogen, Carlsbad, Calif.) were propagated from a cryo-preserved culture by first establishing a 72-ml shaker culture of 1.5×107 cells in FreeStyle™ 293 Expression Medium (Invitrogen, Carlsbad, Calif.). The culture was then expanded to 2000 ml of 2.2×109 cells over a period of 12 days. Cells were grown in baffled conical PEGT flasks with loose caps (Nunc, Denmark) at 37° C., 8% CO2, and 100-125 rpm in an orbital shaker incubator (Multitron II, Infors, Switzerland). The cell density never exceeded 1.4×106 cells/ml before expansion of the culture, and the flask:culture volume was kept at approximately 3:1. Except for the initial 72-ml culture, the provided growth medium was supplemented with vitamin K1 to 5 ppm (Sigma) required for post-translational gamma-carboxylation of factor VII. The expanded HEK293F culture (2000 ml, 2.2×109 cells) was then transfected with a mixture containing 800 μg of plasmid pHOJ344 (1 μg/μl) and 1500 μg of plasmid DNA pHOJ357 (1 μg/μl). Addition of plasmid DNA and 239fectin™ (Invitrogen, Carlsbad, Calif.) to the Opti-MEM® I solution (Invitrogen, Carlsbad, Calif.), as well as mixing of these and the subsequent addition of the formed lipid DNA complexes to the cell culture were performed dropwise while gently swirling the receiving solution and according to manufacturer's instructions. Following incubation of the transfected culture for 96 hours at 37° C., 8% CO2, and 100 rpm, the conditioned growth medium containing the factor VIIa F40C-sTF(1-219) V207C complex was recovered by centrifugation and stored at −80° C. until further processing.
Purification of Factor VII F40C-sTF(1-219) V207C complex—Conditioned medium to which CaCl2 had been added to a concentration of 10 mM was loaded onto a 25-ml column containing the monoclonal antibody F1A2 (Novo Nordisk A/S, Bagsværd, Denmark) coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, GE Healthcare). The column was equilibrated with 50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.5. After washing with equilibration buffer and equilibration buffer containing 2 M NaCl, bound material was eluted with equilibration buffer containing 10 mM EDTA instead of CaCl2. Calcium chloride was subsequently added to the collected peak fraction to a final concentration of 20 mM.
To remove small amounts of free factor VIIa F40C, the preparation was passed over a 1-ml HiTrap NHS column (GE Healthcare) to which 4 mg sTF(1-219) had been coupled according to manufacturer's instructions. Prior to loading, the column was equilibrated in 50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.5. The flow through containing factor VII F40C-sTF(1-219) V207C complex, and devoid of detectable free factor VII F40C and sTF(1-219) V207C, was collected.
To promote activation of the factor VII F40C-sTF(1-219) V207C complex, human factor IXa was added to a final concentration of 0.1 mg/ml. After complete activation as verified by reducing SDS-PAGE, factor VIIa F40C-sTF(1-219) V207C complex was purified by F1A2 Sepharose 4B affinity chromatography as described above, except that a 5-ml column was used and the equilibration buffer was 10 mM MES, 100 mM NaCl, 10 mM CaCl2, pH 6.0. The final protein preparation was stored in aliquots at −80° C.
Active-site titration assay—Active site concentrations of native (wild-type) factor VIIa and factor VIIa F40C-sTF(1-219) V207C were determined from the irreversible loss of amidolytic activity upon titration with sub-stoichiometric levels of D-Phe-Phe-Arg-chloromethyl ketone (FFR-cmk) essentially as described elsewhere (Bock P.E. (1992) J. Biol. Chem., 267, 14963-14973). Briefly, each protein was diluted into 50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.01% Tween 80, pH 7.0 buffer to an approximate concentration of 300 nM. Diluted protein (20 μl) was then combined with 20 μl 1.5 μM sTF in the case of free factor VIIa and 20 μl buffer in the case of factor VIIa F40C-sTF(1-219) V207C, and finally 20 μl 0-1.2 μM FFR-cmk (freshly prepared in buffer from a FFR-cmk stock dissolved in DMSO and stored at −80° C.). After overnight incubation at room temperature, residual amidolytic activity was measured. The activity assay was carried out in polystyrene microtiter plates (Nunc, Denmark) in a final volume of 200 μl assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.01% Tween 80, pH 7.4) containing approx. 10 nM factor VIIa or factor VIIa-sTF complex, corresponding to 10-fold dilutions of the samples. After 15 min pre-incubation at room temperature, 1 mM chromogenic substrate S-2288 was added and the absorbance monitored continuously at 405 nm for 10 min in a SpectraMax™ 340 microplate spectrophotometer equipped with SOFTmax PRO software (v2.2; Molecular Devices Corp., Sunnyvale, Calif.). Amidolytic activity was reported as the slope of the linear progress curves after blank subtraction. Active site concentrations were determined by extrapolation to zero activity, corresponding to the minimal concentration of FFR-cmk completely abolishing amidolytic activity.
In vitro proteolysis assay—Native (wild-type) factor VIIa and factor VIIa F40C-sTF(1-219) V207C were assayed in parallel to directly compare their specific activities. The assay was carried out in a microtiter plate (Nunc, Denmark). Factor VIIa (300 nM) or factor VIIa F40C-sTF(1-219) V207C (15 nM) and human Factor X (0.2 μM) in 100 μl 50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.01% Tween 80, pH 7.4 buffer were incubated for 20 min at ambient temperature. Factor X activation was then stopped by the addition of 50 μl 50 mM HEPES, 100 mM NaCl, 40 mM EDTA, 0.01% Tween 80, pH 7.4 buffer containing 0.5 μM TF8-5G9 antibody blocking the FX binding site on TF (Ruf et al. (1991) Thromb. Haemost., 66, 529-533). The amount of FXa generated was measured by addition of 50 μl of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765) to a final concentration 0.5 mM. The absorbance at 405 nm was measured continuously in a SpectraMax™ 340 microplate spectrophotometer equipped with SOFTmax PRO software (v2.2; Molecular Devices Corp., Sunnyvale, Calif.). Specific amidolytic activities, reported as the slope of the linear progress curves after blank subtraction divided by the protein concentration in the assay, were used to calculate the ratio between the specific proteolytic activities of factor VIIa-sTF complex and wild-type factor VIIa:
Ratio=((A405 nm factor VIIa-sTF complex)/[Factor VIIa-sTF complex])/((A405 nm factor VIIa wild-type)/[Factor VIIa wild-type])
Results are given in Table 2.
SDS-PAGE analysis—Factor VIIa and factor VIIa F40C-sTF(1-219) V207C (approx 1.5 μg of each) were analyzed by non-reducing and reducing SDS-PAGE on a 4-12% Bis-Tris NuPAGE® gel (Invitrogen Life Technologies, Carlsbad, Calif.) run at 200 V for 35 min in MES buffer (Invitrogen Life Technologies, Carlsbad, Calif.) according to manufacturer's recommendations. Gels were washed with water and stained with Simply Blue™ SafeStain (Invitrogen Life Technologies, Carlsbad, Calif.) according to manufacturer's recommendations. The gel is shown in
SEQ ID NO:1—Amino acid sequence (1-406) of native (wild-type) human factor VII. The three-letter indication “GLA” means 4-carboxyglutamic acid (γ-carboxyglutamate)
SEQ ID NO:3—DNA sequence of native (wild-type) factor VII including its pre (signal sequence) and pro-regions (underlined).
ATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCTTGGGCTTCAG
GGCTGCCTGGCTGCAGTCTTCGTAACCCAGGAGGAAGCCCAAGGCGTC
CTGCACCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCG
SEQ ID NO:4 —Amino acid sequence of sTF(1-219)
SEQ ID NO:5—DNA sequence of sTF(1-219) including its signal sequence (underlined)
ATGGAGACCCCTGCCTGGCCCCGGGTCCCGCGCCCCGAGACCGCCGT
CGCTCGGACGCTCCTGCTCGGCTGGGTCTTCGCCCAGGTGGCCGGCG
CTTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAA
Construction of DNA encoding sTF(1-219) W45C, sTF(1-219) D58C, sTF(1-219) G109C, and sTF(1-219) P206C—Mutants of sTF(1-219) are constructed by QuickChange® Site-Directed Mutagenesis using primer pairs (forward and reverse) listed in Table 3 and pHOJ356 as template according to manufacturer's instructions (Stratagene, La Jolla, Calif.). The correct identity of all cloned sequences is verified by DNA sequencing.
Construction of DNA encoding factor VII 543C, factor VII Q64C, factor VII E77C, and factor VII F275C—Mutants of factor VII are constructed by QuickChange® Site-Directed Mutagenesis using primer pairs (forward and reverse) listed in Table 4 and pHOJ300 as template according to manufacturer's instructions (Stratagene, La Jolla, Calif.). The correct identity of all cloned sequences is verified by DNA sequencing.
Co-expression of factor VII S43C-sTF(1-219) P206C, factor VII Q64C-sTF(1-219) G109C, factor VII E77C-sTF(1-219) D58C, and factor VII F275C-sTF(1-219) W45C—Cysteine variants of factor VIIa and sTF(1-219) are co-expressed by transient expression in the Freestyle™ 293 Expression System (Invitrogen, Carlsbad, Calif.) as instructed by the manufacturer and detailed in Example 1.
Immuno-blot detection of factor VII Q64C-sTF(1-219) G109C—Conditioned medium from transient co-expression of factor VII Q64C and sTF(1-219) G109C was analyzed by non-reducing SDS-PAGE on a 4-12% Bis-Tris NuPAGE® gel (Invitrogen Life Technologies, Carlsbad, Calif.) run at 200 V for 35 min in MES buffer (Invitrogen Life Technologies, Carlsbad, Calif.) according to manufacturer's recommendations. Complexes were subsequently detected by immunoblotting as known to people skilled in the art using a mixture of monoclonal mouse anti-human FVIIa and anti-human TF primary antibodies and secondary horse radish peroxidase (HRP) conjugated rabbit anti-mouse antibody. Immune-complexes were detected using the SuperSignal® West Pico Chemiluminescent detection kit (Pierce) and a LAS-3000 Image Reader (FUJIFILM Corporation). Results are shown in
Construction of DNA encoding sTF(1-209) and sTF(1-209) V207C—The DNA coding sequence of TF(1-209) including its signal peptide is amplified from a human whole brain cDNA library (Marathon-ready cDNA; Clontech Laboratories Inc., Mountain View, Calif.) by PCR using Expand High Fidelity PCR system (Roche Diagnostics Corporation, Indianapolis, Ind.) according to manufacturer's recommendations and primers oHOJ152-f (sequence listed in Table 1) and oHOJ178-r (sequence listed in Table 5), introducing flanking NheI and XhoI restriction sites. The purified PCR product is cut with NheI and XhoI and then ligated into the corresponding sites of pCI-neo (Promega, Madison, Wis.). The resulting plasmid then serves as template for the construction of sTF(1-209) V207C using QuickChange® Site-Directed Mutagenesis and primers oHOJ179-f and oHOJ179-r (sequences listed in Table 5) according to manufacturer's Instructions (Stratagene, La Jolla, Calif.). The correct identity of all cloned sequences is verified by DNA sequencing.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06112358.4 | Apr 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/053105 | 3/30/2007 | WO | 00 | 11/4/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60790713 | Apr 2006 | US |
