The present invention relates to novel conjugates between polypeptide variants of protein C and a non-polypeptide moiety, such as PEG or sugar moieties. In particular, the present invention provides novel protein C conjugates having an increased resistance to inactivation by e.g. human plasma and .alpha..sub.1-antitrypsin. Consequently, such conjugates have an increased in vivo half-life. Preferred examples include protein C conjugates, wherein at least one additional in vivo N-glycosylation site has been introduced. The conjugates of the invention are useful for treating a variety of diseases, including septic shock.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates to novel conjugates between polypeptide variants of protein C and a non-polypeptide moiety, to means and methods for preparing such conjugates, to pharmaceutical compositions comprising such conjugates and the use of such conjugates in therapy, in particular for the treatment of a variety of coagulation disorders. The present invention also relates to the polypeptide part of the conjugates of the invention.

Accordingly, in its first aspect the invention relates to a conjugate comprising at least one non-polypeptide moiety covalently attached to a protein C polypeptide that comprises an amino acid sequence which differs from that of a parent protein C polypeptide in at least one introduced and/or at least one removed amino acid residue comprising an attachment group for said non-polypeptide moiety.

In a further aspect the invention relates to a variant of a parent protein C polypeptide, said variant comprising a substitution in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245,V250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387 and H388, with the proviso that the substitution is not selected from the group consisting of T254S, T254A, T254H, T254K, T254R, T254N, T254D, T254E, T254G, T254Q, Y302S, Y302A, Y302T, Y302H, Y302K, Y302R, Y302N, Y302D, Y302E, Y302G, Y302Q, F316S, F316A, F316T, F316H, F316K, F316R, F316N, F316D, F316E, F316G and F316Q.

In an even further aspect, the present invention relates to the polypeptide part of the conjugate of the invention.

In still further aspects the present invention relates to a nucleotide sequence encoding the polypeptide part of the conjugate of the invention, to a nucleotide sequence encoding the polypeptide variant of the invention, to an expression vector comprising the nucleotide sequence of the invention and to a host cell comprising the nucleotide sequence of the invention or comprising the expression vector of the invention.

Still other aspects of the present invention relates to a pharmaceutical composition comprising the conjugate or the variant of the invention as well as to methods of producing and using the conjugates and variants of the invention.

DETAILED DISCLOSURE OF THE INVENTION

Conjugate of the Invention

The conjugates of the present invention are the result of a generally new strategy for developing improved protein C molecules. More specifically, by removing and/or introducing an amino acid residue comprising an attachment group for the non-polypeptide moiety it is possible to specifically adapt the polypeptide so as to make the molecule more susceptible to conjugation to the non-polypeptide moiety of choice, to optimize the conjugation pattern, e.g. to ensure an optimal distribution and number of non-polypeptide moieties on the surface of the protein C molecule and to ensure that only the attachment groups intended to be conjugated is present in the molecule, and thereby obtain a new conjugate molecule, which has APC activity and in addition one or more improved properties as compared to protein C molecules available today. For instance, when the total number of amino acid residues comprising an attachment group for the non-polypeptide of choice is increased or decreased to an optimized level, the renal clearance of the conjugate is typically significantly reduced due to the altered shape, size and/or charge of the molecule achieved by the conjugation. Furthermore, we have found that it is possible to design the attachment of a non-polypeptide moiety to an attachment group in the polypeptide part of the conjugate so that inactivation by human plasma or certain inhibitors, such as alpha-1-antitrypsin, is significantly reduced (see below).

The amino acid residue comprising an attachment group for a non-polypeptide moiety, either it be removed or introduced, is selected on the basis of the nature of the non-polypeptide moiety of choice and, in most instances, on the basis of the method in which conjugation between the polypeptide and the non-polypeptide moiety is to be achieved. For instance, when the non-polypeptide moiety is a polymer molecule such as a polyethylene glycol or polyalkylene oxide derived molecule amino acid residues comprising an attachment group may be selected from the group consisting of lysine, cysteine, aspartic acid, glutamic acid, histidine, and tyrosine, preferably cysteine and lysine, in particular lysine. When the non-polypeptide moiety is a sugar moiety the attachment group is, e.g., an in vivo glycosylation site, preferably an N-glycosylation site.

Whenever an attachment group for a non-polypeptide moiety is to be introduced into or removed from the protein C polypeptide in accordance with the present invention, the position of the polypeptide to be modified is conveniently selected as follows:

The position is preferably located at the surface of the protein C polypeptide, and more preferably occupied by an amino acid residue having more than 25% of its side chain exposed to the surface, such as more than 50% of its side chain exposed to the surface. Such positions have been identified on the basis of an analysis of a 3D structure of the human wild-type APC molecule as described in the Methods section herein. Furthermore, homologous positions in non-human APC polypeptides (including variants thereof) comprising an amino acid sequence being homologous to that of wild-type human protein C may easily be determined by suitable alignment of the respective sequences or 3D structures.

In order to determine an optimal distribution of attachment groups, the distance between amino acid residues located at the surface of the polypeptide is calculated on the basis of a 3D structure of the polypeptide. More specifically, the distance between the CB's of the amino acid residues comprising such attachment groups, or the distance between the functional group (NZ for lysine, CG for aspartic acid, CD for glutamic acid, SG for cysteine) of one and the CB of another amino acid residue comprising an attachment group are determined. In case of glycine, CA is used instead of CB. In the polypeptide part of a conjugate of the invention, any of said distances is preferably more than 8 .ANG., in particular more than 10 .ANG. in order to avoid or reduce heterogeneous conjugation.

The total number of amino acid residues to be altered in accordance with the present invention, e.g. as described in the subsequent sections herein, (as compared to the parent protein C molecule) will typically not exceed 15. The exact number of amino acid residues and the type of amino acid residues to be introduced depends, inter alia, on the desired nature and degree of conjugation (e.g. the identity of the non-polypeptide moiety, how many non-polypeptide moieties it is desirable or possible to conjugate to the polypeptide, where in the polypeptide conjugation should be performed or avoided, etc.). Preferably, the polypeptide part of the conjugate of the invention or the polypeptide of the invention comprises an amino acid sequence which differs in 1 15 amino acid residues from the amino acid sequence shown in SEQ ID NO:4, such as in 1 8 or 2 8 amino acid residues, e.g. in 1 5 or 2 5 amino acid residues. Thus, normally the polypeptide part of the conjugate or the polypeptide of the invention comprises an amino acid sequence which differs from the amino acid sequence shown in SEQ ID NO:4 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

Preferably, the conjugate of the invention has one or more improved properties as compared to wild-type human APC, including increased functional in vivo half-life, increased serum half-life, increased resistance to inhibitors, reduced renal clearance, reduced immunogenicity and/or increased bioavailability. It is contemplated that a conjugate of the present invention offers a number of advantages over the currently available APC products, including longer duration between injections, administration of less protein, and fewer side effects.

Furthermore, a reduced anticoagulant activity might be beneficial for reducing the risk of bleeding while maintaining the anti-inflammatory effect of the APC conjugates. This might be especially important when the conjugate has an extended plasma half-life. These new properties should enhance the anti-inflammatory effect compared to the anticoagulant activity allowing a more effective and safe treatment.

Typically, the conjugate according to the invention has a molecular weight of at least about 67 kDa, preferably at least about 70 kDa, although a lower molecular weight may also give rise to a reduced renal clearance. Polymer molecules, such as PEG, or introduced glycosylation sites have been found to be particularly useful for adjusting the molecular weight of the conjugate.

The conjugate of the invention comprises a sufficient number or type of non-polypeptide moieties to improve one or more of the above mentioned desired properties of the protein C polypeptide. Normally a conjugate of the invention comprises 1 10 first non-polypeptide moieties, in particular 1 8 or 1 5 first non-polypeptide moieties.

The conjugate of the invention may further comprise at least one second non-polypeptide moiety which is different from said first non-polypeptide moiety. For instance, the conjugate of the invention may comprise 1 10 second non-polypeptide moieties, in particular 1 8 or 1 5 second non-polypeptide moieties. For instance, when the first non-polypeptide moiety is a sugar moiety, in particular an in vivo attached sugar moiety, a second non-polypeptide moiety of interest could be a polymer of the PEG type. The in vivo attached sugar moiety may be attached to a naturally occurring in vivo glycosylation site of the polypeptide, or an introduced site.

In a very interesting embodiment of the invention the non-polypeptide moiety is introduced in the active site region of protein C, the rationale being that introduction of a non-polypeptide moiety or moieties in this particular region of the protein C molecule will impair binding of inhibitors (such as alpha-1-antitrypin) to APC while still retaining a substantial APC activity. This, in turn, has the consequence that such conjugates will exhibit a significantly prolonged half-life compared to wild-type human APC since elimination of the inhibitor/APC complex via hepatic receptors is avoided or at least reduced. Selection of amino acid residues, which are located in the active site region of protein C is described in detail in Example 2 herein.

When used herein the term "active site region" is defined with reference to Example 2 herein, where the actual amino acid residues which constitute the active site region are shown.

In a particular preferred embodiment of the invention, an attachment group for a non-polypeptide moiety is introduced in a position of the active site region which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface (see Example 3 herein), i.e. an attachment group for a non-polypeptide moiety is introduced in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, N248, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387, and H388, (H211 and C384 being excluded). Preferably, the introduced attachment group is an attachment group for a sugar moiety, in particular an in vivo N-glycosylation site (see the section entitled Conjugate of the invention where the non-polypeptide moiety is a sugar moiety).

Conjugate of the Invention where the Non-Polypeptide Moiety is a Sugar Moiety

As explained above, in a preferred embodiment the present invention relates to a conjugate comprising at least one introduced glycosylation site, in particular an in vivo N-glycosylation site, covalently attached to a protein C polypeptide that comprises an amino acid sequence which differs from a parent protein C polypeptide, in particular from the amino acid sequence shown in SEQ ID NO:4 or a variant thereof, in at least one introduced glycosylation site.

Preferably, the glycosylation site is introduced in a position, which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface, such as at least 50% of its side chain exposed to the surface. Such amino acid residues are identified in Example 1 herein. It should be understood that when the term "at least 25% (or at least 50%) of its side chain exposed to the surface" is used in connection with introduction of an in vivo N-glycosylation site this term refers to the surface accessibility of the amino acid side chain in the position where the sugar moiety is actually attached. In many cases it will be necessary to introduce a serine or a threonine residue in position +2 relative to the asparagine residue to which the sugar moiety is actually attached (unless, of course, this position is already occupied by a serine or a threonine residue) and these positions, where the serine or threonine residues are introduced, are allowed to be buried, i.e. to have less than 25% of their side chains exposed to the surface.

In order to impair the binding of inhibitors, such as alpha-1-antitrypsin, to APC the glycosylation site is preferably introduced in a position which is within the active site region (defined in Example 2 herein) and which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface (defined in Example 3 herein), i.e. the introduced in vivo N-glycosylation site is preferably selected from the group consisting of D172N+K174S, D172N+K174T, D189N+K191S, D189N+K191 T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T, K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+L219S, K217N+L219T, K218N+L220S, K218N+L220T, L220N+R222S, L220N+R222T, V243N+V245S, V243N+V245T, V245N+P247S, V245N+P247T, S250N, S250N+S252T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, T254N+N256S, T254N+N256T, D255N+D257S, D255N+D257T, L296N, L296N+T298S, Y302N, Y302N+S304T, H303N, H303N+S305T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, T315N+V317S, T315N+V317T, F316N+L318S, F316N+L318T, V334N, V334N+S336T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, 1348N+G350S, 1348N+G350T, L349N+D351S, L349N+D351T, D351N+Q353S, D351N+Q353T, R352N+D354S, R352N+D354T, E357N+D359S, E357N+D359T, G383N+G385S, G383N+G385T, L386N+H388S, L386N+H388T, L387N+N389S, L387N+N389T, H388N+Y390S and H388N+Y390T.

More preferably, the introduced in vivo N-glycosylation site is selected from the group consisting of S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D189N+K191S, D189N+K191T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S305N+E307S, S305N+E307T, E307N+E309S, E307N+E309T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T.

Even more preferably the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S305N+E307T, S336N+M338T, V339T, M338N, G383N+G385T, and most preferably the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191T, K191N+K193T, D214N, T253N+D255T, S305N+E307T, S336N+M338T, M338N, G383N+G385T and L386N+H388T. In a particular preferred embodiment the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191T, D214N and L386+H388T.

As explained above, the increased resistance towards inactivation by alpha-1-antitrypsin and/or human plasma may be determined and assessed by the "Alpha-1-Antritrypsin Inactivation Assay", the "Human Plasma Inactivation Assay I" or the "Human Plasma Inactivation Assay II" disclosed herein.

The conjugate of the invention may contain a single in vivo glycosylation site (in addition to the already present glycosylation sites at positions 97, 248, 313 and 329). However, in order to obtain efficient shielding of protease cleavage sites on the surface of the parent polypeptide and/or to efficiently impair inhibitor binding, it is often desirable that the polypeptide part of the conjugate comprises more than one in vivo glycosylation site, in particular 2 5 (additional) in vivo glycosylation sites, such as 2, 3, 4 or 5 (additional) in vivo glycosylation sites, preferably introduced by one or more of the substitutions described in any of the above lists.

Furthermore, the amino acid sequence of a polypeptide having at least one of the above mentioned in vivo N-glycosylation site modifications may differ from that of the parent polypeptide in that at least one cysteine residue has been introduced as identified above in the section entitled "Conjugate of the invention having a non-polypeptide moiety attached to a cysteine residue", or at least one non-cysteine residue has been introduced as identified above in the section entitled "Conjugate of the invention having a non-polypeptide moiety attaching to a non-cysteine residue".

Moreover, the polypeptide part of the conjugate of the invention may contain additional mutations, which are known to be advantageous. For example, in addition to the glycosylation sites discussed above, the polypeptide part of the conjugate may contain a substitution in a position selected from the group consisting of L194, A195, L228, Y249 and combinations thereof, in particular L194S, L194S+T245S and L194A+T254S (see WO 00/66754). Other examples of preferred additional substitutions include substitution or introduction of one or more glycosylation site(s) at or near positions known to be susceptible to proteolytic degradation. One position that is known to be susceptible to proteolytic degradation is H10 of wild-type human APC (see WO 98/48822).

It will be understood that in order to prepare a conjugate according to this aspect of the invention, the polypeptide must be expressed in a glycosylating host cell capable of attaching sugar moieties at the glycosylation sites or alternatively subjected to in vitro glycosylation. Examples of glycosylating host cells are given in the section below entitled "Coupling to a sugar moiety".

Conjugate of the Invention wherein the Non-Polypeptide Moiety is Attached to a Cysteine Residue

In another embodiment of the invention, the present invention relates to a conjugate comprising at least one non-polypeptide moiety, in particular a polymer molecule, covalently attached to a protein C polypeptide that comprises an amino acid sequence which differs from a parent protein C polypeptide, in particular from the amino acid sequence shown in SEQ ID NO:4 or a variant thereof, in at least one cysteine residue has been introduced and/or removed, in particular introduced. Thus, in an interesting embodiment of the invention the non-polypeptide moiety has cysteine as an attachment group. Preferably, the cysteine attachment group is introduced in a position which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface, such as at least 50% of its side chain exposed to the surface. Such amino acid residues are identified in Example 1 herein. Of particular interest among these positions are positions that in the parent polypeptide are occupied by a T or an S residue, preferably an S residue. In accordance herewith, an interesting cysteine-modified conjugate is one, wherein a cysteine residue has been introduced into at least one position selected from the group consisting of S3, S11, S12, T37, S42, S61, T68, S75, S77, S82, S99, S119, S153, S190, S216, S252, T253, T268, S270, S281, S304, S305, T315, S332, S336, S340, S367, and S416, and more preferably from the group consisting of S3, S11, S12, S42, S61, S75, S77, S82, S99, S119, S153, S190, S216, S252, S270, S281, S304, S305, S332, S336, S340, S367, and S416.

In a similar way as described above (see the section entitled "Conjugate of the invention where the non-polypeptide moiety is a sugar moiety" the cysteine residue is preferably introduced in a position which is within the active site region (defined in Example 2 herein) and which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface (defined in Example 3 herein), i.e. the cysteine residue is preferably introduced in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, V339, M338, I348, L349, D351, R352, E357, G383, E385, L386, L387 and H388. More preferably, the cysteine residue is introduced in a positions selected from the group consisting of D189, S190, K191, D214, K217, K251, S252, T253, Y302, S305, E307, S336, V339, M338, G383 and L386.

The polypeptide part of the conjugate according to this embodiment typically comprises 1 10 introduced cysteine residues, in particular 1 5 or 1 3 introduced cysteine residues, e.g. 1, 2 or 3 introduced cysteine residues.

While the non-polypeptide moiety of the conjugate according to this aspect of the invention may be any molecule which, when using the given conjugation method has a cysteine residue as an attachment group (such as an polymer moiety, a lipophilic group or an organic derivatizing agent), it is preferred that the non-polypeptide moiety is a polymer molecule, e.g. any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule". Preferably, the polymer molecule is selected from the group consisting of linear or branched polyethylene glycol or polyalkylene oxide. Most preferably, the polymer molecule is PEG, such as VS-PEG. The conjugation between the polypeptide and the polymer may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. by using a one step method or by the stepwise manner referred to in said section. When the polypeptide comprises only one conjugatable cysteine residue, this is preferably conjugated to a first non-polypeptide moiety with a molecular weight of at least about 10 kDa or at least about 15 kDa, such as a molecular weight of about 12 kDa, about 15 kDa or about 20 kDa, either directly conjugated or indirectly through a low molecular weight polymer (as disclosed in WO 99/55377). When the conjugate comprises two or more first non-polypeptide moieties, normally each of these has a molecular weight of about 5 kDa, about 10 kDa or about 12 kDa.

The conjugate according to this embodiment may comprise at least one second non-polypeptide moiety, such as 1 10, 1 8, 1 5 or 1 3 such moieties. When the first non-polypeptide moiety is a polyalkylene oxide or PEG derived polymer, the second non-polypeptide moiety is preferably a sugar moiety, in particular an in vivo attached moiety. The sugar moiety may be present at one or more of the naturally-occurring glycosylation sites present in the parent polypeptide, or at an introduced glycosylation site. Suitable introduced glycosylation sites, in particular N-glycosylation sites, are described in the section entitled "Conjugate of the invention wherein the non-polypeptide moiety is a sugar moiety".

Moreover, the polypeptide part of the conjugate of the invention may contain additional mutations, which are known to be advantageous. For example, in addition to the introduced cysteine residues discussed above, the polypeptide part of the conjugate may contain a substitution in a position selected from the group consisting of L194, A195, L228, Y249 and combinations thereof, in particular. L194S, L194S+T245S and L194A+T254S (see WO 00/66754). Other examples of preferred additional substitutions include substitution or introduction of one or more cysteine residue(s) at or near positions known to be susceptible to proteolytic degradation. One position that is known to be susceptible to proteolytic degradation is H10 of wild-type human APC (see WO 98/48822).

Conjugate of the Invention wherein the Non-Polypeptide Moiety is Attached to a Non-Cysteine Moiety

Based on the present disclosure the skilled person will be aware that amino acid residues comprising other attachment groups may be introduced by substitution into the parent polypeptide, using the same approach as that illustrated above with glycosylation sites and cysteine residues. For instance, one or more amino acid residues comprising an acid group (glutamic acid or aspartic acid), tyrosine, serine or lysine may be introduced into the positions discussed above (see the sections entitled "Conjugate of the invention where the non-polypeptide moiety is a sugar moiety" and "Conjugate of the invention wherein the non-polypeptide moiety is attached to a cysteine residue").

Conjugate of the Invention having a Reduced Anticoagulant Activity

Studies have been shown that APC interacts with factor Va and VIIIa through the EGF domains collectively with the protease domain (Zhang et al., Biochemistry 1994; 33; 823 831). This protein-protein interaction is important for the anticoagulant activity because it promotes the contact between APC and these two cofactors. On the other hand, the interaction between APC and EPCR is predominantly determined by the binding of the Gla domain of APC to EPCR (Esmon et al., Haematologica 1999; 84; 363 368). It is also known that the proteolytic activity of APC is important for the anti-inflammatory activity. Therefore, mutations introduced in the EGF domains might only affect the anticoagulant activity of APC without influencing the anti-inflammatory activity. In addition, it is known that serpins binds and inactivates various proteases by binding directly into the catalytic active site. Thus, the inactivation process of APC in circulation by the serpins will not be influenced by these EGF domain mutations and will therefore not affect the plasma half-life of APC.

Thus, in a further aspect the present invention relates to novel variants of parent protein C conjugates, wherein at least one attachment group for a non-polypeptide amino acid residue has been introduced in the EGF-1 and/or the EGF-2 domain and wherein such variants have a decreased anticoagulant activity as compared to human APC and wherein the anti-inflammatory properties have not been substantially changed as compared to human APC.

More particularly, the present invention relates to a conjugate comprising at least one sugar moiety covalently attached to a protein C polypeptide that comprises an amino acid sequence which differs from that of a parent protein C polypeptide in at least one in vivo glycosylation site has been introduced by a substitution selected from the group consisting of H66N, H66N+T68S, 173N, 173N+S75T, S75N, S75N+S77T, D79N+R81S, D79N+R81T, E92N, E92N+S94T, G104N, G104N+T106S, R117N, R117N+S119T, D128N+L130S and D128N+L130T, preferably from the group consisting of H66N, I73N+S75T, S75N+S77T, D79N+R81T, E92N, G104N, R117N+S119T and D128N+L130T.

As explained above such conjugates are believed to exhibit a significantly reduced anticoagulant activity while essentially maintaining the anti-inflammatory effect of human APC.

Thus, in a preferred embodiment, the present invention relates to a conjugate comprising at least one sugar moiety covalently attached to a protein C polypeptide that comprises an amino acid sequence which differs from that of a parent protein C polypeptide in at least one in vivo glycosylation site has been introduced by a substitution selected from the group consisting of H66N, H66N+T68S, 173N, 173N+S75T, S75N, S75N+S77T, D79N+R81S, D79N+R81T, E92N, E92N+S94T, G104N, G104N+T106S, R117N, R117N+S119T, D128N+L130S and D128N+L130T, preferably from the group consisting of H66N, 173N+S75T, S75N+S77T, D79N+R81T, E92N, G104N, R117N+S119T and D128N+L130T, wherein i) the conjugate, in its activated form, has an anticoagulant activity of 0 50% of the human APC anticoagulant activity when tested in the "APC Clotting Assay" described in Example 10 herein, and ii) the ratio between the IC.sub.50-value for the variant (IC.sub.50,variant), in its activated form, and the IC.sub.50-value for the wild-type human APC (IC.sub.50,wt) is less than or equal to 1.20, when determined in accordance with the "APC Anti-inflammatory Assay" described in Example 14 herein.

In interesting embodiments of the invention the conjugate, in its activated form, has an anticoagulant activity of 10 50% of the human APC anticoagulant activity when tested in the "APC Clotting Assay" described in Example 10 herein, such as an anticoagulant activity of 10 40% of the human APC anticoagulant activity, e.g. 10 30% of the human APC anticoagulant activity.

It is preferred that the anti-inflammatory effect of the conjugate according to this aspect of the invention is retained. As indicated above, the anti-inflammatory effect of the conjugate should not be less than 80% of the anti-inflammatory effect of the human APC (expressed as IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.20). Preferably, the anti-inflammatory effect of the conjugate, in its activated form is essentially the same as the anti-inflammatory effect of human APC when determined in the in accordance with the "APC Anti-inflammatory Assay" described in Example 14 herein. For example, it is preferred that the ratio between the IC.sub.50-value for the variant (IC.sub.50,variant), in its activated form, and the IC.sub.50-value for the wild-type human APC (IC.sub.50,wt) is within the following ranges: 0.80.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.20, preferably 0.80.ltoreq.IC.sub.50,variant/IC.sub.50,wt .ltoreq.1.10, e.g. 0.90.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.10, or 0.95.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.05, in particular IC.sub.50,variant/IC.sub.50,wt.apprxeq.1.

It will be understood that the above-mentioned variants may be combined with the modifications described elsewhere in the present application. In particular, the above-mentioned substitutions may be combined with the modifications described herein which give rise to an increased in vivo half-life.

Thus, an interesting conjugate according to the present invention is a conjugate comprising at least one sugar moiety covalently attached to a protein C polypeptide that comprises an amino acid sequence which differs from that of a parent protein C polypeptide in at least two substitutions have been performed, the first substitution being selected from the group consisting of H66N, H66N+T68S, 173N, I73N+S75T, S75N, S75N+S77T, D79N+R81S, D79N+R81T, E92N, E92N+S94T, G104N, G104N+T106S, R117N, R117N+S119T, D128N+L130S and D128N+L130T, preferably from the group consisting of H66N, 173N+S75T, S75N+S77T, D79N+R81T, E92N, G104N, R117N+S119T and D128N+L130T, the second substitution being selected from the group consisting of D172N+K174S, D172N+K174T, D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T, K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+L219S, K217N+L219T, K218N+L220S, K218N+L220T, L220N+R222S, L220N+R222T, V243N+V245S, V243N+V245T, V245N+P247S, V245N+P247T, S250N, S250N+S252T, K251N, K251N+T253S, K251D, K251E, S252N, S252N+T254S, T253N+D255S, T253N+D255T, T254N+N256S, T254N+N256T, D255N+D257S, D255N+D257T, L296N, L296N+T298S, Y302N, Y302N+S304T, H303N, H303N+S305T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, T315N+V317S, T315N+V317T, F316N+L318S, F316N+L318T, V334N, V334N+S336T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, 1348N+G350S, 1348N+G350T, L349N+D351S, L349N+D351T, D351N+Q353S, D351N+Q353T, R352N+D354S, R352N+D354T, E357N+D359S, E357N+D359T, G383N+G385S, G383N+G385T, L386N+H388S, L386N+H388T, L387N+N389S, L387N+N389T, H388N+Y390S and H388N+Y390T, preferably from the group consisting of S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D189N+K191S, D189N+K191T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, K251D, K251E, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S305N+E307S, S305N+E307T, E307N+E309S, E307N+E309T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T, more preferably from the group consisting of D189N+K191T, K191N+K193T, D214N, K251N, K251D, S252N, T253N+D255T, Y302N, S305N+E307T, S336N+M338T, V339T, M338N, G383N+G385T, even more preferably from the group consisting of D189N+K191T, K191N+K193T, D214N, K251D, T253N+D255T, S305N+E307T, S336N+M338T, M338N, G383N+G385T and L386N+H388T, most preferably from the group consisting of D189N+K191T, D214N. K251D and L386+H388T.

It will be understood that such conjugates, in their activated forms, are contemplated to exhibit an increased in vivo half-life, a reduced anticoagulant activity and an essentially unaltered anti-inflammatory effect when assayed in accordance with the test methods described herein.

In particular, it is preferred that such conjugates, as described immediately above, fulfill the below requirements, namely that i) the conjugate, in its activated form, has an anticoagulant activity of 0 50% of the human APC anticoagulant activity when tested in the "APC Clotting Assay" described in Example 10 herein, and ii) the ratio between the IC.sub.50-value for the variant (IC.sub.50,variant), in its activated form, and the IC.sub.50-value for the wild-type human APC (IC.sub.50,wt) is less than or equal to 1.20, when determined in accordance with the "APC Anti-inflammatory Assay" described in Example 14 herein, and iii) the conjugate, in its activated form, has a residual activity of at least 20% when tested in the "Alpha-1-Antitrypsin Inactivation Assay" described in Example 11 herein using an inhibitor concentration of 16.6 .mu.M, or the conjugate, in its activated form and when tested in the "Human Plasma Inactivation Assay I" described in Example 12 herein, has a residual activity of at least 20%, or the ratio between the in vitro half-life of said conjugate, in its activated form, and the in vitro half-life of human APC is at least 1.25 when tested in the "Human Plasma Inactivation Assay II" described in Example 13 herein, or the ratio between the functional in vivo half-life or the serum half-life of said conjugate, in its activated form, and the functional in vivo half-life or serum half-life of human APC is at least 1.25.

In an interesting embodiment such a conjugate, in its activated form, has an anticoagulant activity of 10 50% of the human APC anticoagulant activity when tested in the "APC Clotting Assay" described in Example 10 herein, such as an anticoagulant activity of 10 40% of the human APC anticoagulant activity, e.g. 10 30% of the human APC anticoagulant activity.

It is preferred that the anti-inflammatory effect of the conjugate according to this aspect of the invention is retained. As indicated above, the anti-inflammatory effect of the conjugate should not be less than 80% of the anti-inflammatory effect of the human APC (expressed as IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.20). Preferably, the anti-inflammatory effect of the conjugate, in its activated form is essentially the same as the anti-inflammatory effect of human APC when determined in the in accordance with the "APC Anti-inflammatory Assay" described in Example 14 herein. For example, it is preferred that the ratio between the IC.sub.50-value for the variant (IC.sub.50,variant), in its activated form, and the IC.sub.50-value for the wild-type human APC (IC.sub.50,wt) is within the following ranges: 0.80.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.20, preferably 0.80.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.10, e.g. 0.90.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.10, or 0.95.ltoreq.IC.sub.50,variant/IC.sub.50,wt.ltoreq.1.05, in particular IC.sub.50,variant/IC.sub.50,wt.apprxeq.1.

Moreover, it is preferred that the conjugate fulfills the above-mentioned criteria for inactivation and/or half-life in the "Alpha-1-Antitrypsin Inactivation Assay", "Human Plasma Inactivation Assay I", "Human Plasma Inactivation Assay II", the functional in vivo half-life or the serum half-life, at the levels mentioned in the section entitled "definitions".

Polypeptide Variants of the Invention

In a further aspect the present invention relates to generally novel variants of parent protein C polypeptides. The novel variants are important intermediate compounds for the preparation of conjugates of the invention. In addition, and as will be apparent from the below disclosure and from the examples provided herein, the variants themselves have interesting properties.

Thus, in its broadest aspect the present invention relates to novel variants of a parent protein C polypeptide, where the variants constitute the polypeptide part, more particularly the APC part, of the conjugates of the invention. As will be evident from the examples provided herein, it has been found that some variants, wherein one or more glycosylation sites were introduced, but not utilized, has interesting properties, in particular with respect to increased resistance towards inhibition by alpha-1-antitrypsin and increased resistance towards inactivation by human plasma. These variant comprises at least one substitution in the active site region (as defined in Example 2 herein), in particular they comprise a substitution of an amino acid residue, which is located in the active site region and which has at least 25% of its side chain exposed to the surface (as defined in Example 3 herein). Thus, preferred variants according to this aspect of the invention comprises a substitution in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387 and H388, with the proviso that the substitution is not selected from the group consisting of T254S, T254A, T254H, T254K, T254R, T254N, T254D, T254E, T254G, T254Q, Y302S, Y302A, Y302T, Y302H, Y302K, Y302R, Y302N, Y302D, Y302E, Y302G, Y302Q, F316S, F316A, F316T, F316H, F316K, F316R, F316N, F316D, F316E, F316G and F316Q.

As is evident from the above list of positions, which are located in the active site region and, at the same time, has at least 25% of its side chain exposed to the surface, a significant amount of the these positions are occupied by charged amino acid residues. Analysing the three-dimensional structure of protein C, in particular the above-identified region, it can be observed that at least some of the charged residues interact with each other. For example, K251 is believed to form a salt bridge to D214. Moreover, it can be seen that a cluster of negatively charged amino acid residues (D214, E215 and E357) is present. Without being bound by any particular theory it is contemplated that the charged amino acid residues within the above-identified region, or at least some of the charged amino acid residues within this particular region, are important for capturing and/or binding the substrate/inhibitor. Therefore, amino acid substitutions which are particular interesting according to this aspect of the present invention are constituted by such an amino acid substitutions, wherein a charged amino acid residue, which is located in the active site region and, at the same time, has at least 25% of its side chain exposed to the surface, is substituted with an amino acid residue having no charge, in particular an amino acid residue having no charge but a polar side chain (Gly, Ser, Thr, Cys, Tyr, Asn or Gln), as well as amino acid substitutions, wherein a charged amino acid residue, which is located in the active site region and, at the same time, has at least 25% of its side chain exposed to the surface, is substituted with an amino acid residue having an opposite charge.

Specific examples of amino acid substitutions, wherein the charge of the amino acid residue in question is changed to an opposite charge, include D172K, D172R, D189K, D189R, K191D, K191E, K192D, K192E, K193D, K193E, D214K, D214R, E215K, E215R, K217D, K217E, K218D, K218E, K251 D, K251E, D255K, D255R, R306D, R306E, E307K, E307R, K308D, K308E, E309K, E309R, R312D, R312E, D351K, D351R, R352D, R352E, E357K, E357R, E382K and E382R, such as D214K, D214R, E215K, E215R, K251D, K251E, E357K and E357R, e.g. D214K, D214R, K251D and K251E, in particular K251D.

Other specific examples of amino acid substitutions, wherein the charged amino acid residue in question is substituted with an amino acid side chain having a polar side chain, include D172G/S/T/C/Y/N/Q, D189G/S/T/C/Y/N/Q, K191G/S/T/C/Y/N/Q, K192G/S/T/C/Y/N/Q, K193G/S/T/C/Y/N/Q, D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K217G/S/T/C/Y/N/Q, K218G/S/T/C/Y/N/Q, K251G/S/T/C/Y/N/Q, D255G/S/T/C/Y/N/Q, R306G/S/T/C/Y/N/Q, E307G/S/T/C/Y/N/Q, K308G/S/T/C/Y/N/Q, E309G/S/T/C/Y/N/Q, R312G/S/T/C/Y/N/Q, D351G/S/T/C/Y/N/Q, R352G/S/T/C/Y/N/Q, E357G/S/T/C/Y/N/Q and E382G/S/T/C/Y/N/Q, such as D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K251G/S/T/C/Y/N/Q and E357G/S/T/C/Y/N/Q, e.g. D214Q, E215Q, K251Q and E357Q, in particular K251Q. Another interesting substitution may be K251N+T253A.

Further specific examples of interesting substitutions include the substitutions disclosed in the sections entitled "Conjugate of the invention where the non-polypeptide moiety is a sugar moiety" and "Conjugate of the invention wherein the non-polypeptide moiety is attached to a cysteine residue", in particular the substitutions selected from the group consisting of K251N, S252N, Y302N and S190+K192T, especially K251N and S252N, most preferably K251N.

As will be understood, details and particulars concerning the conjugates of the invention (e.g. activation of protein C, number of substitutions, formulation of conjugates, indications for which the conjugates may be used, increased resistance towards inactivation by alpha-1-antitrypsin and human plasma, etc.) will be the same or analogous to the variant aspect of the invention, whenever appropriate. Thus, statements and details concerning the conjugates of the invention will apply mutatis mutandis to the protein C variants disclosed herein, whenever appropriate.

Non-Polypeptide Moiety of the Conjugate of the Invention

As indicated further above the non-polypeptide moiety of the conjugate of the invention is preferably selected from the group consisting of a polymer molecule, a lipophilic compound, a sugar moiety (by way of in vivo glycosylation) and an organic derivatizing agent. All of these agents may confer desirable properties to the polypeptide part of the conjugate, in particular increased functional in vivo half-life and/or increased plasma half-life. The polypeptide part of the conjugate is normally conjugated to only one type of non-polypeptide moiety, but may also be conjugated to two or more different types of non-polypeptide moieties, e.g. to a polymer molecule and a sugar moiety, to a lipophilic group and a sugar moiety, to an organic derivatizing agent and a sugar moiety, to a lipophilic group and a polymer molecule, etc. The conjugation to two or more different non-polypeptide moieties may be done simultaneous or sequentially.

Methods of Preparing a Conjugate of the Invention

In the following sections "Conjugation to a lipophilic compound", "Conjugation to a polymer molecule", "Conjugation to a sugar moiety" and "Conjugation to an organic derivatizing agent" conjugation to specific types of non-polypeptide moieties is described. In general, a polypeptide conjugate according to the invention may be produced by culturing an appropriate host cell under conditions conducive for the expression of the polypeptide, and recovering the polypeptide, wherein a) the polypeptide comprises at least one N- or O-glycosylation site and the host cell is an eukaryotic host cell capable of in vivo glycosylation, and/or b) the polypeptide is subjected to conjugation to a non-polypeptide moiety in vitro.

It will be understood that the conjugation should be designed so as to produce the optimal molecule with respect to the number of non-polypeptide moieties attached, the size and form of such molecules (e.g. whether they are linear or branched), and the attachment site(s) in the polypeptide. The molecular weight of the non-polypeptide moiety to be used may e.g. be chosen on the basis of the desired effect to be achieved. For instance, if the primary purpose of the conjugation is to achieve a conjugate having a high molecular weight (e.g. to reduce renal clearance) it is usually desirable to conjugate as few high molecular weight non-polypeptide moieties as possible to obtain the desired molecular weight. When a high degree of shielding is desirable this may be obtained by use of a sufficiently high number of low molecular weight non-polypeptide moieties (e.g. with a molecular weight of from about 300 Da to about 5 kDa, such as a molecular weight of from 300 Da to 2 kDa).

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the polypeptide may be any suitable polymer molecule, such as a natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300 100,000 Da, such as about 500 20,000 Da, more preferably in the range of about 500 15,000 Da, even more preferably in the range of about 2 12 kDa, such as in the range of about 3 10 kDa. When the term "about" is used herein in connection with a certain molecular weight, the word "about" indicates an approximate average molecular weight and reflects the fact that there will normally be a certain molecular weight distribution in a given polymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH.sub.2) and a polycarboxylic acid (i.e. poly-COOH). A hetero-polymer is a polymer comprising different coupling groups, such as a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, dextran, including carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vivo half-life and/or serum half-life. Another example of a polymer molecule is human albumin or another abundant plasma protein. Generally, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have various water solubility properties, and are easily excreted from living organisms.

PEG is the preferred polymer molecule, since it has only few reactive groups capable of cross-linking compared to, e.g., polysaccharides such as dextran. In particular, monofunctional PEG, e.g. methoxypolyethylene glycol (mPEG), is of interest since its coupling chemistry is relatively simple (only one reactive group is available for conjugating with attachment groups on the polypeptide). Consequently, the risk of cross-linking is eliminated, the resulting polypeptide conjugates are more homogeneous and the reaction of the polymer molecules with the polypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl-carbonate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e.g. from Shearwater Polymers, Inc., Huntsville, Ala., USA, or from PolyMASC Phammaceuticals plc, UK.

Alternatively, the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference).

Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG and MAL-PEG, including the mPEG forms thereof, and branched PEGs such as PEG2-NHS, including the mPEG forms thereof, and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat. No. 5,643,575, both of which are incorporated herein by reference. Furthermore, the following publications, incorporated herein by reference, disclose useful polymer molecules and/or PEGylation chemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO 97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No. 5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No. 5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO 96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503 and EP 154 316.

The conjugation of the polypeptide and the activated polymer molecules is conducted by use of any conventional method, e.g. as described in the following references (which also describe suitable methods for activation of polymer molecules): R. F. Taylor, (1991), "Protein immobilisation. Fundamental and applications", Marcel Dekker, N. Y.; S. S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Florida, USA; G. T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, N.Y.). The skilled person will be aware that the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the polypeptide (examples of which are given further above), as well as the functional groups of the polymer (e.g. being amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide, vinysulfone or haloacetate). The PEGylation may be directed towards conjugation to all available attachment groups on the polypeptide (i.e. such attachment groups that are exposed at the surface of the polypeptide) or may be directed towards one or more specific attachment groups, e.g. the N-terminal amino group as described in U.S. Pat. No. 5,985,265. Furthermore, the conjugation may be achieved in one step or in a stepwise manner (e.g. as described in WO 99/55377).

It will be understood that the PEGylation is designed so as to produce the optimal molecule with respect to the number of PEG molecules attached, the size and form of such molecules (e.g. whether they are linear or branched), and the attachment site(s) in the polypeptide. The molecular weight of the polymer to be used may e.g. be chosen on the basis of the desired effect to be achieved.

In connection with conjugation to only a single attachment group on the protein (e.g. the N-terminal amino group), it may be advantageous that the polymer molecule, which may be linear or branched, has a high molecular weight, preferably about 10 25 kDa, such as about 15 25 kDa, e.g. about 20 kDa.

Normally, the polymer conjugation is performed under conditions aimed at reacting as many of the available polymer attachment groups with polymer molecules. This is achieved by means of a suitable molar excess of the polymer relative to the polypeptide. Typically, the molar ratios of activated polymer molecules to polypeptide are up to about 1000 1, such as up to about 200 1, or up to about 100 1. In some cases the ration may be somewhat lower, however, such as up to about 50 1, 10 1, 5 1, 2 1 or 1 1 in order to obtain optimal reaction.

It is also contemplated according to the invention to couple the polymer molecules to the polypeptide through a linker. Suitable linkers are well known to the skilled person. A preferred example is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578 3581; U.S. Pat. No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375 378).

Subsequent to the conjugation, residual activated polymer molecules are blocked according to methods known in the art, e.g. by addition of primary amine to the reaction mixture, and the resulting inactivated polymer molecules are removed by a suitable method.

It will be understood that depending on the circumstances, e.g. the amino acid sequence of the polypeptide, the nature of the activated PEG compound being used and the specific PEGylation conditions, including the molar ratio of PEG to polypeptide, varying degrees of PEGylation may be obtained, with a higher degree of PEGylation generally being obtained with a higher ratio of PEG to polypeptide. The PEGylated polypeptides resulting from any given PEGylation process will, however, normally comprise a stochastic distribution of polypeptide conjugates having slightly different degrees of PEGylation.

Coupling to a Sugar Moiety

In order to achieve in vivo glycosylation of a protein C molecule comprising one or more glycosylation sites the nucleotide sequence encoding the polypeptide must be inserted in a glycosylating, eucaryotic expression host. The expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells. In one embodiment the host cell is a mammalian cell, such as a COS cell, a CHO cell, a BHK cell or a HEK cell, e.g. a HEK 293 cell, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. S. cerevisiae or Pichia pastoris, or any of the host cells mentioned hereinafter.

Covalent in vitro coupling of sugar moieties (such as dextran) to amino acid residues of the polypeptide may also be used, e.g. as described, for example in WO 87/05330 and in Aplin et al., CRC Crit Rev. Biochem, pp. 259 306, 1981. The in vitro coupling of sugar moieties or PEG to protein- and peptide-bound Gin-residues can be carried out by transglutaminases (TGases). Transglutaminases catalyse the transfer of donor amine-groups to protein- and peptide-bound Gln-residues in a so-called cross-linking reaction. The donor-amine groups can be protein- or peptide-bound, such as the .epsilon.-amino-group in Lys-residues or it can be part of a small or large organic molecule. An example of a small organic molecule functioning as amino-donor in TGase-catalysed cross-linking is putrescine (1,4-diaminobutane). An example of a larger organic molecule functioning as amino-donor in TGase-catalysed cross-linking is an amine-containing PEG (Sato et al., 1996, Biochemistry 35, 13072 13080).

TGases, in general, are highly specific enzymes, and not every Gln-residues exposed on the surface of a protein is accessible to TGase-catalysed cross-linking to amino-containing substances. On the contrary, only few Gin-residues are naturally functioning as TGase substrates but the exact parameters governing which Gin-residues are good TGase substrates remain unknown. Thus, in order to render a protein susceptible to TGase-catalysed cross-linking reactions it is often a prerequisite at convenient positions to add stretches of amino acid sequence known to function very well as TGase substrates. Several amino acid sequences are known to be or to contain excellent natural TGase substrates e.g. substance P, elafin, fibrinogen, fibronectin, .alpha..sub.2-plasmin inhibitor, .alpha.-caseins, and .beta.-caseins.

Conjugation to an Organic Derivatizing Agent

Covalent modification of the polypeptide may be performed by reacting one or more attachment groups of the polypeptide with an organic derivatizing agent. Suitable derivatizing agents and methods are well known in the art. For example, cysteinyl residues most commonly are reacted with .alpha.-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo-.beta.-(4-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5 7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful. The reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing .alpha.-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione and transaminase-catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group.

Furthermore, these reagents may react with the groups of lysine as well as the arginine guanidino group. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R--N.dbd.C.dbd.N--R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Conjugation to a Lipophilic Compound

The polypeptide and the lipophilic compound may be conjugated to each other, either directly or by use of a linker. The lipophilic compound may be a natural compound such as a saturated or unsaturated fatty acid, a fatty acid diketone, a terpene, a prostaglandin, a vitamine, a carotenoide or steroide, or a synthetic compound such as a carbon acid, an alcohol, an amine and sulphonic acid with one or more alkyl-, aryl-, alkenyl- or other multiple unsaturated compounds. The conjugation between the polypeptide and the lipophilic compound, optionally through a linker may be done according to methods known in the art, e.g. as described by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976 and in WO 96/12505.

Conjugation of a Tagged Polypeptide

The polypeptide may be expressed as a fusion protein with a tag, i.e. an amino acid sequence or peptide stretch made up of typically 1 30, such as 1 20 amino acid residues. Besides allowing for fast and easy purification, the tag is a convenient tool for achieving conjugation between the tagged polypeptide and the non-polypeptide moiety. In particular, the tag may be used for achieving conjugation in microtiter plates or other carriers, such as paramagnetic beads, to which the tagged polypeptide can be immobilised via the tag. The conjugation to the tagged polypeptide in, e.g., microtiter plates has the advantage that the tagged polypeptide can be immobilised in the microtiter plates directly from the culture broth (in principle without any purification) and subjected to conjugation. Thereby, the total number of process steps (from expression to conjugation) can be reduced. Furthermore, the tag may function as a spacer molecule, ensuring an improved accessibility to the immobilised polypeptide to be conjugated. The conjugation using a tagged polypeptide may be to any of the non-polypeptide moieties disclosed herein, e.g. to a polymer molecule such as PEG.

The identity of the specific tag to be used is not critical as long as the tag is capable of being expressed with the polypeptide and is capable of being immobilised on a suitable surface or carrier material. A number of suitable tags are commercially available, e.g. from Unizyme Laboratories, Denmark. For instance, the tag may consist of any of the following sequences -- see Original Patent.

Antibodies against the above tags are commercially available, e.g. from ADI, Aves Lab and Research Diagnostics.

The subsequent cleavage of the tag from the polypeptide may be achieved by use of commercially available enzymes.

Methods of Preparing a Polypeptide Variant of the Invention or the Polypeptide Part of the Conjugate of the Invention

The polypeptide variant of the present invention or the polypeptide part of a conjugate of the invention, optionally in glycosylated form, may be produced by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding the polypeptide and expressing the sequence in a suitable transformed or transfected host. Preferably, the host cell is a gammacarboxylating host cell such as a mammalian cell. However, polypeptides of the invention may be produced, albeit less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.

A nucleotide sequence encoding a polypeptide variant or the polypeptide part of a conjugate of the invention may be constructed by isolating or synthesizing a nucleotide sequence encoding the parent protein C, such as protein C with the amino acid sequence shown in SEQ ID NO:2 and 4 and then changing the nucleotide sequence so as to effect introduction (i.e. insertion or substitution) or removal (i.e. deletion or substitution) of the relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with conventional methods. Alternatively, the nucleotide sequence is prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and assembled by PCR, ligation or ligation chain reaction (LCR) (Barany, PNAS 88:189 193, 1991). The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

Alternative nucleotide sequence modification methods are available for producing polypeptide variants for high throughput screening, for instance methods which involve homologous cross-over such as disclosed in U.S. Pat. No. 5,093,257, and methods which involve gene shuffling, i.e. recombination between two or more homologous nucleotide sequences resulting in new nucleotide sequences having a number of nucleotide alterations when compared to the starting nucleotide sequences. Gene shuffling (also known as DNA shuffling) involves one or more cycles of random fragmentation and reassembly of the nucleotide sequences, followed by screening to select nucleotide sequences encoding polypeptides with desired properties. In order for homology-based nucleic acid shuffling to take place, the relevant parts of the nucleotide sequences are preferably at least 50% identical, such as at least 60% identical, more preferably at least 70% identical, such as at least 80% identical. The recombination can be performed in vitro or in vivo.

Examples of suitable in vitro gene shuffling methods are disclosed by Stemmer et al. (1994), Proc. Natl. Acad. Sci. USA; vol. 91, pp. 10747 10751; Stemmer (1994), Nature, vol. 370, pp. 389 391; Smith (1994), Nature vol. 370, pp. 324 325; Zhao et al., Nat. Biotechnol. 1998, March; 16(3): 258 61; Zhao H. and Arnold, F B, Nucleic Acids Research, 1997, Vol. 25. No. 6 pp. 1307 1308; Shao et al., Nucleic Acids Research 1998, Jan. 15; 26(2): pp. 681 83; and WO 95/17413.

An example of a suitable in vivo shuffling method is disclosed in WO 97/07205. Other techniques for mutagenesis of nucleic acid sequences by in vitro or in vivo recombination are disclosed e.g. in WO 97/20078 and U.S. Pat. No. 5,837,458. Examples of specific shuffling techniques include "family shuffling", "synthetic shuffling" and "in silico shuffling".

Family shuffling involves subjecting a family of homologous genes from different species to one or more cycles of shuffling and subsequent screening or selection. Family shuffling techniques are disclosed e.g. by Crameri et al. (1998), Nature, vol. 391, pp. 288 291; Christians et al. (1999), Nature Biotechnology, vol. 17, pp. 259 264; Chang et al. (1999), Nature Biotechnology, vol. 17, pp. 793 797; and Ness et al. (1999), Nature Biotechnology, vol. 17, 893 896.

Synthetic shuffling involves providing libraries of overlapping synthetic oligonucleotides based e.g. on a sequence alignment of homologous genes of interest. The synthetically generated oligonucleotides are recombined, and the resulting recombinant nucleic acid sequences are screened and if desired used for further shuffling cycles. Synthetic shuffling techniques are disclosed in WO 00/42561.

In silico shuffling refers to a DNA shuffling procedure, which is performed or modelled using a computer system, thereby partly or entirely avoiding the need for physically manipulating nucleic acids. Techniques for in silico shuffling are disclosed in WO 00/42560. Once assembled (by synthesis, site-directed mutagenesis or another method), the nucleotide sequence encoding the polypeptide is inserted into a recombinant vector and operably linked to control sequences necessary for expression of protein C in the desired transformed host cell.

It should of course be understood that not all vectors and expression control sequences function equally well to express the nucleotide sequence encoding a polypeptide described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it or be able to integrate into the chromosome. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleotide sequence encoding the polypeptide, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleotide sequence, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the nucleotide sequence.

The recombinant vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector is one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector, in which the nucleotide sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the nucleotide sequence. The vector is typically derived from plasmid or viral DNA. A number of suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectors are, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) and pCl-neo (Stratagene, La Jola, Calif., USA). Useful expression vectors for yeast cells include the 2.mu. plasmid and derivatives thereof, the POT1 vector (U.S. Pat. No. 4,931,373), the pJSO37 vector described in Okkels, Ann. New York Acad. Sci. 782, 202 207, 1996, and pPICZ A, B or C (Invitrogen). Useful vectors for insect cells include pVL941, pBG311 (Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance And Expression of the Human Gene In Animal Cells", Cell, 45, pp. 685 98 (1986), pBluebac 4.5 and pMelbac (both available from Invitrogen). Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pBR322, pET3a and pET12a (both from Novagen Inc., Wis., USA), wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages.

Other vectors for use in this invention include those that allow the nucleotide sequence encoding the polypeptide to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol., 2, pp. 1304 19 (1982)) and glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication. When the host cell is a yeast cell, suitable sequences enabling the vector to replicate are the yeast plasmid 2.mu. replication genes REP 1 3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125 130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For Saccharomyces cerevisiae, selectable markers include ura3 and leu2. For filamentous fungi, selectable markers include amdS, pyrG, arcB, niaD and sC.

The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of the polypeptide of the invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter.

A wide variety of expression control sequences may be used in the present invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate-early gene promoter (CMV), the human elongation factor 1.alpha. (EF-1.alpha.) promoter, the Drosophila minimal heat shock protein 70 promoter, the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, SV40 or adenovirus E1b region polyadenylation signals and the Kozak consensus sequence (Kozak, M. J Mol Biol 1987 Aug. 20;196(4):947 50).

In order to improve expression in mammalian cells a synthetic intron may be inserted in the 5' untranslated region of the nucleotide sequence encoding the polypeptide. An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (available from Promega Corporation, Wis., USA).

Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the P10 promoter, the Autographa californica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter, and the SV40 polyadenylation sequence. Examples of suitable control sequences for use in yeast host cells include the promoters of the yeast .alpha.-mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydrogenase genes, the ADH2 4c promoter, and the inducible GAL promoter. Examples of suitable control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, an A. niger .alpha.-amylase, A. niger or A. nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3 terminator. Examples of suitable control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system, and the major promoter regions of phage lambda.

The presence or absence of a signal peptide will, e.g., depend on the expression host cell used for the production of the polypeptide to be expressed (whether it is an intracellular or extracellular polypeptide) and whether it is desirable to obtain secretion. For use in filamentous fungi, the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase. The signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral .alpha.-amylase, A. niger acid-stable amylase, or A. niger glucoamylase. For use in insect cells, the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the Lepidopteran manduca sexta adipokinetic hormone precursor, (cf. U.S. Pat. No. 5,023,328), the honeybee melittin (Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al., Protein Expression and Purification 4, 349 357 (1993) or human pancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262 272,1997). A preferred signal peptide for use in mammalian cells is that of hFVII or the murine Ig kappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods 152:89 104). For use in yeast cells suitable signal peptides have been found to be the .alpha.-factor signal peptide from S. cereviciae (cf. U.S. Pat. No. 4,870,008), a modified carboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887 897), the yeast BAR1 signal peptide (cf. WO 87/02670), the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127 137), and the synthetic leader sequence TA57 (WO98/32867). For use in E. coli cells a suitable signal peptide have been found to be the signal peptide ompA (EP581821).

The nucleotide sequence of the invention encoding a protein C polypeptide variant, whether prepared by site-directed mutagenesis, synthesis, PCR or other methods, may optionally include a nucleotide sequence that encode a signal peptide. The signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide. The signal peptide may be homologous (e.g. be that normally associated with human protein C) or heterologous (i.e. originating from another source than human protein C) to the polypeptide or may be homologous or heterologous to the host cell, i.e. be a signal peptide normally expressed from the host cell or one which is not normally expressed from the host cell. Accordingly, the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.

Any suitable host may be used to produce the polypeptide or polypeptide part of the conjugate of the invention, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. Examples of bacterial host cells include grampositive bacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gramnegative bacteria, such as strains of E. coli. The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111 115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823 829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209 221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742 751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771 5278). Examples of suitable filamentous fungal host cells include strains of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and U.S. Pat. No. 5,679,543. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147 156 and WO 96/00787. Examples of suitable yeast host cells include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. Polymorpha or Yarrowia. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182 187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920: and as disclosed by Clontech Laboratories, Inc, Palo Alto, Calif., USA (in the product protocol for the Yeastmaker.TM. Yeast Transformation System Kit). Examples of suitable insect host cells include a Lepidoptora cell line, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (U.S. Pat. No. 5,077,214). Transformation of insect cells and production of heterologous polypeptides therein may be performed as described by Invitrogen. Examples of suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/0), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. Also, the mammalian cell, such as a CHO cell, may be modified to express sialyltransferase, e.g. 1,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335, in order to provide improved glycosylation of the protein C polypeptide.

In order to increase secretion it may be of particular interest to produce the polypeptide of the invention together with an endoprotease, in particular a PACE (Paired basic amino acid converting enzyme) (e.g. as described in U.S. Pat. No. 5,986,079), such as a Kex2 endoprotease (e.g. as described in WO 00/28065).

Methods for introducing exogeneous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000. These methods are well known in the art and e.g. described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells are conducted according to established methods, e.g. as disclosed in (Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, N.J., USA and Harrison M A and Rae I F, General Techniques of Cell Culture, Cambridge University Press 1997).

In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, ultra-filtration, extraction or precipitation.

The polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation) or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Pharmaceutical Compositions and Use

In a further aspect, the present invention relates to a pharmaceutical composition comprising a conjugate of the invention or a variant of the invention and a pharmaceutically acceptable carrier or excipient. In the present context, the term "Pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the patients to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

In a still further aspect, the present invention relates to a conjugate of the invention, a variant of the invention or a pharmaceutical composition of the invention for use as a medicament. More particularly, the conjugates, variants or pharmaceutical compositions of the invention may be used for the manufacture of a medicament for the treatment of stroke; myocardial infarction; after venous thrombosis; disseminated intravascular coagulation (DIC); sepsis; septic shock; emboli, such as pulmonary emboli; transplantation, such as bone marrow transplantation; burns; pregnancy; major surgery/traum or adult respiratory stress syndrome (ARDS), in particular for the treatment of septic shock.

The present invention also relates to a method for treating or preventing a disease selected from the group consisting of stroke; myocardial infarction; after venous thrombosis; disseminated intravascular coagulation (DIC); sepsis; septic shock; emboli, such as pulmonary emboli; transplantation, such as bone marrow transplantation; burns; pregnancy; major surgery/traum and adult respiratory stress syndrome (ARDS), the method comprising administering to a patient in need thereof an effective amount of a conjugate of the invention, of a variant according to the invention, or of a pharmaceutical composition according to the invention, in particular for treating or preventing, especially treating, septic shock.

A "patient" for the purposes of the present invention includes both humans and other mammals. Thus the methods are applicable to both human therapy and veterinary applications.

The polypeptide variants and conjugates of the invention will be administered to patients in an effective dose. By "effective dose" herein is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose will depend on the disorder to be treated, and will be ascertainable by one skilled in the art using known techniques. As mentioned above, in the treatment of severe sepsis 24 .mu.g/kg/h of human APC is administered for 96 hours, which corresponds to a total amount of protein of about 230 mg for a patient having a body weight of about 100 kg. The conjugates and variants of the present invention are, due to their increased plasma half-lives, contemplated to have a higher efficacy due to the extended action-time in plasma. This increased efficacy may, for example, be estimated by calculating the area under the curve (AUC) in the "Human Plasma Inactivation assay II" or by measuring the serum half-life. The increased efficacy means that the effective dose needed to obtain the desired effect for a particular disorder will be smaller (less protein need to be administered) than the effective dose of human APC. In addition, the increased plasma half-life will also allow treatment where the APC variants or conjugates are used regularly with a given time-period. Thus, these new properties will permit the use of a reduced amount and/or and less frequent administration, such as bolus injections, of the compounds of the invention. For example, the compounds of the invention may be administered by a either a bolus or infusion or as a combination thereof with doses which range from 1 .mu.g/kg body weight as a bolus every 2.sup.nd hour for several days (e.g. for 96 hours) to 1 mg/kg body weight as a bolus once every 4.sup.th day. Preferably, as low a dose as possible is administered as less frequent as possible, e.g. 1 500 .mu.g/kg body weight, preferably 1 250 .mu.g/kg body weight, such as 1 100 .mu.g/kg body weight, more preferably 1 50 .mu.g/kg body weight is administered as a bolus every 4 96 hour, e.g. every 8 96 hour, such as every 16 96, every 24 96 hour, every 40 96 hour, every 48 96 hour, every 56 96 hour, every 72 96 hour.

Compounds of the invention, which are preferred are such compounds where the ratio between the AUC of said compound, in its activated form, and the AUC of human APC is at least 1.25 when tested in the "Human Plasma Inactivation Assay II" described in Example 13 herein. Preferably, the ratio is at least 1.5, such as at least 2, e.g. at least 3, more preferably the ratio is at least 4, such as at least 5, e.g. at least 6, even more preferably the ratio is at least 7, such as at least 8, e.g. at least 9, most preferably the ratio is at least 10.

The polypeptide variant or conjugate of the invention can be used "as is" and/or in a salt form thereof. Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.

The pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the polypeptide or conjugate of the invention, either concurrently or in accordance with another treatment schedule. In addition, the polypeptide, conjugate or pharmaceutical composition of the invention may be used as an adjuvant to other therapies.

The pharmaceutical composition of the invention may be formulated in a variety of forms, e.g. as a liquid, gel, lyophilized, or as a compressed solid. The preferred form will depend upon the particular indication being treated and will be readily able to be determined by one skilled in the art.

The administration of the formulations of the present invention can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implantation. In some instances the formulations may be directly applied as a solution or spray.

Parenteral Compositions

An example of a pharmaceutical composition is a solution designed for parenteral administration. Although in many cases pharmaceutical solution formulations are provided in liquid form, appropriate for immediate use, such parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris. Preservatives are added to retard microbial growth, and are typically added in amounts of e.g. about 0.1% 2% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, .alpha.-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as "wetting agents") may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the polypeptide. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic.RTM. polyols, polyoxyethylene sorbitan monoethers (Tween.RTM.-20, Tween.RTM.-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

Sustained Release Preparations

Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the polypeptide or conjugate, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the ProLease.RTM. technology or Lupron Depot.RTM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37.degree. C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

 

Claim 1 of 16 Claims

1. A variant of a parent human protein C polypeptide, the variant comprising a sequence which (a) differs from the parent human protein C polypeptide sequence SEQ ID NO:4 in 1 to 15 amino acid residues, and (b) wherein the Asp residue at position 214 is substituted by an amino acid residue having a polar side chain or by an amino acid residue having an opposite charge to Asp, wherein the variant in activated form exhibits an amidolytic activity.

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