WO2002081520A2

WO2002081520A2 - Single chain dimeric polypeptides derived from the vegf family

Info

Publication number: WO2002081520A2
Application number: PCT/DK2002/000233
Authority: WO
Inventors: Thomas Peter Boesen; Torben Halkier
Original assignee: Maxygen Holdings Ltd.
Priority date: 2001-04-06
Filing date: 2002-04-08
Publication date: 2002-10-17
Also published as: AU2002338313A1; WO2002081520A3

Abstract

The invention relates to a single-chain dimeric polypeptide which binds to an extracellular ligand-binding domain of a VEGF type 2 receptor (KDR) or a VEGF type 3 receptor (Flt-4), the polypeptide comprising two receptor-binding sites of which one is capable of binding to a ligand-binding domain of the receptor and one is incapable of effectively binding to a ligand-binding domain of the receptor, and wherein at least one monomer of the dimeric polypeptide is derived from VEGF, VEGF-C or VEGF-D, whereby the single-chain dimeric polypeptide is capable of binding to the receptor, but incapable of activating the receptor. The polypeptide functions as a receptor antagonist for prevention or treatment of a disease or condition involving increased signal transduction from or increased activation of the KDR and/or Flt-4 receptor, e.g. to inhibit angiogenesis or lymphangiogenesis.

Description

SINGLE CHAIN DIMERIC POLYPEPTIDES

FIELD OF THE INVENTION

The present invention relates to single chain dimeric receptor antagonist polypeptides comprising at least one monomer derived from the VEGF family, in particular at least one monomer derived from VEGF, VEGF-C or VEGF-D. The polypeptides are able to bind to a VEGF type 2 or 3 receptor but are not able to activate such receptors.

BACKGROUND OF THE INVENTION

Angiogenesis

Angiogenesis is the sprouting of blood capillaries from pre-existing blood vessels, while vasculogenesis is the de novo development of blood vessels through differentiation of early endothelial cells during embryonic development.

VEGF is a mitogen that is highly specific for vascular endothelial cells (Dvorak et al. (1995), Am. J. Pathol. 146, 1029-1039). VEGF is a potent angiogenic/vasculogenic factor involved in the development of the vascular system and in the differentiation of endothelial cells as shown by the lethality of targeted disruption of even one allele of the VEGF gene (Carmeliet et al. (1996), Nature 380, 435-439; Ferrara et al. (1996), Nature 380, 439-442). VEGF is sometimes also called vascular permeability factor (VPF) or Vasculotropin.

Angiogenesis/vaculogenesis is, however, not only important in the physiological processes of embryogenesis and wound healing. It is also involved in pathological processes such as tumour growth, metastasis, diabetic retinopathy and rheumatoid arthritis. It is for instance well established that tumour microvessel density and vascular permeability influence the prognosis in various forms of cancer with a good correlation between vascularisation, metastasis, malignancy and survival rates.

An important role for VEGF as a mediator of tumour angiogenesis is suggested by a number of observations. High levels of VEGF are produced by various types of tumours with the result that capillaries are clustered along VEGF-producing tumour cells, and it has been found that VEGF expression/overexpression correlates well with the induction of neo ascularisation in tumours in a number of different cancers and in many cases also with a poor prognosis. It has also been shown that tumour angiogenesis and subsequent tumour growth are inhibited in vivo when VEGF signaling is inhibited by various means. Thus, there is a large body of evidence showing that neutralising the action of VEGF results in an inhibition of tumour angiogenesis and an inhibition of tumour growth.

The VEGF molecule

At present, five different human VEGF mRNA species have been identified coding for VEGF isoforms containing 121, 145, 165, 189, and 206 amino acid residues, respectively (Leung et al. (1989), Science 246, 1306-1309; Keck et al. (1989), Science 246, 1309-1312; Tischer et al., (1991), J. Biol. Chem. 266, 11947-11954; Houck et al. (1991), Mol. Endocrinol. 5, 1806-1814; Poltorak et al. (1997), J. Biol. Chem. 272, 7151-7158). VEGF₁₆₅ is the most abundantly expressed isoform followed by VEGF₁₂₁ as the second most abundantly expressed isoform. VEGF₁₂₁ may be considered a functional fragment of any of the other isoforms and the four first mentioned isoforms may be considered functional fragments of the VEGF₂₀₆ isoform. The five VEGF mRNA species are most likely produced by alternative splicing of exons 1-5, 6a, 6b, 7 and 8. All five isoforms share a common N-terminal region of 115 amino acid residues (encoded by exons 1-5) and the six C-terminal amino acid residues encoded by exon 8. The three longest isoforms share the same 50 C-terminal residues.

All the VEGF isoforms are bioactive. Thus, not unexpectedly, it is the common N- terminal region of 115 amino acid residues shared by all five isoforms that contains the structural information required for recognition by and binding to the two VEGF receptors (see below) (Keyt et al. (1996), J. Biol. Chem. 271, 7788-7795). This has been clearly shown as 110 amino acid residue N-terminal fragments of VEGFι₆₅ and VEGF₁₈ , generated through cleavage by the protease plasmin, have been shown to be endothelial cell mitogens, and to bind to both VEGF receptors (Keyt et al., supra; Houck et al. (1992), J. Biol. Chem. 267, 26031-26037). The five isoforms differ with respect to binding to heparin, heparan sulfate, and the extracellular matrix (ECM). In summary, the gene for VEGF contains coding sequences for domains/regions that confer receptor-binding, heparin/ECM-binding, and heparin-binding. Differential use of this genetic information results in five VEGF isoforms with different binding capabilities for heparin and the ECM and in consequence with different bioavailability and therefore different bioactivity. VEGF has been purified from a variety of species as a disulfide-bonded apparently homodimeric protein with a relative molecular weight around 45 kDa as estimated from SDS- PAGE. In accordance with this the monomer (which is biologically inactive) has an estimated molecular weight of 23 kDa. The amino acid sequence of human VEGF contains one potential N-glycosylation site at Asn75, and studies have shown that glycosylated and non-glycosylated VEGF have the same biological activity.

The disulfide bonding of the cysteine residues in VEGF₁₆₅ has been deduced from X- ray crystallography for the N-terminal receptor-binding site (Muller et al. (1997), Proc. Natl. Acad. Sci. USA 94, 7192-7197) and from N-terminal amino acid sequencing of tryptic fragments of the C-terminal heparin-binding domain (Keck et al. (1997), Arch. Biochem. Biophys. 344, 103-113). In the receptor-binding site, the three intra-chain disulfide-bonds Cys26-Cys68, Cys57-Cysl02, and Cys61-Cysl04 form a so-called cystine knot motive (see below). Cys51 and Cys60 are engaged in the two inter-chain disulfide bonds holding the two anti-parallel monomers covalently together. Thus, Cys51 in one monomer forms a disulfide bond with Cys60 in the other monomer and vice versa. In the heparin-binding domain of VEGF₁₆₅ the four disulfide bonds are Cysll7-Cysl35, Cysl20-Cysl37, Cysl39-Cysl58, and Cysl46-Cysl60. Based on the amino acid sequence homology to PDGF, VEGF was included in the superfamily of cystine knot growth factors (Sun et al. (1995), Annu. Rev. Biophys. Biomol. Struct. 24, 269-291). This has further been confirmed by the three-dimensional structure of the receptor-binding site of VEGF. The topology of the VEGF monomer is similar to that observed in PDGF (Oefher et al. (1992), EMBO. J. 11, 3921-3926), although upon alignment of the two amino acid sequences only 19% of the positions are occupied by identical amino acid residues.

The VEGF monomer contains a total of seven β strand segments (βl to β7) and two α-helical segments (αl and α2). The most prominent and central feature in the structure of the VEGF monomer is a central highly irregular antiparallel four-stranded β sheet comprising strands βl, β3, β5, and β6. This four-stranded β sheet displays the characteristic cystine knot at one end. The cystine knot consists of two disulfide bonds forming a covalently linked ring structure between two adjacent β strands (β3 and β7) together with a third disulfide bond penetrating this ring and connecting the beginning of two other β strands (βl and β4).

VEGF dimerizes in an antiparallel side-by-side fashion and the monomers are, as already mentioned, covalently linked by two disulfide bonds. Structural elements at the opposite end of the monomer from the cystine knot are involved in formation of a hydrophobic core across the monomer-monomer subunit interface. The amino acid residues involved in this hydrophobic core are derived from the loop connecting strands βl and β3, the end of strand β5, the beginning of strand β6, and the loop connecting strands β5 and β6, all from one monomer in combination with the N-terminal α-helix (αl) of the other monomer. As will be described below, this hydrophobic core is important in receptor binding.

The VEGF receptors As mentioned above, VEGF is presumably a vascular endothelial cell-specific growth factor as it appears to be inactive on fibroblasts, keratinocytes, vascular smooth muscle cells, lens epithelial cells, corneal endothelial cells, adrenal cortical cells, and granulosa cells (Ferrara et al. (1989), Biochem. Biophys. Res. Commun. 161, 851-858; Gospodarowicz et al. (1989), Proc. Natl. Acad. Sci. USA 86, 7311-7315). VEGF exerts its effects on vascular endothelial cells through at least two receptors known as Flt-1 (fins-like tyrosine kinase 1, also known as VEGF receptor 1) and KDR (kinase domain receptor or kinase-insert domain-containing receptor, also known as VEGF receptor 2). Both receptors are tyrosine kinases.

The amino acid sequences of human Flt-1 (Shibuya et al. (1990), Oncogene 5, 519- 524) as well as of human KDR (Terman et al. (1992), Biochem Biophys. Res. Commun. 187, 1579-1586; Terman et al. (1991), Oncogene 6, 1677-1683) are known and show that both proteins comprising more than 1300 amino acid residues are composed of 7 extracellular immunoglobulin-like (Ig-like) domains containing the ligand-binding domains, a transmembrane domain and an intracellular tyrosine kinase domain. Thus, the two VEGF receptors are homologous. Overall, identical amino acid residues occupy approximately 40% of the homologous positions, but this percentage is much higher in large parts of the tyrosine kinase domain. A number of studies imply that KDR but not Flt-1 plays the important role in VEGF-induced mitogenesis.

The three-dimensional structure of Ig-like domain 2 from Flt-1 has been determined in complex with the receptor-binding domain of VEGF (residues 8-109) (Wiesmann et al.

(1997), Cell 91, 695-704) giving information about the receptor-ligand interactions. The three dimensional structure of a variant of the kinase domain from KDR has also recently been solved (McTigue et al. (1999), Structure Fold. Des. 15, 319-330). Further, an additional VEGF receptor has been identified on endothelial cells and various tumour cells (Gitay-Goren et al. (1992), J. Biol. Chem. 267, 6093-6098; Gitay-Goren et al. (1993), Biochem. Biophys. Res. Commun. 190, 702-708; Gitay-Goren et al. (1996), J. Biol. Chem. 271, 5519-5523; Soker et al. (1996), J. Biol. Chem. 271, 5761-5767; Omura et al. (1997), J. Biol. Chem. 272, 23317-23322). Interestingly, this receptor binds VEGF₁₆₅ but not VEGF , and apparently it does so through the exon 7-encoded domain as this is the only difference between the two proteins (Soker et al. (1996), supra; Soker et al. (1997), J. Biol. Chem. 272, 31582-31588). This VEGF receptor has through purification and expression cloning from a tumour cell line been identified as neuroρilin-1 (Soker et al. (1998), Cell 92, 735-745). Neuroρilin-1 is expected to be a co-receptor for VEGF₁₆₅, which is supported by the observation that KDR binds VEGF₁₆₅ more efficiently in cells expressing neuropilin-1 than in cells not expressing neuropilin-1 (Soker et al. (1998), supra).

The VEGF: VEGF receptor interaction

The interaction between the receptor-binding site of VEGF and the two VEGF receptors Flt-1 and KDR has been studied in a variety of ways, giving information on different levels.

Many studies have had the aim of identifying the VEGF-binding parts of the VEGF receptors. Especially the VEGF-binding domain of Flt-1 has been investigated, and although minor differences exist among the studies it may be concluded that Ig-like domain 2 from Flt- 1 is necessary for binding of VEGF although not sufficient for VEGF-binding with wild type Flt-1 affinity (Wiesmann et al. (1997), supra; Davis-Smyth et al. (1996), EMBO J. 15, 4919- 4927; Cunningham et al. (1997), Biochem. Biophys. Res. Commun. 231, 596-599; Barleon et al. (1997), J. Biol. Chem. 272, 10382-10388; Tanaka et al. (1997), Jpn. J. Cancer Res. 88, 867-876; Herley et al. (1999), Biochem. Biophys. Res. Commun. 262, 731-738). The general observation is that Ig-like domains 1-3 bind VEGF with wild type Flt-1 affinity.

For KDR, Ig-like domains 2-3 are sufficient for VEGF-binding with wild type KDR affinity (Fuh et al. (1998), J. Biol. Chem. 273, 11197-11204).

It is interesting to note, however, that the monomer/dimer status of the VEGF receptors have different significance for the affinity for VEGF of the two receptors. In many of the studies aimed at clarifying the minimal domain requirements for VEGF-binding by the receptors, the receptor constructs were expressed as fusion proteins between the receptor-Ig- like-domains in question and parts of the heavy chain from IgG. Through the IgG-part these constructs dimerise and the binding constants determined are thus the binding constants for the interaction between dimeric VEGF and these predimerised VEGF receptor constructs. In some studies monomeric VEGF receptor constructs have also been made (Fuh et al. (1998), supra; Wiesmann et al. (1997), supra) and here a striking difference between the two VEGF receptors was found. The difference in VEGF-binding affinity for monomeric and predimerised Flt-1 constructs were found to be minimal (approximately 2-fold weaker VEGF- binding by monomeric than by predimerised Flt-1 constructs) (Wiesmann et al. (1997), supra). For the KDR constructs a different situation was found as monomeric KDR constructs have a 100-fold weaker VEGF-binding than predimerised KDR constructs (Fuh et al. (1998), supra). The most detailed information about the interactions between VEGF and a VEGF receptor has been obtained from the three dimensional structure of the complex between the receptor-binding domain of VEGF and the Ig-like domain 2 from Flt-1 (Wiesmann et al. (1997), supra). From the structure it can be seen that the receptor-binding sites in VEGF are at the poles of the dimer and formed at the interface between the VEGF monomers. The contact surface in the VEGF dimer is divided about 65%/35% between the two monomers. In one monomer, the contact surface involves amino acid residues 16-27 (the N-terminal helix), 61-66 (the loop between strand β3 and β4), and 103-106 (strand β7). In the second monomer, the contact surface involves amino acid residues 46-48 (strand β2) and 79-91 (strand β5-loop- strand β6). In Flt-1 Ig-like domain 2 the contact surface is comprised by amino acid residues from the N-terminal bulge, strand βa', part of strands βg and βf, the loop connecting strands βc and βc', and the helical turn connecting strands βe and βf.

Alanine-scanning mutagenesis has also been attempted previously as a means for elucidating the receptor-binding site in VEGF (Muller et al. (1997), supra; Keyt et al. (1996), J. Biol. Chem. 271, 5638-5646). In the first study, one site in VEGF of major importance for KDR-binding was found to be defined by Arg82, Lys84 and His86, while a site of major importance for Flt-1-binding was found to be defined by Asp63, Glu64 and Glu67. Interestingly, the site of major importance for binding to one receptor was found to be of minor (but detectable) importance for the binding to the other receptor (Keyt et al. (1996), supra). Along the same lines it was observed that introduction of an N-glycosylation site at position 82 in VEGF through the substitutions Arg82Asn, Ile83Leu, Lys84Ser influenced the binding to KDR significantly but not the binding to Flt-1. More elaborate alanine-scanning mutagenesis of VEGF using phage display led to the proposal that the KDR-binding site was defined by amino acid residues Phel7, Ile43, Ile46, Glu64, Gln79, Ile83, lys84 and Pro85 (Muller et al. (1997), supra).

Comparing the results of the alarύne-scanning mutagenesis and the conclusions based hereupon with the knowledge obtained from the three dimensional structure of the complex between the receptor-binding domain of VEGF and Ig-like domain 2 from Flt-1, they are not completely in agreement. Of the VEGF amino acid residues that were found to be of moderate to great importance for tight binding to KDR, 5 are buried in the interface with Flt-1 Ig-like domain 2 in the complex, suggesting that the binding sites for KDR and Flt-1 are very similar (Wiesmann et al. (1997), supra). This is not the interpretation based upon the alanine- scanning mutagenesis data alone, as these suggested separate binding sites for Flt-1 and KDR on VEGF. The reason for this discrepancy is not presently known. Nevertheless, it is contemplated that the binding sites for Flt-1 and KDR are indeed somewhat overlapping, and that both of the above techniques may provide valuable information for producing VEGF variants.

Lymphangiogenesis and VEGF homologs

Lymphangiogenesis, formation of new lymphatic vessels from existing vessels, is a process similar to angiogenesis. Lymphangiogenesis has had less focus than angiogenesis in relation to cancer therapy, but many results indicate that it is involved in tumor growth and/or metastasis and that inhibition of lymphangiogenesis may be beneficial to cancer patients, see e.g. (Fielder, W. et al. (1997), Leukemia, 11, 1234-1237; Valtola, R. et al. (1999), Am. J. Pathol., 154, 1381-1390; Tsurusaki, T. et al. (1999), Br. J. Cancer, 80, 309-313; Yonemura, Y. et al. (1999), Clin. Cancer Res., 5, 1823-1829; Ohta, Y. et al. (1999), Br. J. Cancer, 81, 54- 61; Skobe, M. et al. (1999), J. Invest Dermatol., 113, 1047-1053; Bunone, G. et al. (1999), Am. J. Pathol., 155, 1967-1976; Fellmer, P. T. et al. (1999), Surgery, 126, 1056-1061). Two growth factors related to VEGF, namely VEGF-C ( Joukov, V. et al. (1996)

EMBO J., 15, 290-298; Joukov, V. et al. (1996), EMBO J., 15, 1751) and VEGF-D (Yamada, Y. et al. (1997), Genomics, 42, 483-488; Achen, M. G. et al. (1998), Proc. Natl. Acad. Sci. U. S. A, 95, 548-553), stimulate lymphangiogenesis by activating a tyrosine kinase receptor, Flt-4 (Kukk, E. et al. (1996), Development, 122, 3829-3837; Jeltsch, M. et al. (1997), Science, 276, 1423-1425; Oh, S. J. et al. (1997), Dev. Biol., 188, 96-109). Both VEGF-C and VEGF-D also activate KDR, and angiogenic activity of VEGF-C, at least during embryonic development, has also been reported (Lymboussaki, A. et al. (1999), Circ. Res., 85, 992-999; Lymboussaki, A. et al. (1998), Am. J. Pathol., 153, 395-403; Witzenbichler, B. et al. (1998), Am. J. Pathol., 153, 381-394; Yonekura, H. et al. (1999), J. Biol. Chem., 274, 35172-35178). Thus, a large body of evidence indicates that inhibition of lymphangiogenesis may be useful as treatment for certain cancers and that this in some cases may be achieved by inhibiting the activity of Flt-4. BRIEF DISCLOSURE OF THE INVENTION

It has now been found that it is possible to modify polypeptide ligands that bind to the VEGF type 2 and 3 receptors in a way that does not destroy the ability of the ligand to bind to the receptor, but destroys the ability of the ligand to activate the receptor. Since these receptors belong to the class of oligomeric cellular receptors that depend on oligomerisation and/or conformational changes to be activated, binding of the single chain polypeptides of the invention without activation of the receptors allows the single chain polypeptides to function as effective antagonists.

Accordingly, in one aspect, the invention relates to a single-chain dimeric polypeptide which binds to an extracellular ligand-binding domain of a VEGF type 2 receptor (KDR) or a VEGF type 3 receptor (Flt-4), the polypeptide comprising two receptor-binding sites of which one is capable of binding to a ligand-binding domain of the receptor and one is incapable of effectively binding to a ligand-binding domain of the receptor, and wherein at least one monomer of the dimeric polypeptide is derived from VEGF, VEGF-C or VEGF-D, whereby the single-chain dimeric polypeptide is capable of binding to the receptor, but incapable of activating the receptor.

Other aspects of the invention relate to a nucleotide sequence encoding such a single- chain dimeric polypeptide, an expression vector comprising such a nucleotide sequence, a recombinant host cell comprising the nucleotide sequence or expression vector, and methods for producing such nucleotide sequences and single-chain dimeric polypeptides. Further aspects of the invention relate to compositions comprising the single-chain dimeric polypeptides as well as use of the single-chain dimeric polypeptides for the preparation of medicaments and for the prevention or treatment of diseases or conditions in which reduced signal transduction from a VEGF type 2 or type 3 receptor is desired.

BRIEF DESCRIPTION OF THE SEQUENCES AND DRAWINGS

SEQ ID NO:l is the amino acid sequence of VEGF₁₂₁, the monomer used in single- chain VEGF.

SEQ ID NO:2 is the amino acid sequence of a proposed single-chain VEGF-C monomer comprising the VEGF homology domain of VEGF-C.

SEQ ID NO:3 is the amino acid sequence of a proposed single-chain VEGF-D monomer comprising the VEGF homology domain of VEGF-D. SEQ ID NO:4 is the DNA sequence encoding single-chain VEGF, α-factor prepro peptide, peptide linker and flanking restriction sites for cloning.

SEQ ID NO:5 is the DNA sequence encoding single-chain VEGF-C, α-factor prepro peptide, peptide linker and flanking restriction sites for cloning. SEQ ID NO:6 is the amino acid sequence of the unprocessed precursor of human

VEGF-C (from SWISS-PROT).

SEQ ID NO: 7 is the amino acid sequence of the unprocessed precursor of human VEGF-D (from SWISS-PROT).

Figure 1 compares HUVEC proliferation activity of VEGF₁₂₁ and single-chain VEGF Figure 2 shows the results of agonist assays of VEGF₁₂₁ and double-Arg variants

Figure 3 shows the results of antagonist assays of double-Arg variants

Figure 4 shows the result of a BIAcore assay on single-chain VEGF and antagonist.

DETAILED DISCLOSURE OF THE INVENTION In the present context, the term "polypeptide" is understood to indicate a mature protein or a precursor form thereof as well as a functional fragment thereof which essentially has retained the ability of the mature protein to bind the VEGF type 2 or type 3 receptor. A functional fragment may for instance be an N- and/or C-terminal truncated form of a full- length polypeptide, or an isoform, in particular a native isoform, of a full-length polypeptide. The polypeptides of the invention are derived from or otherwise made so as to mimic the structure and function of the parent polypeptides, which in their native form are dimers, i.e. they are composed of two monomeric subunits which are connected by disulfide bonds. The term "derived" is intended to indicate that the monomeric polypeptide subunit is prepared to mimic structural and/or functional properties of the corresponding native or parent polypeptide in question. As used herein, the term "derived" is intended to encompass polypeptides which have an altered amino acid sequence compared to the relevant native polypeptide as well as, in certain cases, polypeptides wherein one or possibly both monomers have the same amino acid sequence as the respective native monomeric polypeptide(s). For example, the single chain polypeptide of the invention may comprise a monomer having the native sequence of VEGF-C together with a monomer having one or more amino acid alterations compared to the native VEGF-C. Both monomers of such a single chain polypeptide are considered to be "derived from" native VEGF-C as used herein, even though in such a case one of the monomers may in fact be identical in amino acid sequence to the native polypeptide.

As used herein, "VEGF" refers to proteins that are also known in the literature as "VEGF-A", i.e. the VEGF isoforms containing 121, 145, 165, 189 or 206 amino acid residues as described above, in contrast to "VEGF-C" and "VEGF-D". In the present specification, amino acid positions of VEGF are indicated with reference to the sequence of VEGF_{1 1} (SEQ ID NO:l). It will be clear that any of the known VEGF isoforms may be used in the context of the single-chain dimeric polypeptides of the invention, and that when other isoforms than VEGF₁₂₁ are used, the amino acid positions referred to herein should be understood as being the corresponding positions in the VEGF isoform in question (based on a sequence alignment).

It will further be understood that for any of the polypeptides referred to herein that may be used for preparing a single-chain dimeric polypeptide of the invention, it will be possible to perform other alterations than those specifically disclosed herein in connection with e.g. modification of a receptor-binding site. In particular, one or both of the monomers in a dimeric single-chain polypeptide may be altered, relative to the native or parent polypeptide, by e.g. truncation of the N-terminal and/or C-terminal, or by other deletions, insertions or substitutions.

Typically, the amino acid sequence of the "derived" polypeptide is at least 60% identical to that of said native polypeptide, normally at least 70% identical, such as at least 80% or even at least 90% or 95% identical. Also, the "derived" polypeptide may share a number of functional and/or structural properties with said native polypeptide, in particular one or more of the properties discussed herein, especially in terms of receptor-binding properties and/or oligomer association properties and/or conformation of a receptor-binding site and/or an association domain thereof. Typically, the monomeric polypeptide is encoded by the same nucleotide sequence as the corresponding native monomer or from a nucleotide sequence which is able to express a polypeptide with the same amino acid sequence as the corresponding native monomer, or from any such nucleotide sequence which has been modified so that the monomeric polypeptide is expressed with one or more desirable mutations, e.g. as described herein. Preferably, the monomeric polypeptides used in the present invention are of mammalian origin, in particular of human origin.

The term "parent polypeptide" is used about the usually native dimeric polypeptide, which in accordance with the present invention is provided in a modified single-chain form. The monomers may be identical (in which case the polypeptide is termed a "homomer") or different (in which case the polypeptide is termed a "heteromer"). According to the invention, the polypeptides are provided in single-chain form, which means that the monomers are linked by peptide bonds, optionally through a linker peptide, rather than being linked by non- covalent bonds or disulfide bonds. Accordingly, the single-chain polypeptides of the invention are expressed as one polypeptide from a single nucleotide sequence rather than being expressed as single monomer molecules which are assembled to a dimeric polypeptide only after expression. Each parent monomer may be a wild-type monomeric polypeptide or a variant thereof, for instance a mutein form of the wild-type monomeric polypeptide which has been prepared by substitution or deletion of one or more amino acid residues thereof and/or insertion of one or more additional amino acid residues therein.

In the present context, the term "dimeric polypeptide" is merely intended to indicate that the polypeptide is of a type which is dimeric in its native state since, strictly speaking, the single-chain form of the polypeptide cannot be said to be dimeric. The terms "dimer" and "dimeric" are used in the same manner. The term "signaling polypeptide" denotes a polypeptide that interacts with a cellular receptor so as to activate the receptor and thereby provide a signal initiating a signal transduction cascade in the cell carrying the receptor. Such a polypeptide is often also termed a ligand.

In the present context, an "antagonist" is a molecule which is capable of binding to a desired receptor but incapable of mediating correct conformational changes of the receptor molecules necessary to result in an activated complex, whereby ligand-mediated receptor activation is substantially inhibited. In order to efficiently inhibit receptor activation, the single chain polypeptide must be capable of binding to a ligand-binding domain of a receptor with a sufficiently high affinity to compete with the endogenous ligand.

One important advantage of the present invention is that by providing dimeric polypeptides comprising two receptor-binding sites in single-chain form expressed from one continuous nucleotide sequence it is possible to selectively modify one receptor-binding site and leave the other binding site intact through asymmetrical mutagenesis. Further, it is also possible in this manner to modify both binding sites in different ways, i.e. so that one of the binding sites is altered to provide it with increased binding affinity compared to the native dimeric polypeptide, while the other site is modified so as to render it incapable of effectively binding and activating the receptor. This is in contrast to the non-single-chain situation, wherein monomers are expressed individually from the same or different genes and subsequently assembled in the cell. For instance, in the case of a homodimer, modification of the gene encoding the monomer through mutagenesis will always be symmetrical. Accordingly, the production of an oligomeric non-single-chain polypeptide with one intact and one modified receptor-binding site requires that the monomers must be modified and produced separately before in vitro recombination. Following recombination, purification of the desired dimeric non-single-chain polypeptide has to be carried out in order to separate it from non-desired dimer. Thus, it is very laborious, if possible at all, to produce a preparation of dimeric non-single-chain polypeptides each of which has one intact and one modified receptor-binding site, since this requires that monomers having the appropriate receptor- binding sites be assembled correctly.

In addition, the single-chain form of the polypeptides more readily lends itself to production by recombinant DNA techniques in that the polypeptides may be expressed from a single gene rather than being assembled in the cell from two individual monomers (homomers) or, in the case of heteromers, expressed from two individual genes and assembled in the cell as is often the case with oligomeric polypeptides in nature. Single-chain polypeptides may have the added advantage of greater stability upon administration, for instance against degradation by proteolytic enzymes present in the body, e.g. in plasma, so that they may exhibit a longer half-life in vivo.

In the present context the term "active receptor-binding site" is intended to indicate a receptor-binding site which is capable of binding to a ligand-binding domain of the cellular receptor in question, i.e. in particular a VEGF type 2 or 3 cellular receptor. The active receptor-binding site has a sufficient affinity towards the ligand-binding domain of the receptor to effect binding between the receptor-binding site and the receptor and thereby to block the receptor from binding to, e.g., a native dimeric polypeptide (an endogenous ligand), thus preventing subsequent activation by the native polypeptide. This affinity is preferably the same as or higher than the affinity of the native polypeptide. The term "inactive receptor-binding site" is intended to indicate a receptor-binding site which renders the single-chain dimeric polypeptide incapable of activating the receptor. Usually, an inactive receptor-binding site is incapable of effectively binding to a ligand- binding domain of the cellular receptor in question. The term "incapable of effectively binding" is intended to indicate that the interaction between the binding site of the ligand and the binding site of the receptor is unable to mediate correct receptor oligomerisation or other conformational change required for activation and thus to trigger a signal transduction cascade within the cell. This can be due to an inability of the receptor-binding site to recognize and/or bind the ligand-binding site on the receptor or due to an imperfect binding, which does not permit correct interaction between the intracellular parts of the oligomerised receptor subunits. Accordingly, the inactive receptor-binding site may exhibit affinity towards the receptor, which however, under normal concentrations of receptor-binding site, is insignificant as a means of inducing receptor oligomerisation. Preferably, the inactive receptor-binding site has no affinity towards the receptor and is thus incapable of binding thereto. Although the term "receptor-binding site" is used in connection with the inactive receptor-binding site, strictly speaking, the inactive "receptor-binding site" may not be capable of binding to the receptor. Thus, in the case of an inactive receptor-binding site, the term "receptor-binding site" is merely used to reflect that said site is derived from an active receptor-binding site in accordance with the present invention, typically by modifying one or more amino acid residues thereof or by addition of a non-polypeptide moiety, said modification leading to the inactivation of the binding site.

The term "ligand-binding domain" refers to the part or parts of a cellular receptor which is/are involved in specific recognition of and interaction with a receptor-binding site of an endogenous ligand. Analogously, the "receptor-binding site" is understood as a number of amino acid residues in a polypeptide involved in binding to the ligand-binding domain of the receptor. Normally, the receptor-binding site comprises 1-50 amino acid residues, such as 5-30 or 10-25 amino acid residues. The amino acid residues in question may be located in sequence, but are more often placed in spatial proximity to each other as a result of the folding of the polypeptide. The receptor-binding site of interest for the present invention includes amino acid residues originating from both of the monomers of the dimeric polypeptide. More specifically, the receptor-binding sites may be located at interfaces between the monomeric constituents of the dimeric polypeptide.

As used herein, the terms "first monomer" and "second monomer" are used to differentiate between the two monomers of a dimeric polypeptide and, unless otherwise indicated or apparent from the context, are not intended to give the position of an individual monomer with respect to the N- and C-termini. Where a specific position is intended, reference will often be made to the N- or C-terminal monomer.

The two receptor-binding sites in the single-chain dimeric polypeptides of the invention may be "symmetrical" in the sense that amino acid residues of the first monomer taking part in formation of the first receptor-binding site are substantially identical to the amino acid residues of the second monomer taking part in formation of the second receptor- binding site. Alternatively, the receptor binding sites may be non-symmetrical.

As indicated above, the VEGF type 2 and 3 receptors comprise subunits of a type requiring oligomerisation or other conformational changes to be activated, (a process which is normally referred to as "receptor oligomerisation", "receptor clustering" or "receptor aggregation"). Binding of one of the native dimeric polypeptides results in a conformational change of the receptor which leads to an interaction between the effector domain and one or more intracellular molecules (termed "effectors" herein) to effect a physiological change in the cell. According to the invention, modifications affecting a receptor-binding site may be carried out within the receptor-binding site itself, i.e. in a manner involving at least one of the amino acid residues forming part of the receptor-binding site, or may be carried out outside the receptor-binding site, but in a region of the polypeptide where such modification influences the folding and consequently the three-dimensional structure of the receptor- binding site or otherwise blocks access to the receptor-binding site so that the binding affinity of ligand binding to the receptor is significantly reduced and is insufficient to effect receptor activation.

While the inactive receptor-binding site is different from that of a corresponding native binding site, the active receptor-binding site may be unmodified, i.e. be constituted by the amino acid residues which are also found in the corresponding native binding site, or may be modified, e.g. to have an increased affinity towards a ligand binding domain of the receptor. In a preferred embodiment, the active binding site has such an increased binding affinity.

In a preferred embodiment, the polypeptide of the invention thus comprises one receptor-binding site with at least one modification that results in increased receptor-binding activity (i.e. increased affinity) of the modified receptor-binding site compared to a corresponding polypeptide without said modification. In this case, the active receptor-binding site will have an increased binding affinity compared to the parent polypeptide, thereby allowing an improved binding of the polypeptide to the receptor, and thus an improvement in the effect obtained by the inactive binding site.

The different monomers of a heteromer may originate from the same parent polypeptide or from different polypeptides. Thus, although one monomer of the present single chain dimeric polypeptides will be derived from VEGF, VEGF-C or VEGF-D, preferably VEGF-C or VEGF-C, the second monomer may be derived from the same polypeptide as the first monomer, or it may be derived from a different polypeptide..The single chain dimeric polypeptide may thus comprise:

1. two monomers derived from VEGF-C,

2. two monomers derived from VEGF-D,

3. two monomers derived from VEGF, 4. one monomer derived from VEGF-C, VEGF-D or VEGF, and one monomer derived from a different polypeptide selected from VEGF-C, VEGF-D and VEGF, or

5. one monomer derived from VEGF-C, VEGF-D or VEGF, and one monomer that is not derived from any of VEGF-C, VEGF-D and VEGF. In the latter case, the other monomer may be derived from a polypeptide selected from the group consisting of VEGF-B, P1GF, PDGF-A, PDGF-B, PDGF-C, SCDGF-A, SCDGF-B and other members of the cystine-knot growth factor family. Preferred other monomers include those derived from P1GF or VEGF-B.

It is contemplated that in cases where one of the monomers of the single-chain dimeric polypeptide is derived from an isoform of VEGF, the other monomer may be derived from the same isoform of VEGF, a different isoform of VEGF, or a different member of the cystine- knot growth factor family. In the case of a single-chain polypeptide derived from e.g. VEGF₁₂₁ and VEGF₁₆₅, it is preferred that the monomer derived from VEGF₁₂₁ is at the N- terminal and the monomer derived from VEGFι₆₅ is at the C-terminal. In a particular embodiment, one monomer of the single-chain polypeptide is derived from a VEGF polypeptide and the other monomer is derived from a different polypeptide belonging to the cystine-knot growth factor family, preferably VEGF-B, VEGF-C, VEGF-D or P1GF, more preferably VEGF-B or P1GF.

When combining monomers derived from different polypeptides, it may be necessary to change amino acid residues of a monomer association domain required for assembly of the two monomers, e.g. as described in WO 96/40774. In the present context the term "association domain" is intended to indicate amino acid residues which line the contact points between monomers and which are essential for obtaining a proper assembly/conformation of an active single-chain dimeric polypeptide of the invention. Accordingly, the single-chain dimeric polypeptide of the invention may be a heterodimeric polypeptide wherein at least one of the monomers is modified in an association domain thereof so as to enable association of said monomer to the other monomer of the heterooligomeric polypeptide in order to obtain an active single-chain dimeric polypeptide. For instance, amino acid residues of an association domain of a first monomer to be modified may be replaced by amino acid residues of an association domain of a second monomer to which the first monomer is to associate. Inactivation of a receptor-binding site

In general, there are a number of different possible approaches to obtaining a single- chain dimeric polypeptide of the invention that is able to bind but not activate a cellular receptor selected from the VEGF type 2 and VEGF type 3 receptors. These different approaches include heterodimeric constructs in which the two monomeric polypeptide units are derived from different polypeptides, as well as constructs in which where the individual monomeric units are derived from the same polypeptide. In either case, one or both of the monomeric units will typically have one or more changes compared to the respective native or parent polypeptides from which they are derived. In the case of heterodimeric constructs, one or more changes will normally be required in order to obtain sufficient binding affinity to the target receptor and/or in order to be able to form stable dimers in which the two monomers are structurally compatible. If the heterodimeric polypeptide shows full or partial agonist activity, one or more changes will normally be required in order to reduce this agonist activity. In the case of single-chain constructs in which the two monomers are derived from the same polypeptide, one or more changes will be required in order to render one of the two receptor binding sites incapable of effectively binding the receptor. As will be explained in further detail below, possible changes that may be performed in one or both of the monomers include amino acid substitutions, additions or deletions and/or providing for attachment of one or more non-polypeptide moieties to the polypeptide. In one embodiment, the single-chain polypeptide of the invention is one wherein the inactive receptor-binding site is rendered inactive due to steric hindrance. For instance, steric hindrance is achieved when the receptor-binding site is blocked by a non-polypeptide moiety or is blocked by any part, preferably the side chain, of one or more amino acid residues which have been introduced (by insertion or more preferably by substitution) into one or more positions located in the receptor-binding site so that the relevant part of the amino acid residue(s) hinders binding to the ligand-binding domain of the receptor. For example, introduction of one or more bulky amino acid residues into the receptor-binding site may provide this effect.

Thus, in one embodiment, suitable modification of the polypeptide of the invention is advantageously effected by modification of the polypeptide in a least one position of a receptor-binding site. The polypeptide may be a variant (mutant form) of a native or wild-type ligand for the given receptor which furthermore is provided in single-chain form. Modification may be accomplished by suitable deletion, insertion, substitution or addition of one or more amino acid residues within the receptor-binding site. The modification should be of a type which essentially renders the receptor-binding site inactive as defined herein, but should, on the other hand, not be so extensive as to substantially alter the conformation of the other receptor-binding site of the polypeptide and thereby render it incapable of binding to the ligand-binding domain of another receptor subunit. In addition to the above-described modification of one receptor-binding site, the other receptor-binding site may be modified to become capable of more effectively binding to the ligand-binding domain of another receptor subunit as compared to the unmodified, native receptor-binding site.

The total number of amino acid residues to be altered in accordance with the present invention (as compared to the amino acid sequence of the receptor-binding site of the parent polypeptide) will typically not exceed 15. A receptor-binding site of the single-chain polypeptide thus preferably comprises an amino acid sequence which differs in 1-15 amino acid residues from the amino acid sequence of the corresponding receptor-binding site in the parent polypeptide in question, 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 comprises an amino acid sequence which differs from the amino acid sequence of the receptor-binding site of the parent polypeptide in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

Inactivation of a receptor-binding site by amino acid residue modification

Introduction, in particular by substitution, of an amino acid residue may be accomplished with any natural or synthetic amino acid residue, but in order to inactivate a receptor-binding site it is preferably one which significantly alters the properties of the receptor-binding site, such as substitution of a non-charged amino acid by a charged amino acid (e.g. arginine, lysine, glutamic acid or aspartic acid), substitution of a non-aromatic amino acid by an aromatic amino acid with a bulky side chain (e.g. phenylalanine, tryptophan, tyrosine), substitution of a non-hydrophobic amino acid by a hydrophobic amino acid (e.g. leucine, isoleucine, valine), substitution of a non-polar amino acid by a polar amino acid (glutamine or asparagine), substitution of a small amino acid (e.g. glycine, alanine, serine or threonine) by a bulkier amino acid (such as methionine or any of the amino acids mentioned above), or other suitable substitution. One type of substitution useful for the present purpose is substituting one or more amino acid residues in the receptor-binding site of a given polypeptide by an amino acid residue occupying ah equivalent position in a homologous polypeptide ("homologous" in the sense that the polypeptides belong to the same family of polypeptides and exhibit a certain degree of sequence similarity, i.e. a sufficient sequence identity to allow alignment of the respective sequences). For instance, when the monomer to be modified is VEGF-C or VEGF-D, the amino acid substitution is one wherein one or more amino acid residues of a receptor-binding site are replaced with the amino acid residues occupying equivalent positions in VEGF. It is contemplated that such substitution does not impair the overall conformational structure of the single-chain dimeric polypeptide, but is sufficient to contribute to inactivation of or as such to inactive the receptor-binding site thereof.

The terms "homology" and "identity" as used in connection with amino acid sequences are used in their conventional meanings. Amino acid sequence homology/identity is conveniently determined from aligned sequences (aligned by use of the algorithm CLUSTALW, version 1.8, June 1999, (Thompson et al. (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions- specific gap penalties and weight matrix choice, Nucleic Acids Research, 22:4673-4680) using default parameters) or provided from the PFAM families database version 4.0 (Mp://pfam.wustl.eduΛ (Nucleic Acids Res 1999 Jan 1; 27(l):260-2) by use of GENEDOC version 2.5 (Nicholas, K.B., Nicholas H.B. Jr., and Deerfield, D.W. II. 1997 GeneDoc:

Analysis and Visualization of Genetic Variation, EMBNEW.NEWS 4:14; Nicholas, K.B. and Nicholas H.B. Jr. 1997 GeneDoc: Analysis and Visualization of Genetic Variation).

The term "polypeptide family" is used in its conventional meaning, i.e. to indicate a group of polypeptides which are related to each other by having an amino acid sequence which exhibits a sufficient degree of identity to allow alignment of the sequences. Polypeptide families are available, e.g. from the PFAM families database, version 4.0, or the PROSITE data base (Hofmann et al., The PROSITE database, its status in 1999 Nucleic Acids Res. 27:215-219(1999)) or may be prepared by use of a suitable computer program such as CLUSTALW. Furthermore, the protein sequence family may be provided from recursive searches in protein sequence databases like SWISS-PROT or TrEMBL (Bairoch A., Apweiler R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999 Nucleic Acids Res. 27:49-54 (1999)) using well established sequence search/comparison algorithms like FASTA (Pearson W.R. and Lipman D.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 85. 2444-2448), BLAST (Altshul, S.F. et al. (1991) Nucleic Acids Res. 25. 3389-3402), PSI- BLAST (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: A new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.) or from searches in nucleotide sequence databases like EMBL (Stoesser et al., Nucleic Acids Research, 1999, 27(l):18-24) or GENEBANK (Benson et al., Nucleic Acids Res 1999, 27(1):12-17) using equally well established search algorithms. An overview of these methods can be found in Trends Guide to Bioinformatics (1998), Elsevier Science.

A model structure may easily be constructed by the skilled person on the basis of the known three-dimensional structure of another member of the polypeptide family to which the polypeptide of interest belongs, i.e. in the present context the cystine-knot growth factor family. In order to be able to construct a model structure it is normally desirable that the polypeptide of interest displays at least 30% sequence identity with the polypeptide with the known three-dimensional structure. The model structure may be constructed using any suitable software known in the art, for example the software Modeller (Andrej Sali, Roberto Sanchez, Azat Badretdinov, Andras Fiser, and Eric Feyfant, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA) or the software WHAT IF; A molecular modeling and drug design program (G.Vriend, J. Mol. Graph. (1990) 8, 52-56).

The term "equivalent position" is intended to indicate a position in the amino acid sequence of a given polypeptide which is homologous (i.e. corresponding in position in either primary or tertiary structure) to a position in the amino acid sequence of another polypeptide belonging to the same polypeptide sequence family. Where possible, the "equivalent position" is conveniently determined on the basis of an alignment of members of the polypeptide sequence family in question or alternatively on the basis of superimposed three-dimensional structures, e.g. using CLUSTALW. Preferred amino acid residue modifications in monomers derived from VEGF, VEGF-

C and VEGF-D are described in further detail in the Examples section below.

Inactivation of a receptor-binding site by introduction of an attachment group for a non- polypeptide moiety In accordance with the invention, as an alternative to amino acid residue modification as described above and in the examples below, a receptor-binding site may be blocked by a non-polypeptide moiety. The non-polypeptide moiety is conjugated or otherwise coupled to the single-chain polypeptide through an attachment group of an amino acid residue which is located so as to allow the conjugated non-polypeptide moiety to block the receptor-binding site. The non-polypeptide moiety may for instance be a polymer molecule, a carbohydrate (or oligosaccharide) molecule, a lipophilic molecule or an organic derivatizing agent. The "polymer molecule" is a molecule formed by covalent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein. The term "polymer" may be used interchangeably with the term "polymer molecule", and may also be termed a "macromolecular moiety". The term is intended to cover carbohydrate molecules, although, normally, the term is not intended to cover the type of carbohydrate molecule which is attached to the polypeptide by in vivo N- or O-glycosylation (as further described below). Except where the number of non-polypeptide moieties, e.g. polymer molecules, is expressly indicated, every reference to e.g. a "polymer" or "polymer molecule" contained in a single- chain polypeptide of the invention or otherwise used in the present context shall be understood to be a reference to one or more such polymer molecule(s).

The term "attachment group" is intended to indicate a functional group of the polypeptide, in particular of an amino acid residue thereof or an oligosaccharide moiety, capable of attaching a non-peptide moiety such as a polymer molecule, a lipophilic molecule or an organic derivatizing agent. Useful attachment groups and their matching non-peptide moieties are apparent from the table below.

For in vivo N-glycosylation, the term "attachment group" is used in an unconventional way to indicate the amino acid residues constituting an N-glycosylation site (with the sequence N-X'-S/T/C-X", wherein X' is any amino acid residue except proline, X" any amino acid residue which may or may not be identical to X and which preferably is different from proline, N is asparagine, and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine). Although the asparagine residue of the N- glycosylation site is where the oligosaccharide moiety is attached during glycosylation, such attachment cannot be achieved unless the other amino acid residues of the N-glycosylation site are present. Accordingly, when the non-peptide moiety is an oligosaccharide moiety and the conjugation is to be achieved by N-glycosylation, the term "amino acid residue comprising an attachment group for the non-peptide moiety" as used in connection with alterations of the amino acid sequence of the polypeptide of interest is to be understood as meaning that one or more amino acid residues constituting an N-glycosylation site are to be altered in such a manner that either a functional N-glycosylation site is introduced into the amino acid sequence or removed from said sequence.

Conjugation to PEG or another polymer 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 1000-15,000 Da, even more preferably in the range of about 2000-12,000 Da, such as about 3000-10,000. Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). A hetero-polymer is a polymer which comprises 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 blocking a receptor-binding site, and optionally 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, e.g., to polysaccharides such as dextran, and the like. 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. Suitable activated polymer molecules are commercially available, e.g. from Shearwater Corp., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, 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 Corp. 2001 Catalog (Polyethylene Glycol and Derivatives for Biomedical Applications, 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, and branched PEGs such as PEG2-NHS and those disclosed in US 5,932,462 and US

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: US 5,824,778, US 5,476,653, WO 97/32607, EP 229,108, EP 402,378, US 4,902,502, US 5,281,698, US 5,122,614, US 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, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, US 5,736,625, WO 98/05363, EP 809 996, US 5,629,384, WO 96/41813, WO 96/07670, US 5,473,034, US 5,516,673, EP 605 963, US 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, Boca Raton; 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 or most 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 (US 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 in addition to or as an alternative to attachment of PEG or another non-polypeptide moiety with the aim of blocking a receptor binding site, one or more non-polypeptide moieties may be attached to a single-chain dimeric polypeptide of the invention for other purposes, e.g. to provide an increased half-life in vivo or to reduce immunogenicity. When the polypeptide is conjugated to one or more non-polypeptide moieties for a purpose other than blocking of a receptor-binding site, such non-polypeptide moiety/moieties will for obvious reasons normally be attached to an amino acid residue located outside of the receptor-binding site. Preferably, such non-polypeptide moieties are attached to one or more amino acid residues whose side chain is exposed at the surface of the dimeric polypeptide.

PEGylation will be designed in each individual case 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, for instance taking into consideration whether the primary purpose of the conjugation is to block a receptor-binding site, or whether there is an additional or alternative purpose, e.g. to reduce clearance by means of a high molecular weight or to provide epitope shielding in order to reduce immunogenicity. For example, for reduced clearance, e.g. by renal clearance, it may be desirable to conjugate relatively few high molecular weight polymer molecules to obtain the desired molecular weight, while epitope shielding obtained by use of a sufficiently high number of lower molecular weight polymer molecules (e.g. with a molecular weight of about 5000 Da) is often advantageous, for instance 2-8, such as 3-6 such polymer molecules.

Normally, the polymer conjugation is performed under conditions aiming at reacting all or at least most available polymer attachment groups with polymer molecules. Typical 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 ratio may be somewhat lower, however, such as up to about 50-1, 10-1 or 5-1.

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. An example is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; US 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.

Specific PEGylation strategies include, for example: 1) a single PEG molecule attached to the N-terminal of the polypeptide and no other PEG molecules, e.g. a linear or branched PEG molecule with a molecular weight of about 20 kDa or more, the polypeptide optionally further comprising one or more oligosaccharide moieties attached to an N-linked or O-linked glycosylation site of the polypeptide or carbohydrate moieties attached by in vitro glycosylation; 2) a PEG molecule attached to lysine residues in the polypeptide available for PEGylation, e.g. a linear or branched PEG molecule with a molecular weight of about 5-12 kDa; and 3) a PEG molecule attached to lysine residues in the polypeptide available for PEGylation, and in addition to the N-terminal amino acid residue of the polypeptide.

Conjugation to an oligosaccharide moiety

Conjugation to an oligosaccharide moiety may take place in vivo or in vitro. In order to achieve in vivo glycosylation of a single-chain polypeptide 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 CHO cell, BHK or HEK cell, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae or Pichia pastoris, or any of the host cells mentioned hereinafter.

Covalent in vitro coupling of glycosides (such as dextran) to amino acid residues of the single-chain polypeptide may also be used, e.g. as described in WO 87/05330 and in Aplin et al., CRC Grit Rev. Biochem., pp. 259-306, 1981. Furthermore, in vitro coupling of oligosaccharide moieties or PEG to protein- and peptide-bound Gin-residues can be carried out by fransglutaminases (TGases). Transglutaminases catalyse the transfer of donor amine-groups to protein- and peptide-bound Gin-residues in a so-called cross-linking reaction. The donor-amine groups can be protein- or peptide-bound e.g. as the ε-amino-group in Lys-residues or can be part of a small or large organic molecule. An example of a small organic molecule functioning as an amino-donor in TGase-catalysed cross-linking is putrescine (1,4-diaminobutane). An example of a larger organic molecule functioning as an amino-donor in TGase-catalysed cross-linking is an amine-containing PEG (Sato et al., Biochemistry 35, 13072-13080). TGases, in general, are highly specific enzymes, and not every Gin-residue 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 function naturally 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, α₂-plasmin inhibitor, α-caseins, and β-caseins.

Altering conjugation sites in a receptor-binding site

If an amino acid residue comprising an attachment group for the non-polypeptide moiety in question is located in the vicinity of the receptor-binding site, modification may simply be achieved by conjugation of the non-polypeptide moiety of choice to the attachment group in question. Subsequently, the resulting conjugated single-chain polypeptide is tested for its capability to bind to a ligand-binding domain of the receptor, and incapability of effecting signal transduction. It may be necessary to remove such amino acid residue(s) from the second receptor-binding site, preferably by conservative amino acid substitution, to ensure that the non-polypeptide moiety is conjugated only to the intended amino acid residue, and not to amino acid residues located in the vicinity of other receptor-binding site for which no reduction of receptor-binding capability is intended. Alternatively, an amino acid residue comprising an attachment group for the non-polypeptide moiety of choice may be introduced, preferably by substitution, within the receptor-binding site to be modified, either by use of site-directed mutagenesis or by random mutagenesis. When site-directed mutagenesis is used, the actual position(s) to be modified are conveniently selected on the basis of an analysis of the three-dimensional structure of the receptor-binding site to be modified. When random mutagenesis is used, it is normally limited to amino acid residues of the receptor-binding site to be modified. The site-directed or random mutagenesis is normally accompanied by a suitable screening of the resulting polypeptide variants. Preferably, the variants resulting from site-directed or random mutagenesis are conjugated to the non-polypeptide moiety of choice prior to screening.

When an amino acid residue comprising an attachment group is to be introduced into a receptor-binding site or in the vicinity thereof so that a non-polypeptide moiety conjugated to said amino acid residue inactivates the receptor-binding site, it is usually sufficient that such amino acid residue is introduced in only one of the monomers contributing to the receptor- binding site. Furthermore, when an amino acid residue comprising an attachment group is to be introduced into or in the vicinity of the receptor-binding site this should preferably be done so that the attachment group for the non-polypeptide moiety is exposed at the surface of the polypeptide and thereby rendered accessible for conjugation to the non-polypeptide moiety. The latter may be evaluated in a model or 3D structure of the single-chain polypeptide or of the receptor-binding site.

As explained above, in order to avoid conjugation to the non-polypeptide moiety of choice in regions of the single-chain polypeptide where such conjugation is not desirable, the single-chain polypeptide may be further modified so as to have removed, preferably by substitution, more preferably by conservative substitution, one or more amino acid residues comprising an attachment group for said non-polypeptide moiety. For instance, it may be important to remove such amino acid residue if it is present in the intact receptor-binding site of the single-chain polypeptide. It is also contemplated to improve binding of the polypeptide to a receptor through the intact, active receptor-binding site by substitution or insertion of one or more amino acid residues in said receptor-binding site so as to obtain a stronger binding affinity to said receptor. It is at present assumed that suitable amino acid substitutions may be conservative substitutions. Examples of such conservative substitutions are substitution within the group of basic amino acids (such as arginine, lysine or histidine), acidic amino acids (such as glutamic acid or aspartic acid), polar amino acids (such as glutamine or asparagine), hydrophobic amino acids (such as leucine, isoleucine or valine), aromatic amino acids (such as phenylalanine, tryptophan or tyrosine) and small amino acids (such as glycine, alanine, serine or threonine). Preferred substitutions in the present invention may in particular be chosen from among the conservative substitution groups listed in the table below.

Conservative substitution groups:

1 Alanine (A) Glycine (G) Serine (S) Threonine (T)

2 Aspartic acid (D) Glutamic acid (E)

3 Asparagine (N) Glutamine (Q)

4 Arginine (R) Histidine (H) Lysine (K)

5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V)

6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Peptide linker

The monomers composing the polypeptide may be linked by a peptide bond, or may be connected by a suitable linker peptide. Choosing a suitable linker for a specific case where two polypeptide chains are to be connected depends on various parameters, e.g. the nature of the two polypeptide chains (e.g. whether they naturally form a dimer or not), the distance between the N- and C-termini to be connected if known from three-dimensional structure determination, and/or the stability of the linker towards proteolysis and oxidation. Furthermore, the linker may contain amino acid residues that provide flexibility. The linker peptide may therefore predominantly include the following amino acid residues: Gly, Ser, Ala or Thr. The linker peptide should have a length which is adequate to link two monomers in such a way that they assume the correct conformation relative to one another so that they retain the desired activity as antagonists of a given receptor. A suitable length for this purpose is a length of at least one and not more than about 30 amino acid residues, such as a sequence of about 5-20 amino acid residues, in particular about 10-15 amino acid residues. Likewise, the amino acid residues selected for inclusion in the linker peptide should exhibit properties that do not interfere significantly with the activity of the polypeptide. Thus, the linker peptide should on the whole not exhibit a charge which would be inconsistent with the activity of the polypeptide, or interfere with internal folding, or form bonds or other interactions with amino acid residues in one or more of the monomers which would seriously impede the binding of the polypeptide to the ligand-binding domain of the receptor in question.

Specific linkers for use in the present invention may be designed on the basis of known naturally occurring as well as artificial polypeptide linkers (see, e.g., Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995), Protein Eng. 8, 725-731 ;

Robinson & Sauer (1996), Biochemistry 35, 109-116; Khandekar et al. (1997), J. Biol. Chem. 272, 32190-32197; Fares et al. (1998), Endocrinology 139, 2459-2464; Smallshaw et al. (1999), Protein Eng. 12, 623-630; US 5,856,456). For instance, linkers used for creating single-chain antibodies, e.g. a 15mer consisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Gly Ser)₃), are contemplated to be useful in the present invention.

Furthermore, phage display technology as well as selective infective phage technology can be used to diversify and select appropriate linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al. (1998), Protein Eng. 11, 405-410). Also, Arc repressor phage display has been used to optimize the linker length and composition for increased stability of the single-chain protein (Robinson and Sauer (1998), Proc. Natl. Acad. Sci. USA 95, 5929-5934).

Another way of obtaining a suitable linker is by optimizing a simple linker, e.g. ((Gly₄Ser)_n), through random mutagenesis. It will be clear from the present specification that whatever the nature of the linker, it should be one which is not readily susceptible to cleavage by e.g. proteases or chemical agents, since cleavage of the multimeric polypeptide to result in two or more monomeric units is not desired in the present context.

The monomeric polypeptides to be linked according to the invention may be provided in truncated form, e.g. having 1-10, such as 1-5 of the amino acid residues of either the N- or C-terminal deleted relative to the corresponding wild-type polypeptide, and the truncated monomeric polypeptides may be fused to each other directly or linked through a peptide linker as discussed above.

Reducing immunogenicity

One problem that may be encountered when using a single-chain multimeric polypeptide of the invention is that the polypeptide or parts thereof, e.g. a peptide linker used in the construction of the polypeptide, may be recognized as a foreign, undesirable substance by the immune system of an individual treated with the polypeptide. Accordingly, it may be desirable to shield epitopes or other immunogenic determinants giving rise to an immune response in an individual treated with the polypeptide. For this purpose it may be desirable that the single-chain dimeric polypeptide of the invention comprises one or more non-polypeptide moieties that are located so as to shield any amino acid changes as compared to the native polypeptide. The presence of such moieties may also increase the functional in vivo half-life of the polypeptide of the invention. Accordingly, in a further embodiment the single-chain dimeric polypeptide of the invention is one wherein a non-polypeptide moiety is conjugated to an amino acid residue of a linker peptide or an amino acid residue of at least one monomer constituent of the single-chain dimeric polypeptide so as to reduce the immunogenicity of the single-chain dimeric polypeptide, and in particular any linker peptide part thereof. The conjugation may be achieved by any of the methods disclosed above. In one embodiment, a naturally occurring TGase substrate sequence is introduced into or replaces the linker connecting the monomers in the single-chain dimeric polypeptide, whereby the single-chain polypeptide may be modified using the highly specific TGase- catalysed cross-linking (described above).

The term "reduced immunogenicity" is intended to indicate that the conjugate gives rise to a measurably lower immune response than a reference molecule, such as a wild-type polypeptide, as determined under comparable conditions. The immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology Edition, Blackwell) for further definition of immunogenicity). Normally, reduced antibody reactivity will be an indication of reduced immunogenicity. The reduced immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.

Increasing half-life

The term "functional in vivo half-life" is used in its normal meaning, i.e. the time in which 50% of a given functionality of the conjugate is retained. As an alternative to determining functional in vivo half-life, "serum half-life" may be determined, i.e. the time in which 50% of the conjugate circulates in the plasma or bloodstream prior to being cleared. Determination of serum half-life is often more simple than determining the functional in vivo half-life, and the magnitude of serum half-life is usually a good indication of the magnitude of functional in vivo half-life. Alternative terms for serum half-life include "plasma half-life", "circulating half-life", "serum clearance", "plasma clearance" and "clearance half-life". The conjugate is normally cleared by the action of one or more of the reticuloendothelial systems (RES), kidney, spleen or liver, receptor mediated elimination or by specific or unspecific proteolysis. Normally, clearance depends on size (relative to the cutoff for glomerular filtration), charge, attached carbohydrate chains, and the presence of cellular receptors for the protein. Suitable methods for determining the functional in vivo half-life or serum half-life are well known in the art.

The term "increased" as used about the functional in vivo half-life or serum half-life is used to indicate that the relevant half-life of the conjugate is statistically significantly increased relative to that of a reference molecule as determined under comparable conditions. As explained above, an increased half-life may typically be obtained by conjugation to one or more non-polypeptide moieties, e.g. by means of PEGylation or in vivo glycosylation.

Methods for preparing polypeptides of the invention The present invention further relates to a method for preparing a single-chain dimeric polypeptide of the invention, which method comprises culturing a recombinant host cell comprising a single nucleotide sequence encoding said polypeptide in a suitable culture medium under conditions permitting expression of the nucleotide sequence and recovering the resulting polypeptide from the cell culture. In further aspects the invention relates to a nucleotide sequence encoding a single chain dimeric polypeptide of the invention, an expression vector comprising said nucleotide sequence and a recombinant host cell comprising said sequence or said vector. Nucleotide sequence encoding a polypeptide of the invention and its preparation

As used herein the term "nucleotide sequence" is intended to indicate a consecutive stretch of two or more nucleotides of cDNA, genomic DNA, synthetic DNA or RNA origin. The nucleotide sequence encoding the polypeptide of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). For the present purpose, the nucleotide sequence encoding the polypeptide is preferably of vertebrate origin, i.e. derived from genomic DNA or cDNA library of the relevant tissue. In particular, the nucleotide sequence may be of mammalian origin, in particular human origin.

The nucleotide sequence of the invention encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matches et al., EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors. Furthermore, the nucleotide sequence may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleotide sequence, in accordance with standard techniques.

The nucleotide sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202, US 4,683,195 or Saiki et al., Science 239 (1988), 487-491.

In cases where the dimeric polypeptide comprises two monomeric units having an identical or highly homologous amino acid sequence, it is preferred, in order to avoid recombination between the nucleotide sequences that encode the individual monomeric units, that codon differences between these coding sequences are maximized.

Mutagenesis methods

In a further aspect, the invention relates to a method for producing a nucleotide sequence encoding a single-chain dimeric polypeptide of the invention, wherein a single nucleotide sequence encoding the single-chain dimeric polypeptide is subjected to mutagenesis so as to render at least one receptor-binding site of the encoded polypeptide inactive and/or to increase the binding affinity of a receptor-binding site towards a ligand- binding domain of a receptor relative to a corresponding binding site of an unmodified single- chain dimeric polypeptide or relative to the wild-type polypeptide.

Suitable mutations may be introduced by, e.g., site-directed mutagenesis as described by Sambrook et al., or by random mutagenesis or DNA shuffling, e.g. as described below followed by screening for sequences coding for polypeptides with the desired activity. Screening may be carried out by an assay method as described below. Random mutagenesis (whether performed in the whole nucleotide sequence or one or more selected regions thereof) may be performed by any suitable method. For example, random mutagenesis is performed using a suitable physical or chemical mutagenizing agent, a suitable oligonucleotide, PCR generated mutagenesis or any combination of these mutagenizing agents/methods according to state of the art technology, e.g. as disclosed in WO 97/07202.

Error prone PCR generated mutagenesis, e.g. as described by J.O. Deshler (1992), GATA 9(4): 103-106 and Leung et al., Technique (1989) Vol. 1, No. 1, pp. 11-15, is particularly useful for mutagenesis of longer peptide stretches (corresponding to nucleotide sequences containing more than 100 bp) or entire genes, and are preferably performed under conditions that increase the misincorporation of nucleotides.

Random mutagenesis based on doped or spiked oligonucleotides is of particular use for mutagenesis of one or more regions containing shorter nucleotide sequences (normally containing less than 100 nucleotides per region). Mutagenesis of several regions is conveniently conducted by using several doped oligonucleotides and combining them by PCR. Doped or spiked oligonucleotides may also be used for random mutagenesis of nucleotide sequences encoding longer peptide stretches or entire genes when it is desirable to be able to control the random mutagenesis to a higher extent than what is possible with error prone PCR generated mutagenesis.

Conveniently, random mutagenesis of one or more selected regions of a nucleotide sequence encoding the polypeptide of interest is performed using PCR generated mutagenesis, in which one or more suitable oligonucleotide probes which flank the area to be mutagenized are used. Preferably, for mutagenesis of selected peptide stretches doped or spiked oligonucleotides are used. The doping or spiking can be designed to introduce any kind of amino acid residue and/or to avoid a codon for an unwanted amino acid residue (by lowering the amount of or completely avoiding the nucleotides resulting in this codon). The doping may be designed on the basis of the skilled person's intelligent consideration of nucleotide doping (in accordance with generally known principles), by use of a suitable algorithm, e.g. a computer program which is based on the algorithm described by Siderovski DP and Mak TW, Comput. Biol. Med. (1993) Vol. 23, No. 6, pp. 463-474 or Jensen et al. Nucleic Acids

Research, 1998, Vol. 26, No. 3 or by using trinucleotides (Sondek, J. and Shortle, D., Proc. Natl. Acad. Sci, USA, Vol. 89, pp. 3581-3585, April 1992; Kayushin et al., Nucleic Acids Research, 1996, Vol. 24, No. 19, pp. 3748-3755; Virnekas et al., Nucleic Acids Research, 1994, Vol. 22, No. 25; WO 93/21203). The doped or spiked oligonucleotide can be incorporated into the nucleotide sequence encoding the polypeptide of interest by any published technique using e.g. PCR, LCR or any DNA polymerase or ligase.

Random mutagenesis may be performed in two, three, four, five, six or more regions at the same time by synthesizing doped oligonucleotides covering each region and assembling the oligonucleotides by state of the art technologies, for example by a PCR method. One convenient PCR method involves a PCR reaction wherein the nucleotide sequence encoding the polypeptide of interest is used as a template and the doped oligonucleotides are used as primers. In addition, cloning primers localized outside the targeted regions may be used. The resulting PCR product can either be directly cloned into an appropriate expression vector or gel purified and amplified in a second PCR reaction using the cloning primers and cloned into an appropriate expression vector.

Besides substitutions the random mutagenesis may also cover random introduction of insertions or deletions. Preferably, the insertions are made so as to be in reading frame, e.g. by performing multiple introduction of three nucleotides as described by Hallet et al., Nucleic Acids Res. 1997, 25(9):1866-7 and Sondek and Shortle, Proc Natl. Acad. Sci USA 1992, 89(8):3581-5.

The nucleotide sequence(s) or nucleotide sequence region(s) to be mutagenized are typically present on a suitable vector such as a plasmid or a bacteriophage, which as such is incubated with or otherwise exposed to the mutagenizing agent. The nucleotide sequence(s) to be mutagenized may also be present in a host cell either by being integrated into the genome of said cell or by being present On a vector harboured in the cell. Alternatively, the nucleotide sequence to be mutagenized is in isolated form. The nucleotide sequence is preferably a DNA sequence such as a cDNA, genomic DNA or synthetic DNA sequence. In one embodiment the random mutagenesis is accompanied by conjugation to a non- polypeptide moiety. More specifically, a modified conjugated single-chain polypeptide of the invention may be prepared by a) expressing a random mutagenized library of nucleotide sequences encoding a parent polypeptide in single-chain form, b) conjugating one or more non-polypeptide moieties to the polypeptide variants expressed in step a), c) screening the resulting conjugates for antagonist activity or receptor-binding, but not activating capability, d) selecting polypeptide conjugates having such capability, and e) optionally subjecting the nucleotide sequence encoding the polypeptide part of a polypeptide conjugate selected in step d) to one or more repeated cycles of steps a)-d). The above method for random mutagenesis and conjugation is further described in PCT/DK00/00371 (WO 01/04287). In accordance with this embodiment the polypeptide conjugate can be prepared in a high throughput screening system allowing production and screening of a high number of different polypeptides in a short time. This is in particular suitable in the following situations:

• obtaining an improved binding affinity

• altering receptor specificity • creating partial antagonists

• reducing/eliminating intrinsic activity of agonists

• identifying optimal linkers.

Nucleotide sequence modification methods suitable for producing polypeptide variants for high throughput screening further include for instance methods which involve homologous cross-over such as disclosed in US 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, Mar; 16(3): 258-61; Zhao H. and Arnold, FB, 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 US 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 modeled 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.

Shuffling and high throughput screening, for example family shuffling combined with high throughput screening using e.g. FACS (Fluorescent Activated Cell Sorting), is a particularly preferred method that is well suited for producing novel proteins with desired binding characteristics.

When using random mutagenesis as outlined above, the expression step a) can be conducted in any suitable manner, and conveniently as described further below. Suitably, the random mutagenized library is prepared by subjecting a nucleotide sequence encoding the parent polypeptide in single-chain form to random mutagenesis so as to create a large number of mutated nucleotide sequences. The random mutagenesis may be entirely random, both with respect to where in the nucleotide sequence the mutagenesis occurs and with respect to the nature of mutagenesis. Alternatively, the random mutagenesis may be conducted so as to randomly mutate one or more selected regions of the polypeptide, in particular a receptor-binding site thereof. The library is typically present in a host cell, from which expression is achieved. Of particular interest is a host cell which is capable of a reasonable transformation frequency such as bacterium, e.g. E. coli, yeast, e.g. S. cereviciae, or fungus. Alternatively, a high throughput transfection system of mammalian cells or other cells capable of a desirable post-translational modification (such as in vivo glycosylation) may be employed and examples include CHO (Chinese Hamster Ovary) and COS and BHK (Baby Hamster Kidney) cells.

Conjugation step b) is conveniently conducted as described above in connection with conjugation to a polymer or an oligosaccharide moiety. The screening step c) is an important element of the method according to this embodiment of the invention. The screening is conveniently conducted as a primary screening for receptor-binding, but not activating capability, e.g. based on the principles disclosed in the assays described below.

In a preferred embodiment as many as possible of steps a-d) are performed in a high throughput screening system. In particular, it is preferred that steps a)-d) are performed in a robotized system, wherein the expression from the random mutagenized library of nucleotide sequences is achieved in microtiter plates, the resulting supernatant is transferred to a different microtiter plate, preferably under conditions allowing immobilization of the polypeptides, and optionally under conditions where the receptor-binding site of the polypeptide is blocked, e.g. by a suitable receptor, receptor analogue or antibody, and/or the polypeptide is provided with a tag, e.g. a His tag known in the art (such as His-His-His-His- His-His; Met-Lys-His-His-His-His-His-His; Met-Lys-His-His-Ala-His-His-Gln-His-His; or

Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln (all available from Unizyme Laboratories, Denmark); the optionally immobilized, blocked and/or tagged polypeptides are subjected to conjugation to a non-polypeptide moiety while present in the microtiter plate and the resulting polypeptide conjugates present in the microtiter plate are subjected to the relevant screening. Subsequently, selected positive polypeptide conjugates are subjected to further characterization including secondary screening.

Vectors and expression It should of course be understood that not all vectors and expression control sequences function equally well to express the nucleotide sequence encoding the polypeptide described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art will be able to 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, CA, USA) and pCI-neo (Stratagene, La JoUa, CA, USA). 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., WI, 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 Ml 3 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof, the POT1 vector (US 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 pBluebac 4.5 and pMelbac (both available from Invitrogen). 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., US 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 2μ 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 filamentous fungi, selectable markers include amdS, pyrG, arcB, niaP, 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, 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 lα (EF-lα) 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, S V40 or adenovirus Elb 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 of interest. An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (available from Promega Corporation, WI, USA).

Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the P 10 promoter, the Autographa californica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculo virus 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 α-mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydogenase genes, the ADH2-4c promoter and the inducibie 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 α- 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. To direct a polypeptide of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the polypeptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide. The secretory signal sequence may be that normally associated with the polypeptide or may be from a gene encoding another secreted protein.

For secretion from yeast cells, the secretory signal sequence may encode any signal peptide, which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell. The signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the α-factor signal peptide (cf. US 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), and the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

In addition, to obtain an efficient secretion, a pro-peptide encoding sequence may be inserted downstream of the signal sequence and upstream of the nucleotide sequence encoding the polypeptide. A pro-peptide may be the yeast α-factor pro-peptide (the use of which is described in e.g. US 4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP 163 529) or a synthetic pro-peptide (WO 89/02463, WO 92/11378 or WO98/32867).

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 α-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. US 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). For use in mammalian cells, a suitable signal sequence is the murine Ig kappa light chain signal sequence (Coloma, M (1992) J. Imm. Methods 152:89-104) or the signal sequence naturally associated with the nucleotide sequence encoding the polypeptide. The procedures used to ligate the DNA sequences coding for the polypeptide of the invention, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.ci ). Any suitable host may be used to produce the polypeptide, including bacteria, fungi

(including yeasts) and higher eukaryotic cells (including plant, insect and mammalian cells, and other appropriate animal cells and cell lines), as well as transgenic animals or plants. Examples of bacterial host cells include gram-positive bacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gram-negative 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 ox A. nidulans, Fusarium or Trichoderma. Fungal cells maybe 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 US 5,679,543. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. 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; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920. 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. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are disclosed by Clontech Laboratories, Inc, Palo Alto, CA, USA (in the product protocol for the Yeastmaker™ Yeast Tranformation System Kit), and by Reeves et al., FEMS Microbiology Letters 99 (1992) 193-198, Manivasakam and Schiestl, Nucleic Acids Research, 1993, Vol. 21, No. 18, pp. 4414-4415 and Ganeva et al., FEMS Microbiology Letters 121 (1994) 159- 164.

Examples of suitable insect host cells include a Lepidoptora cell line, such as Spodopterafrugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (US 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/O), 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, ockville, Maryland. 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, New Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997). In order to produce a glycosylated polypeptide a eukaryotic host cell, e.g. of the type mentioned above, is preferably used.

In the production method 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 the periplasmic space or from various cell lysates. The polypeptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilised metal ion affinity chromatography, or the like, dependent on the type of polypeptide in question.

Pharmaceutical composition of the invention and its use

In a further aspect the single-chain dimeric polypeptide is used for the manufacture of a medicament for treatment of conditions in which antagonism of the VEGF type 2 or type 3 receptor is desired, as well as methods of treatment of a human or other mammal in need thereof comprising administering to the mammal an effective amount of such a polypeptide. The single-chain dimeric polypeptide of the invention is normally administered in a composition including one or more pharmaceutically acceptable carriers or excipients. "Pharmaceutically acceptable" means a carrier or excipient that does not cause any untoward effects in patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art, and the polypeptide or conjugate of the invention can be formulated into pharmaceutical compositions by well-known methods (see e.g. 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)). Pharmaceutically acceptable excipients that may be used in compositions comprising the polypeptide or conjugate of the invention include, for example, buffering agents, stabilizing agents, preservatives, isotonifiers, non- ionic surfactants or detergents ("wetting agents"), antioxidants, bulking agents or fillers, chelating agents and cosolvents.

The pharmaceutical composition of the polypeptide or conjugate of the invention may be formulated in a variety of forms, including liquids, e.g. ready-to-use solutions or suspensions, gels, lyophilized, or any other suitable form, e.g. powder or crystals suitable for preparing a solution. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art.

The pharmaceutical composition containing the polypeptide or conjugate of the invention may be administered intravenously, intramuscularly, intraperitoneally, intradermally, subcutaneously, sublingualy, buccally, intranasally, transdermally, by inhalation, or in any other acceptable manner, e.g. using PowderJect® or ProLease® technology or a pen injection system. The preferred mode of administration will depend upon the particular indication being treated and will be apparent to one of skill in the art. The pharmaceutical composition of the invention may be administered 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 of the invention, either concurrently or in accordance with any other acceptable treatment schedule. In addition, the polypeptide or pharmaceutical composition of the invention may be used as an adjunct to other therapies.

The present invention will be further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

VEGF-based single-chain antagonist

The invention has been illustrated by creating a VEGF-based KDR antagonist comprising a single-chain form of VEGF_{1 1} with a fourteen residue linker between the monomers and arginine residues substituted for Glu-64 in the first (N-terminal) monomer and Ile-46 in the second (C-terminal) monomer. The antagonist was able to completely inhibit VEGF induced HUVEC proliferation in vitro and displayed reduced affinity for the KDR receptor in BIAcore binding analysis in accordance with model stating that the antagonist has only one active receptor-binding site.

Gene synthesis and creation of a single-chain dimer

Gene synthesis, cloning, transfection and expression was performed by methods well known in the art. Protocols specific for Pichia pastoris expression were used as described by Invitrogen. The Pichia pastoris X-33 strain and the pPICZαA vector were obtained from Invitrogen.

The DNA sequence of the synthetic gene for single-chain VEGF including the fourteen residue linker with the amino acid sequence GSTSGSGKSSEGKG along with the DNA coding the prepro-peptide of S. cerevisiae α-factor (see e.g. US 4,870,008, US 4,546,082) and flanking restriction sites for cloning is listed as SEQ ID NO: 4. The codon composition of this gene has been optimized towards the expression host to improve protein expression while maximizing the difference between the codon usage of the two monomers to reduce recombination events. This fragment was synthesized by running two times thirty-five cycles of PCR on a template created by mixing equimolar amounts in the range 0.02-0.5 pmol/μl of seventy bases DNA oligos covering the whole DNA fragment with twenty basepair overlap, alternating sense and anti-sense oligos. The first PCR was run without end- primers and 1-5 μl of this PCR reaction was used in a secondary 100 μl PCR reaction in the presence of 1.2 pmol/μl of each end-primer. The PCR reactions were run using the pwo polymerase from Roche Molecular Biochemicals (Switzerland). The fragment was subsequently digested with Hindlll and Xbal restriction enzymes, ligated into pPICZαA and cloned into E. coli. Plasmid was purified from bacterial culture using plasmid purification kits from Qiagen and verified by DNA sequencing. The plasmids were then transfected into Pichia pastoris X-33 yeast.

Expression and purification

Single-chain VEGF and variants hereof were expressed using Pichia pastoris X-33 yeast cultures created as described above, according to Invitrogen protocols, and single-chain VEGF or variants were found to be present in the culture supernatants upon harvest by centrifugation. The presence of single-chain VEGF or variants was determined by Western blotting using polyclonal, antigen-purified anti-VEGF antibodies from R&D Systems (MN, USA).

Single-chain VEGF or variants were purified from the culture supernatants by two column chromatography cation-exchange steps in 100 mM acetic acid buffer, pH 4.4, using ToyoPerl 550C cation-exchange resin (TosoHaas, Germany) for the first step and Resource S 1 ml prepacked cation-exchange columns (Amersham Pharmacia Biotech, Sweden) for the second step, in both cases eluting the protein with a 0-1.5 M NaCl gradient and determining the fractions containing single-chain VEGF or variant by Western blotting as described above or by SDS-PAGE. For initial screening of variants, only the first cation-exchange step was carried out.

The purified protein was concentrated and buffer-changed to PBS using VivaSpin columns (VivaScience, MA, USA) and aliquots were stored at -20 °C. The purity, identity and quantity of the protein was determined by SDS-PAGE, N-terminal sequencing, MALDI- TOF mass spectrometry and amino acid analysis using methods generally known in the art. In both single-chain VEGF and variants, the three N-terminal residues of the first monomer were found to be removed during post-translational processing. According to MS, the preparations contain mixtures of non-, mono- and di-glycosylated VEGF. In vitro assay

The ability of single-chain VEGF or variants to stimulate HUVEC (human umbillical vein endothelial cell) proliferation or inhibit VEGF₁₂₁ stimulated HUVEC proliferation was evaluated by seeding 3-5000 HUVEC's (primary HUVEC's passage number 1-5 from PromoCell, Germany) per well in 96 well tissue culture clusters in DMEM/F-12/2% FBS and adding single-chain VEGF, variants and VEGF₁₂₁ in varying concentrations. The cultures were incubated at 37 °C with 5% CO₂ for approximately 48 hours and the relative number of viable cells in each well was determined by adding WST-1 Cell Proliferation Reagent from Roche Molecular Biochemicals and measuring the developing color by determining absorption at 450 nm according to the manufacturer's instructions. Results from assays on single-chain VEGF and variants were analysed using GraphPad Prism 3 statistical software and compared to VEGF₁₂₁ standard curves.

Single-chain VEGF variants

Single-chain VEGF displayed an activity identical to that of wild type VEGF₁₂₁ in HUVEC proliferation assays, see Figure 1.

According to published pharmacological data on VEGF variants (Fuh, G. et al. (1998), J. Biol. Chem., 273, 11197-11204; Muller, Y. A. et al. (1997), Proc. Natl. Acad. Sci. U. S. A, 94, 7192-7197), the residues most important for the interaction with KDR are Phe-17, Tyr-45, Ile-46, Glu-64, Gin- 79 and Ile-83. Based on these data, the following double-arginine substitutions were prepared in single-chain VEGF such that the two arginines were substituted into the same receptor-binding site:

I2:46R, I2:83R ("2:83" designating the 83^rd residue in the C-terminal monomer) I2:46R, Y2:45R I2:46R, Q2:79R I2:46R, E1:64R

Figures 2 and 3 display representative agonist and antagonist proliferation assay results for these four variants. As shown in Figure 3, the (I2:46R, El :64R) variant is able to fully inhibit VEGF_{1 1} induced HUVEC proliferation, whereas none of the other variants are able to inhibit proliferation, and as shown in Figure 2, the (I2:46R, El :64R) variant displays only marginal agonist activity at low concentrations which is inhibited at higher concentrations, while the other variants display full agonist activity similar to that of wild type VEGF₁₂₁. It should be noted that although the five positions selected for mutation appear to be equally important from the results in (Fuh, G. et al. (1998), supra; Muller, Y. A. et al. (1997), supra), they are clearly not equally well suited for the purpose of creating an antagonist. Part of this may of course be that the net charge change of the (I2:46R, El :64R) variant is +3 whereas it is +2 for the other variants.

BIAcore assay

Using the principle of surface plasma resonance (SPR), it is possible to directly measure the interaction of two proteins. For example, the BIAcore series of SPR machines detects in real time the interaction of a chip immoblized protein and a soluble protein sample flowed over the chip surface. The interaction is displayed in the form of a sensogram with binding (increased Response Units) occurring in presence of soluble protein and dissociation (decreased Response Units) occurring in the absence of soluble protein.

The interaction between VEGF single chain proteins and a KDR/Fc chimera (R&D Systems) was analysed using a BIAcore 3000 analyser (BIAcore, Uppsalla, Sweden). KDR/Fc was coupled to a BIAcore CM5 chip and equivalent molar amounts (133nM) of wild type single-chain and the (I2:46R, E1:64R) variant were injected over the receptor. The resulting sensograms are shown in Figure 4. Two observations can be made: first, at the same molar concentration, less (I2:46R, E1:64R) variant is bound than wild type single-chain. This is indicative of lower affinity to the dimeric receptor. Second, the rate of dissociation of the variant is faster than the wild type single-chain. The observation of lower affinity to a dimeric VEGF receptor supports the hypothesis that receptor binding of the variant is mediated via one rather than two binding sites.

Further development

As described above, the antagonist created has a low (approximately 25% of wild type) efficacy in HUVEC proliferation assays at low concentrations. Although this efficacy is very low and observed only in a very narrow concentration range, it may be advantagous to reduce this activity, which may accomplished e.g. by introducing more arginines into the receptor-binding sites. Previous studies of the interactions between VEGF and its receptors indicate that the regions of interest are K16-25, 143-S50, N62-E67 and T77-I91. As seen from the results with the double-Arg variants above, it may be difficult to accurately predict which positions are likely to reduce the agonist activity of the single-chain variants, so the best approach may be to perform an "arginine-scan" of the regions, similar to the classical alanine- scans well known in the art.

A KDR antagonist as described above may be suboptimal for therapeutic use if e.g. the potency is too low or the in vivo half-life is too short. In that case, the potency of the antagonist may be increased by raising the affinity of the intact receptor binding site using e.g. DNA shuffling as described above. Since single-chain VEGF has a molecular weight of approximately 30 kDa and therefore is likely to have a relatively fast renal clearance, it may be desirable to improve the in vivo half-life. This may be addressed by e.g. directed PEGylation as described in WO 01/04287.

Example 2

VEGF-C based single-chain antagonist

The invention may be further illustrated by a single-chain polypeptide comprising a first and a second VEGF-C monomer with one receptor-binding site inactivated. The sequence homology between VEGF, VEGF-C and VEGF-D as well as the fact that all three growth factors bind to and activate KDR indicate that an antagonist can be prepared in a manner similar to that used for creating an antagonist based on VEGF. An antagonist based on VEGF-C may have the further advantage over an antagonist based on VEGF that since VEGF-C binds to both KDR and Flt-4, the VEGF-C based antagonist would be expected to inhibit both angiogenesis and lymphangiogenesis. VEGF-C may also be used as a scaffold for creating antagonists inhibiting only angiogenesis or only lymphangiogenesis by altering the active receptor-binding site to make it either KDR specific or Flt-4 specific, e.g. using DNA shuffling.

Selecting positions for altering the receptor binding site No structural data has been published on VEGF-C, so regions comprising the receptor- binding site are predicted from the homology with VEGF. This suggests that the regions of interest are: K18-Q28, T46-P53, N65-Q70 and S80-P96 (numbering according to SEQ ID NO: 2). Preferably, one or more of the following positions should be altered: K18, S19, 120, D21, N47, T48, F49, F50, K51, N65, S66, E67, G68, L69, K81, T82 and 186, more preferably S 19, T48, F49, F50, K51 , E67, K81 , T82 and 186, most preferably S 19, T48, F49, E67 and 186.

Preferred substituents are charged residues (D, E, K, R), bulky residues (F, Y, W, H), β-branched residues (I, S, T), glycine or proline, more preferably one of E, R, G or P. In general, preferred types on substitutions would be uncharged residues to E or R, positively charged residues to E, negatively charged residues to R, and G to one of A, L, I, S, T, F, W or P, preferably L, S, R or P.

Examples of preferred substitutions are, in one monomer, one or more of S19R, S19E and E67R, and/or in the other monomer, one or more of T48R, T48E, F49R, F49E, I86R and I86E.

Most preferably, an antagonist with substitutions analogous to those of the single- chain VEGF antagonist may be prepared, i.e. (El :67R), (El :67R, N2:47R), (El :67R, T2:48R), (E1:67R, F2:49R), (E1:67R, F2:50R).

Creating a VEGF-C based antagonist

Gene synthesis and cloning, expression and purification is carried out as for the VEGF based antagonist, although other heterologous expression systems well known in the art may be used as alternatives to the Pichia pastoris expression system. Also, purification of the variants may require different types of chromatography and other variations in standard protein chemistry methods; such methods are well known to persons skilled in the art.

The single-chain VEGF-C may be based on a subset of the wild type VEGF-C protein corresponding to the receptor-binding domain of VEGF, called the "VEGF homology domain" of VEGF-C. Based on sequence alignment, a 140 residue fragment is proposed as a starting point. The amino acid sequence of this fragment is listed as SEQ ID NO: 2. However, it may be beneficial to alter the length of the monomer. The DNA sequence of the synthetic gene for single-chain VEGF-C based on the 140 residue monomer is listed as SEQ ID NO: 5.

Initially, the screening of single-chain VEGF-C variants may be carried out in a HUVEC proliferation assay similar to that used for single-chain VEGF variants, since VEGF- C also binds to KDR. Flt-4 specific in vitro assays may be used as well. When a single-chain VEGF-C variant with antagonist properties has been identified, it is preferred to carry out further development to increase potency by e.g. DNA shuffling or to increase in vivo half-life by e.g. PEGylation. The DNA shuffling may be directed at increasing the affinity for either or both of KDR and Flt-4, so it may be possible to create KDR-specific, Flt-4 specific or KDR/Flt-4 promiscuous antagonists. Example 3

VEGF-D based single-chain antagonist

VEGF-D binds to and activate both KDR and Flt-4, as does VEGF-C, so creation of a VEGF-D based antagonist may be carried out in the same manner as described above for VEGF-C.

Specifically, the suggested VEGF homology domain of VEGF-D is listed in SEQ ID NO: 3. Residue numbering is relative to this sequence.

As for VEGF-C, the regions selected for mutation are based on homology with VEGF, and the regions found in VEGF-D are K8-Q18, T36-P43, N55-Q60 and S70-L86. Preferably, one or more of the following positions would be altered: K8, V9, S10, Dl 1, T38, F39, F40, K41, N55, E56, E57, S58, L59, K71, Q72, 176, more preferably V9, T38, F39, F40, K41, E57, K71, Q72, 176, most preferably V9, T38, F39, E57, 176.

Examples of preferred substitutions include, in one monomer, one or more of V9E, V9R and E57R, and/or in the other monomer, one or more of T38E, T38R, F39E, F39R, I76E and I76R.

Most preferably, an antagonist with substitutions analogous to those of the single- chain VEGF antagonist may be prepared, i.e. (El :57R), (El :57R, N2:37R), (El :57R, T2:38R), (E1:57R, F2:39R), (E1:57R, F2:40R).

Example 4 Heterodimer antagonist

The invention may be further illustrated by a single-chain heterodimeric polypeptide comprising one monomer derived from VEGF, VEGF-C or VEGF-D and another monomer, which is different from the first monomer, derived from a homologous growth factor, e.g. one of VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A, PDGF-B, PDGF-C, SCDGF-A, SCDGF-B or another member of the cystine-knot growth factor family, preferably VEGF, VEGF-B, VEGF-C, VEGF-D or P1GF, most preferably VEGF-B or P1GF. The receptor- binding sites of the cystine-knot growth factors comprise residues from both monomers, but since the two receptor-binding sites are formed from different regions of each monomer, a heterodimer will have two different receptor-binding sites, either of which may or may not have affinity towards the target receptor and as such may function as a receptor antagonist and as a starting point for development of therapeutically useful compounds. Heterodimers comprising one monomer that binds the target receptor in its native form and one monomer that does not bind the target receptor in its native form are more likely to have receptor- binding sites with different affinities, so that a VEGF/P1GF heterodimer would be likely to antagonize KDR, and a VEGF-C/P1GF heterodimer would be likely to antagonize both KDR and Flt-4. However, not all combinations would be expected to be able to successfully form stable dimers since the two monomers must be structurally compatible. Experiments may be performed to determine heterodimers that can be formed, and two different monomers that are not able to form dimers may be altered so that they are able to form dimers, e.g. by DNA shuffling or by site-directed mutagenesis, preferably changing one or more residues of the receptor binding site to the corresponding residue(s) of homologous growth factors which are able to form dimers with the relevant monomer. Examples in the literature of formation of cystine-knot growth factor heterodimers include PDGF-A PDGF-B and VEGF/P1GF dimers (Meyer-Ingold, W. et al. (1995), Cell Biol. Int., 19, 389-398; Cao, Y. et al. (1996), J. Biol. Chem., 271, 3154-3162).

A gene encoding the single-chain heterodimer may be synthesized by a method similar to that used for synthesizing the gene encoding single-chain VEGF, and expression of the protein may be carried out using any one of a number of heterologous expression systems commonly used in the art. Cellular in vitro assays, e.g. HUVEC proliferation assays or similar assays, depending on the target receptor, can be carried out to evaluate the activity of the heterodimer, and candidates with desired antagonist properties can be further developed by e.g. DNA shuffling to improve potency and e.g. PEGylation to increase in vivo half-life.

Description of the Figures

Figure 1

Single-chain VEGF was compared to VEGF₁₂₁ from R&D Systems in a HUVEC proliferation assay. The assays were carried out as described in the "VEGF based single-chain antagonist" example, and the absorption from the WST-1 Cell Proliferation Reagent (Roche Molecular Biochemicals) was plotted against the logarithm of the concentration of growth factor given in μg/ml. As seen from the graph, VEGF₁₂₁ and single-chain VEGF have very similar potency and efficacy in the HUVEC proliferation assay.

Figure 2

HUVEC proliferation was stimulated with VEGF₁₂₁ from R&D Systems and different single-chain VEGF variants as described for Figure 1. The results are representative of at least three independent assays. The cell density is plotted against the logarithm of the dilution of stock solutions of varying concentrations. Therefore, the potency of the variants cannot be determined from these plots.

Figure 3 HUVEC antagonist assay with background stimulation with 7 ng/ml VEGF₁₂₁ from

R&D Systems antagonized with increasing concentrations of single-chain VEGF variants. The results are representative of at least three independent assays.

Figure 4

BIAcore analysis of the interaction of single-chain VEGF and the (I2:46R, E1:64R) variants of single-chain VEGF with KDR/Fc chimera.

Claims

1. A single-chain dimeric polypeptide which binds to an extracellular ligand-binding domain of a VEGF type 2 receptor (KDR) or a VEGF type 3 receptor (Flt-4), the polypeptide comprising two receptor-binding sites of which one is capable of binding to a ligand-binding domain of the receptor and one is incapable of effectively binding to a ligand-binding domain of the receptor, and wherein at least one monomer of the dimeric polypeptide is derived from VEGF, VEGF-C or VEGF-D, whereby the single-chain dimeric polypeptide is capable of binding to the receptor, but incapable of activating the receptor.

2. The polypeptide of claim 1 , wherein a second monomer of the dimeric polypeptide is derived from a polypeptide selected from the group consisting of VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A, PDGF-B, PDGF-C, SCDGF-A, SCDGF-B and other members of the cystine-knot growth factor family, preferably P1GF or VEGF-B

3. The polypeptide of claim 1 , wherein each monomer of the dimeric polypeptide is derived from VEGF-C, and wherein at least one of the monomers has an altered amino acid sequence compared to VEGF-C as set forth in SEQ ID NO:2.

4. The polypeptide of claim 1 , wherein each monomer of the dimeric polypeptide is derived from VEGF-D, and wherein at least one of the monomers has an altered amino acid sequence compared to VEGF-D as set forth in SEQ ID NO:3.

5. The polypeptide of claim 1 , wherein the polypeptide is a heterodimer.

6. The polypeptide of any of claims 1 -3 and 5, comprising a monomer derived from VEGF-C with at least one mutation, compared to VEGF-C as set forth in SEQ ID NO:2, in at least one region selected from the group consisting of K18 - Q28, T46 - P53, N65 - Q70 and S80 - P96.

7. The polypeptide of claim 6, wherein said monomer derived from VEGF-C has at least one mutation at an amino acid residue position selected from the group consisting of Kl 8,

SI 9, 120, D21, N47, T48, F49, F50, K51, N65, S66, E67, G68, L69, K81, T82 and 186; preferably from the group consisting of SI 9, T48, F49, F50, K51, E67, K81, T82 and 186; and more preferably selected from the group consisting of SI 9, T48, F49, E67 and 186.

8. The polypeptide of claim 1 , comprising a first monomer derived from VEGF-C having at least the mutation E67R, and optionally comprising a second monomer derived from

VEGF-C having at least one mutation selected from the group consisting of N47R, T48R, F49R and F50R.

9. The polypeptide of any of claims 1 -2 and 4-5, comprising a monomer derived from VEGF-D with at least one mutation, compared to VEGF-D as set forth in SEQ ID NO:3, in at least one region selected from the group consisting of K8 - Ql 8, T36 - P43, N55 - Q60 and S70 - L86.

10. The polypeptide of claim 9, wherein the monomer derived from VEGF-D has at least one mutation at an amino acid residue position selected from the group consisting of K8, V9,

S10, Dll, T38, F39, F40, K41, N55, E56, E57, S58, L59, K71, Q72 and 176; preferably from the group consisting of V9, T38, F39, F40, K41, E57, K71, Q72 and 176; and more preferably selected from the group consisting of V9, T38, F39, E57 and 176.

11. The polypeptide of claim 1 , comprising a first monomer derived from VEGF-D having at least the mutation E57R, and optionally comprising a second monomer derived from VEGF-D having at least one mutation selected from the group consisting of N37R, T38R, F39R and F40R.

12. The polypeptide of claim 1 , comprising a first monomer derived from a VEGF isomer, said monomer optionally having at least one mutation, compared to VEGF₁₂₁ as set forth in SEQ ID NO:l, in at least one region selected from the group consisting of K16-Y25, 143-S50, N62-E67 and T77-I91.

13. The polypeptide of any of claims 1-12, wherein a monomer derived from VEGF,

VEGF-C or VEGF-D is altered compared to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3, respectively, by at least one substitution to introduce a charged residue (D, E, K, R), a bulky residue (F, Y, W, H), a β-branched residue (I, S or T) or G or P; preferably E, R, G or P.

14. The polypeptide of claim 13 , comprising at least one substitution of an uncharged residue to E or R, a positively charged residue to E, a negatively charged residue to R, or G to A, L, I, S, T, F, W or P.

15. The polypeptide of claim 3, comprising, in one VEGF-C-derived monomer, at least one substitution selected from the group consisting of S19R, S19E and E67R, and/or in the other VEGF-C-derived monomer, at least one substitution selected from the group consisting of T48R, T48E, F49R, F49E, I86R and I86E.

16. The polypeptide of claim 4, comprising, in one VEGF-D-derived monomer, at least one substitution selected from the group consisting of SI OR, S10E and E57R, and/or in the other VEGF-D-derived monomer, at least one substitution selected from the group consisting of T38R, T38E, F39R, F39E, I76R and I76E.

17. The polypeptide of any of the preceding claims, comprising a modified receptor- binding site that is incapable of effectively binding to the ligand-binding domain of the receptor due to steric hindrance.

18. The polypeptide of claim 17, wherein the modified receptor-binding site differs from its parent receptor-binding site by deletion, substitution and/or insertion of one or more amino acid residues at one or more positions of said parent receptor-binding site.

19. The polypeptide of claim 17, wherein the modified receptor-binding site is blocked by a non-polypeptide moiety.

20. The polypeptide of claim 19, which is modified by introduction of an amino acid residue comprising an attachment group for a non-polypeptide moiety in such a manner that a non-polypeptide moiety conjugated to said attachment group blocks a receptor-binding site.

21. The polypeptide of claim 20, wherein the amino acid residue comprising an attachment group for a non-polypeptide moiety is introduced in a receptor-binding site.

22. The polypeptide of any of claims 19-21, wherein the non-polypeptide moiety is an oligosaccharide moiety or a polymer.

23. The polypeptide of any of claims 1 -22, comprising a receptor-binding site with at least one modification that results in increased receptor-binding affinity of said modified receptor- binding site compared to a corresponding polypeptide without said modification.

24. The polypeptide of any of claims 1 -23 , wherein the monomers are linked by a linker peptide.

25. The polypeptide of any of the preceding claims, comprising at least one non- polypeptide moiety bound to an attachment group of the polypeptide, wherein said attachment group is part of an amino acid residue located outside of the receptor-binding sites.

26. A nucleotide sequence encoding a single-chain dimeric polypeptide according to any of claims 1-25.

27. An expression vector comprising a nucleotide sequence according to claim 26.

28. A recombinant host cell comprising a nucleotide sequence according to claim 26 or an expression vector according to claim 27.

29. A method for producing a nucleotide sequence according to claim 26, wherein a single nucleotide sequence encoding the single-chain polypeptide is subjected to mutagenesis so as to render one receptor-binding site of the polypeptide encoded by said nucleotide sequence incapable of effectively binding to a ligand-binding domain of the cellular receptor, and/or to provide one receptor-binding site with increased binding affinity.

30. The method of claim 29, wherein the mutagenesis comprises at least one DNA shuffling cycle.

31. A method for preparing a single-chain dimeric polypeptide according to any of claims 1-25, comprising culturing a recombinant host cell according to claim 28 comprising a single nucleotide sequence encoding said polypeptide in a suitable culture medium under conditions permitting expression of the nucleotide sequence and recovering the resulting polypeptide from the cell culture.

32. The method of claim 31 , which further comprises conjugating the polypeptide to a non-polypeptide moiety.

33. A composition comprising a single-chain dimeric polypeptide according to any of claims 1-25 together with at least one pharmaceutically acceptable excipient or vehicle.

34. Use of a single-chain dimeric polypeptide according to any of claims 1-25 for the preparation of a medicament for the prevention or treatment of a disease or condition involving increased signal transduction from or increased activation of a VEGF type 2 or type 3 receptor.

35. A method for treating a disease or condition involving increased signal transduction from or increased activation of a VEGF type 2 or type 3 receptor, comprising administering to a patient in need thereof an effective amount of a polypeptide according to any of claims 1-25 or a composition according to claim 33.