DE10026713A1 - Mutein a chain of a protein from the superfamily of the growth factor TGF-beta - Google Patents

Abstract

The invention relates to muteins of a chain of a protein from the superfamily of growth factor TGF- beta having antagonistic and/or partially agonistic activity. The factors of the TGF- beta superfamily each perform their own specific function in the organism. An overexpression of these proteins can have serious consequences for the affected patient, e.g. ectopic bone formation or psoriasis. In order to be able to confront these pathophysiological effects, inhibitors for these factors should be developed. To this end, the invention provides muteins of a chain of a protein from the superfamily of growth factor TGF- beta , which exhibit antagonistic and/or partially agonistic activity after the formation of a homodimer, whereby the muteins are modified at one or more positions which, in an unaltered protein, are involved in a low-affinity binding of the protein to the receptor thereof. The inventive muteins are of interest for the prophylaxis and treatment of illnesses, which are caused by an overexpression of factors of the TGF- beta superfamily.

Description

The present invention relates to muteins of a chain of a protein from the Superfamily of the growth factor TGF-β with antagonistic and / or partial agonistic activity, derivatives of a protein from the TGF-β superfamily, pharma Zeutische compositions containing the muteins and / or derivatives of the invention include and the nucleic acids coding for the muteins or derivatives thereof.

The TGF- (transforming growth factor) -β protein family includes one large number of structurally related polypeptide growth factors, each of which a fascinating range of cellular processes, including cell proliferation, Cell line determination, differentiation, mobility, adhesion and cell death regulated. The Factors become correspondingly complex, temporal and tissue-specific patterns expresses and play an important role in development, homeostasis and Repair of almost all tissues in eukaryotic organisms, from the Fruit fly to human. Overall, these factors are for one essential part of the intracellular signals responsible for cell fate determine.

A number of works in recent years have been carried out to elucidate the TGF-β Signal transduction path led. Signal transduction involves receptor Serine kinases on the cell surface, their substrates, the SMAD proteins Phosphorylation migrate to the nucleus, where they transcribe the target gene in Activate collaboration with DNA binding partners. The multifunctional nature of TGF-β and the other factors belonging to the TGF-β superfamily appears the interaction of different receptors, SMAD proteins and DNA to bind binding proteins. Disorders of this signal transduction path are the Cause of various forms of human carcinoma and developmental disorders.  

The TGF-β superfamily comprises different subfamilies, each with two to four Members. A detailed overview of the different subfamilies and their Properties is e.g. B. in Massague (1998).

The following Table I gives an overview of some of the most important ones to date known about 20 members of the TGF-β superfamily known as "bone morphogenetic proteins "and" growth and differentiation factors "the new formation and regeneration of Control tissues in the adult organism as well as early and late steps of the Embryonic development are significantly involved.

Table I

Although the primary amino acid sequences of members of the TGF-β superfamily, such as from the table it can be seen that the correspondence between them is sometimes relatively small have, there are common proteins of the different subfamilies structural features. So z. B. all proteins of the superfamily dimers, which consist of two mostly identical monomers are constructed. Another commonality is Signal transduction pathway: all TGF-β-like protein factors signal via cellular receptors made up of two different types of serine kinase receptor chains are assembled. The type I chain has a cytoplasmic GS box and a serine kinase that activates the cellular SMAD1 and -5 signaling proteins. The guy II chain activates a type I receptor serine kinase by transphosphorylation of GS Box segment. The small receptor ectodomains of type I or type II chains, each 120 to 150 amino acids long, show very little among themselves Similarity, and even different chains of the same type are relatively few preserved. A common feature of all known TGF-β- receptor chains Superfamily, however, are four conserved disulfide bridges; additional disulfide bridges and the positions of a few amino acid residues appear for either Type I or Type II receptor proteins to be characteristic.

However, the members of the TGF-β superfamily disintegrate in terms of theirs Binding mechanism in 2 groups, the TGF-β- / activin-like proteins and those of BMP (bone-morphogenetic protein) -2-like proteins.

For the namesake of the superfamily, d. H. TGF-β, is an ordered sequential Mechanism of binding to its cellular receptors has been described. Accordingly, an external ligand initially binds to the type II receptor chain and then type I receptor is recruited from the membrane into the complex. Accordingly, the type II chain is the TGF-β receptor with high affinity. For the interaction of activins with their receptors appears to be comparable Mechanism to give. The members of the TGF-β superfamily, their binding mechanism coincides with that described for TGF-β collectively referred to as TGF-β / activin-like proteins. Become one of them all members of the TGF-β superfamily, the N-terminal a 4th disulfide bridge  exhibit. According to current knowledge, these are all TGF-β1, TGF-β2, TGF-β3 Activins and inhibins as well as BMP-11 and GDF-8.

However, BMP-2 binds to its cellular receptors Mechanism established from that for the TGF-β / activin-like proteins differs. In contrast to the situation with TGF-β, the high affinity receptors are for BMP-2 the type I chains BMPR-IA, BMPR-IB and possibly also ActR-I. Type II Chains themselves can bind dissolved BMP-2 as well, but by far lower affinity. From this it was concluded that the order of the type I and Type II receptor interaction with BMP-2 compared to that established for TGF-β Order is reversed. The proteins obtained for the TGF-β / -activin-like proteins Findings cannot therefore be based on BMP-2 and factors with a similar mechanism, such as B. BMP-4, -5, -6 and -7, GDF-5, -6, -7 (BMP-2-like factors) become.

To date, no epitope has been identified for any of the Type I or Type II receptors corresponding ligands have been localized and characterized. For TGF-β1 and activin A mutant proteins have been constructed and analyzed that altered one exhibit biological activity and receptor binding affinity. For BMP-2 are synthetic peptides have been described which correspond to the loops of BMP-2 and which inhibit the BMP-2 activity (EP 691 349).

All factors of the TGF-β superfamily, including the closely related representatives of one Subgroup, perform their own specific function in the organism, which can be found in the cell- and stage-specific expression of the genes and the effects of Shows mutations. Inactivation of BMP or GDF genes can occur in mammals to die in various embryonic stages (BMP-2, BMP-4) or in the lead perinatal period (BMP-7); Furthermore, there are specific changes to the Development of skeletal elements (GDF-5, BMP-5) have been observed. By Overexpression of the proteins can lead to ectopic bone formation (BMP-2, BMP-4 and others), psoriasis (BMP-6), new neurite formation (GFD-5) or for the regeneration of ischemic kidney damage (BMP-7). An overexpression of GDF-8  (Myostatin) may cause muscle wasting. The through is also problematic TGF-β induced growth of the extracellular matrix in fibrosis, scarring or Cirrhosis.

A pathophysiological effect of the proteins from the TGF-β superfamily to be able to prevent or counteract, it would be highly desirable Develop inhibitors for these factors.

The object of the invention is therefore to provide means with which pathophysiological effects of members of the TGF-β superfamily decreased can be.

According to the invention, this object is achieved by mutein a chain of a protein from the superfamily of the growth factor TGF-β, the mutein after formation a homodimer has antagonistic and / or partially agonistic activity, wherein the mutein is changed at one or more position (s) which are in the unchanged protein involved in low affinity binding to its receptor is / are.

According to the invention, an antagonist is understood to mean proteins which are linked to the Receptors for the natural proteins of the super family of the growth factor Bind TGF-β, but with their binding the normal biological subsequent reactions do not trigger. After binding an antagonist, there is no signal transduction instead of. "Partially agonistic activity" in connection with the present Invention understood an activity that to a certain extent is normal biological follow-up reactions, but the extent of these follow-up reactions is far behind which a subsequent reaction triggered by a natural protein lags behind. Partially agonistic activity is therefore associated with the present invention understood an activity that is less than 80%, preferred less than 50% and particularly preferably less than 25% of the activity of the corresponding natural protein. The activity can z. B. by the  C2C12 test can be determined, the following in the chapter "Material and methods" is described.

A "low affinity bond" is used in connection with the BMP-2-like Subfamily understood a bond that only occurs when the concentration of the ligand of 10 nM or more, often even more than 100 nM or 1 µM, to a half maximum Saturation of the receptor protein leads. In contrast, a "high affinity Binding "is understood to mean a bond that already occurs at a concentration of the ligand of less than 10 nM, often even less than 1 nM, to a half maximum Saturation of a receptor protein leads. The saturation depending on the Ligand concentration can be measured using a biosensor system, as described in Example 4.

In combination with the subfamily of TGF-β / activin-like proteins, a "High affinity" binding also binds the ligand to receptor type II in Absence of receptor type I, as evidenced by chemical reactions in whole cells Cross-linking with radioactive labeled ligands can be demonstrated. In contrast in addition, the "low affinity" binding of the ligand to the receptor chain I is absent the type II chain is possible with very little efficiency and is in the presence of type II Chain reinforced. This can also be done by cross-linking with radioactively labeled Check ligands (see e.g. Massague; 1998; Wuytens et al., 1999).

Under an "unchanged protein" a growth factor from the TGF-β- Superfamily understood in the form in which it is naturally found in a mammal is found and has biological activity. The indication "on one or more Position (s) "means that only one amino acid may have been changed in the mutein, but that, e.g. B. where more extensive deletions have been carried out, also a larger number of amino acids can be changed. In preferred Embodiments are between 1 to 50, particularly preferably 1 to 25 or 1 to 10, most preferably changed between 1 to 5 amino acids.

According to the invention, the muteins can be a deletion of one or more amino acids have, the deletion of several amino acids on several positions of the  Protein chain. However, muteins in which one or multiple amino acids are substituted by other amino acids, the multiple Amino acids can be adjacent or not adjacent. One is preferred non-conservative substitution, particularly preferred substitution by a Amino acid with a different charge or size. Here is the following explained: Basically, four physicochemical groups are divided into the the naturally occurring amino acids are classified. To the group of basic amino acids include arginine, lysine and histidine. To the group of acidic Amino acids include glutamic acid and aspartic acid. The uncharged / polar Amino acids include glutamine, asparagine, serine, threonine and tyrosine. They don't polar amino acids include methionine, phenylalanine, tryptophan, cysteine, glycine, Alanine, valine and proline, leucine and isoleucine. A non-conservative substitution in this context means the exchange of a given amino acid by an amino acid from another physicochemical group. Especially it is preferred to replace an amino acid of a first group with one Amino acid of a second group, the amino acids of the second group being one have a different charge than the amino acids of the first group.

It is also preferred to replace a large amino acid with one of the small ones Amino acids glycine, alanine or serine. It is also preferred to exchange one of the small amino acids through one of the large amino acids tryptophan, tyrosine, Phenylalanine, leucine, isoleucine or glutamine.

In a third embodiment, one or more amino acids are inserted. Multiple amino acid insertions can be at one or more positions Positions of the chain occur.

In a further embodiment, one or more of the specified Amino acid residues chemically modified. The modification can be e.g. B. the act covalently with one or more residues resulting from the following Group selected are: carboxylic acids, amines, polyethylene glycol, biotin and sugar (DeSantis et al., 1999).  

In a preferred embodiment, the mutein is from a chain of a BMP-2 similar protein derived. The family of BMP-2-like proteins includes BMP-2 subfamily, BMF-5 subfamily and GDF-5 subfamily (see classification of these Families of Massague (1998)). As can also be seen from Table I, the Members of the BMP-2 subfamily have an identity of 92% among themselves, while the Members of the BMP-5 and the GDF-5 subfamily have an identity of 54 to 61% have on BMP-2. For the members of these three subfamilies this will be assumed that they followed the reaction mechanism mentioned above for BMP-2 has been proven to follow, d. that is, unlike the TGF-βs or Activins initially with high affinity for the type I chains BMPR-IA, BMPR-IB and possibly also bind ActR-I.

Surprisingly, it has now been found that muteins with a partially agonistic or antagonistic action are those muteins in which at least one amino acid from the binding epitope for the natural BMP receptor in the protein chain derived from BMP-2, BMP-4, BMP-5 etc. II deleted, substituted or modified or at least one amino acid inserted in the binding epitope. In the context of the present invention, the inventors have determined the binding epitopes for all receptors involved. The amino acid positions of BMP-2, which determine binding affinity for the BMPR-IA or BMPR-II receptor chains, were found to form two non-overlapping sets. In Fig. 1 it is shown that these determinants BMP-2 sequence are distributed over the whole. The spatial model in Figure 5 shows that the functional residues form two separate epitopes on the surface of the homodimeric BMP-2 molecule.

In the first epitope, which contains residues from both subunits, the determinants for the BMPR-IA interactions are arranged. In Fig. 5 the amino acid residues of the first epitope on a first subunit are shown in italics, while the amino acid residues of the first epitope on a second subunit characterized by normal letters. The epitope is highly discontinuous and includes residues from the β1 sheet, the loop in front of the helix α3 and the helix α3 from one monomer as well as parts of the large ω loop between the sheets β2 and β3 and the sheet β8 of the other monomer. One of the monomers thus contributes the residues V26, D30 and W31 from the β-sheet regions β-2 and β3 and the residues K101 and Y103 from the β-sheet region β8. The other monomer contributes residues I62, L66, N68 and S69 from helix α3 and residues F49, P50, A52 and H54 from the area before helix α3. Because of the spatial structure of the monomer, this epitope is referred to as a "wrist" epitope: the monomers are compared with an open hand, in which the central helix α3 represents the wrist and 2 β sheets arranged side by side represent the 4 fingers; loops 1 and 2 correspond to the fingertips of each pair of fingers. The N-terminal segment is located at the position of the thumb. Consequently, the first epitope, which is arranged around the central α-helix, is on the wrist (wrist). It has dimensions of approximately 2 x 2.5 to 3 nm. This extension is compatible with the function as a high-affinity interaction site.

The second epitope that is on the back of the hand near the outer finger segments is arranged for the low affinity binding of BMP-2 to the BMPR-II responsible. It is composed only of amino acid residues from a subunit and is also known as the "Knuckle" epitope. The amino acid residues A34 and H39 are arranged in the β-sheets β-3 and β-4, the amino acid residues S88 and L90 im Leaflet β-7 and L100 in leaflet β-8. The inventors take the amino acid residue E109 indicates that it is another amino acid residue important for contact. The second epitope appears to be much smaller than the first epitope, since there are many amino acid residues on the Limits of the second epitope with no apparent effects on receptor binding or the biological activity can be modified. However, at the moment it can it cannot be excluded that the epitope contains additional functional amino acid residues contains.

The two epitopes are functionally and spatially separated. From all Binding determinants were found to be either for BMPR-I (Type I) or BMPR-II / ActR-II (Type II) are specific. In the case of BMP-2, antagonistic Muteins can only be found for the Knuckle epitope. The different epitopes are defined by binding determinants and by neutral residues from each other delimited. They do not form overlapping areas on the surface of the established 3-  dimensional structure of BMP-2. However, it cannot be excluded that while type I and type II receptor binding cooperative effects occur. The wrist Epitope and the Knuckle epitope are separated from each other only by the thickness of a β-sheet separated, which can change the conformation after binding to the ectodomain and on this way can convey cooperative effects. The spatial separation The epitope further suggests that everyone with symmetry contiguous parts of the dimeric BMP-2 molecule a pair of functional epitopes which contains two independent wrist epitopes and two independent knuckle Epitopes can bind a total of 4 receptor chains. A complex between one BMP-2 and two BMPR-IA ectodomains have already been identified (Kirsch et al., 2000 (c)).

However, none of the epitopes has the typical charge patterns recently found for Receptors have been discussed (Griffith et al. 1996).

Without wishing to be bound by this theory, it is believed that the BMP-2 antagonists of the invention are most likely a result of the ordered sequential binding mechanism that causes receptor activation. According to the model, the antagonist blocks the high-affinity type I receptor chain with its intact wrist epitope, and the Knuckle epitope modified by substitution, deletion, modification or insertion prevents the subsequent oligomerization with low-affinity type II receptor chains. The comparatively low IC _{50 of} the antagonists and their efficient competition with BMP-2 for receptor binding indicate that it is predominantly the type I chains that control the binding of BMP-2 to the entire receptor complex, possibly by changing the rate of association for Determine BMP-2. The half-life of the complex between BMP-2 and the type 1 receptor of more than 30 minutes most likely leads to the fact that the binding to the cellular receptor is irreversible. An interesting observation in this connection is the low residual activity of the highly antagonistic muteins A34D and L90A in the C2C12 test, if one takes into account that the binding to the ectodomains of the type II receptor chains is only about 5 to 15-fold reduced. The simultaneous binding of two type II chains may be necessary for efficient receptor activation, so that a decrease in binding affinity has a greater impact.

Since the other BMP-2-like proteins have their corresponding receptors after the activate the same mechanism as that shown for BMP-2, d. i.e., about a High affinity wrist epitope and a low affinity knuckle epitope can also antagonistic muteins of these proteins through amino acid substitutions in the knuckle Epitope can be generated.

In preferred embodiments, one or more of the amino acid residues which form the surface-exposed regions from the β-sheet structures β-3, β-4, β-7, β-8 or β-9 are changed. These residues exposed to the surface are as follows:
β-3: V33, A34
between β-3 and β-4: P35, P36;
β-4: G37, Y38, H39;
after β-4: F41, Y42;
β-6: T82, E83, L84, S85;
β-7: A86, I87, S88, L90;
β-8: K97, V98, V99, L100;
β-9: V107, E109, G110.

In a first embodiment, one or more of the specified Amino acid residues deleted individually or in groups of up to 5 amino acids. Preferably amino acids are deleted for which an interaction with the BMP Receptor II is occupied or its deletion affects the conformation of the "Knuckle" epitopes. By combining a substitution with an insertion and / or deletion or by combining an insertion with a deletion further muteins are produced which may have a reduced affinity for the Have BMP receptor II.  

Another possibility, starting from the known sequence of a monomer for BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6 or GDF-7, too to come antagonistic or partially agonistic muteins consists in the Insertion of one or more amino acids into the surface exposed areas of the "Knuckle" epitopes. In principle, these amino acids must also serve the purpose weaken or prevent binding to BMP receptor II.

In a preferred embodiment, the mutein is a chain of a BPM-2-like protein, one or more of the following amino acids from BMP-2 or these corresponding amino acids from another BMP-2-like protein being substituted by other amino acids :
V33, A34, P35, P36, G37, Y38, H39, F41, Y42, T82, E83, L84, S85, A86, I87, S88, L90, K97, V98, V99, L100, V107, E109 and G110.

The following Table II gives an overview of preferred substitute amino acids for the mentioned amino acid residues:  

Table II

It is known from the literature that the different members of the BMP subfamilies 2 and 5 and of the GDF subfamily 5 , even if there is overall a relatively low homology between these proteins, have the same arrangement of the cysteines which are decisive for the tertiary structure. Accordingly, taking these conserved positions into account, the amino acid positions corresponding to a particular amino acid in BMP-2 can be determined for the other members of the subfamilies mentioned. For example, the BMP-2 position V33 in BMP-7 corresponds to an isoleucine, A34 is also alanine, P35 a proline, P36 a glutamate, H39 an alanine, S88 a serine, L90 a leucine, V98 a valine, L100 a leucine and E109 an arginine. Fig. 6, a run with the program "Multalin" Alignment shows the sequences of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, GDF-5, GDF-6, GDF 7, GDF-3, GDF-1, BMP-10, GDF-2, BMP-15, GDF-9B, GDF-9, BMP-3, GDF-10, Act-A, Act-B, Act-C, BMP-11, GDF-8, TGF-β1, TGF-β2, TGF-β3, Inh-a, MIS and GDNF, from which the amino acids corresponding to a particular BMP-2 amino acid in other members of the TGF-β superfamily are taken can. The positions determined by comparison with BMP-2 can also be changed by substitution, deletion or chemical modification. Likewise, the epitopes can lose binding affinity by insertion, insertions being preferred immediately before or after the indicated positions.

The change in positions mentioned for BMP-2 in members of the subfamilies BMP-5 and GDF-5 through a non-conservative substitution, deletion or chemical Modification leads to muteins with modified ones, as a rule reduced binding properties for BMPR-II or for another type II Receptor.

The invention further relates to muteins that have at least 50% identity on the Amino acid level with a growth factor from the TGF-β superfamily and also have antagonistic and / or partially agonistic activity. So are also includes muteins whose amino acid sequence differs from the amino acid sequence the corresponding natural protein chains also in areas that are suitable for a  antagonistic or partially agonistic activity are not decisive, differs.

The term "identity" known to those skilled in the art denotes the degree of relatedness shaft between two or more DNA molecules or two or more polypeptide Molecule determined by the match between the sequences. The percentage of "identity" results from the percentage of identical areas in two or more sequences considering gaps or others Sequence specifics.

The identity of related polypeptides or DNA molecules can be determined using known methods. As a rule, special computer programs with algorithms that take account of the special requirements are used. Preferred methods for determining identity initially produce the greatest agreement between the sequences examined. Computer programs for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12 ( 12 ): 387 ( 1984 ); Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul, S. et al., J. Molec Biol 215 : 403/410 ( 1990 )). The BLAST X program can be obtained from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Handbook, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul, S., et al., J. Mol. 215: 403/410 ( 1990 )). The well-known Smith Waterman algorithm can also be used to determine identity.

Preferred parameters for sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol 48: 443-453 (1970)
Comparative matrix: BLOSUM62 from Henikoff & Henikoff, PNAS USA 89 (1992), 10915-10919
Gap penalty: 12
Gap length value
(Gap Length Penalty): 2

The GAP program is also suitable for use with the above parameters net. The above parameters are the default parameters for Amino acid sequence comparisons, with gaps at the ends of the homology value reduce. With very small sequences compared to the reference sequence, it can it may still be necessary to increase the expected value up to 100,000 and if necessary reduce the word length (wordsize) down to 2.

Further exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices including those mentioned in the program manual, Wisconsin package, version 9 , September 1997, can be used. The selection will depend on the comparison to be carried out and also on whether the comparison is carried out between pairs of sequences, GAP or Best Fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred.

A match of 50% determined with the above-mentioned algorithm is obtained referred to as 50% identity. The same applies to higher degrees of identity.

In preferred embodiments, the muteins according to the invention have one Identity of 60% or more, e.g. B. more than 70% or 80%, with the sequence of one Chain of mature human BMP-2-like protein. The sequence for mature human BMP-2 is found e.g. B. in Celeste et al. (1990). Muteins with are even more preferred more than 90, 95 or 97% identity.

As mentioned above, in the case of BMP-2, the epitope is a low affinity bond enters the "knuckle" epitope, while the epitope, which has a high affinity bond with the receptor that is "wrist" epitope. The muteins according to the invention can continue to be derived from a protein of the TGF-β / activin family. In this case However, it is surprisingly shown that changes in the  "Knuckle" epitope, but changes in the "wrist" epitope to muteins with lead antagonistic and / or partially agonistic activity. Accordingly, in Muteins according to the invention which are derived from a protein of the TGF-β / activin family are derived, one or more amino acids are modified from the "wrist" epitope.

The TGF-β / Aktivin family includes TGF-β1, TGF-β2, TGF-β3, all activins, inhibins, BMP-11 and GDF-8. Previously known activins include, for example, activin βA, Aktivin βB, Aktivin βC and Aktivin βE. According to today's, inhibins include State of knowledge of the inhibins βA, βB and βC.

Antagonists are also in the case of the muteins derived from the TGF-β / activin family or partial agonists primarily by changing amino acids in the areas exposed to surface, d. H. those areas that are attached to involved in the receptor. This is essentially the Helix in front of the sheet structure β1, the sheet structure β1, the long loop between the leaflet structures β2 and β3, the loop in front of the helix α3, the helix α3 and the Folder structure β8. As previously described for the BMP-2-like proteins, such changes through deletions, substitutions or modifications can be brought about, as well as by the insertion of one or more amino acids. The Possibilities given above in connection with the BMP-2-like proteins can be implemented accordingly.

In preferred embodiments, at least one of the following amino acids is changed, ie deleted, substituted and / or modified and / or one or more amino acids are inserted, the position information relating to BMP-2:
K5, S13, V26, G27, W28, N29, D30, W31, P48, F49, P50, A52, D53, H54, N59, I62, V63, L66, N68, S69, V70, K101, Y103.

As can be seen in FIG. 6, this position information in TGF-β1 corresponds to:
Y6, N14, L28, G29, W30, K31, missing, W32, P49, Y50, I51, S53, missing, missing, Q57, K60, V61, L64, N66, Q67, H68, E99, L101.

The 3-dimensional structure of the complex between BMP-2 and the type I receptor BMPR-IA (Kirsch, et al .; 2000 (c)) shows that these residues are essential at the receptor con are involved.

The muteins according to the invention can also if they are derived from TGF-β- / activin Similar growth factors are derived, in addition, for binding to the Receptor not essential regions are changed. Muteins with an identity of at least 50% with a chain of a TGF-β / activin-like growth factor antagonistic and / or partial agonistic activity are also included. Such muteins can e.g. B. also come from other mammals, for example Mouse, rat, rabbit, guinea pig, beef, pork or sheep. As long as such Muteins in the C2C12 cell test show an antagonistic and / or partially agonistic activity have, they are also the subject of the invention. Muteins with are preferred an identity of 60% or more, e.g. B. more than 70% or 80% identity with a Chain of a mutein from the TGF-β / active family. Muteins are even more preferred with more than 90, 95 or 97% identity.

In a further embodiment, muteins according to the invention are provided by generate major changes in the underlying molecules, e.g. B. by an insertion is introduced in addition to a substitution. Possible muteins of both Subfamilies of the TGF-β superfamily also contain one in addition to a deletion Insertion, or in addition to a substitution, a deletion, or at least one Substitution, deletion or insertion in connection with a chemical modification. Of course, 2 changes of one type, e.g. B. a substitution two different positions, alone or in combination with changing a second Type, e.g. B. an insertion at another location. Likewise, more than 2 Types of changes are combined, e.g. B. can in addition to a substitution both a deletion in one place and an insertion in another available.  

The person skilled in the art knows how to produce the muteins. In addition to a conventional one Protein synthesis, e.g. B. the Merrifield synthesis, lend themselves to substitution, deletion and insertion mainly recombinant methods. Based on the known Genes can be specifically inserted mutations, e.g. B. by the site-specific mutagenesis dependent on oligonucleotides. Fragments can be deleted or be used. Alternatively, DNA sequences coding for the muteins de novo can be synthesized.

Chemical modifications are also known in the art introduced. The implementation of chemical modifications to protein chains is e.g. B. described in DeSantis & Jones (1999).

In further preferred embodiments, the mutein described above is also with covalently linked to a target-specific molecule. This can e.g. B. a heparin be binding epitope that an increased binding to the Glycosaminoglycane der extracellular matrix or the cell surface (see e.g. PCT / EP00 / 00637). Under the proviso that this target-specific molecule is an antibody, z. B. targeted signal transduction in those cells that are inhibited Have surface protein that is recognized by the antibody. Target specificity can However, the mutein is not only conferred by covalent binding to an antibody are, but possibly also by covalent binding to a ligand, which is specific for a receptor which only occurs on the target cell.

In preferred embodiments, muteins are covalently linked to them target-specific molecule by recombinant expression of a fusion protein that optionally a spacer between the mutein coding sequence and contains target-specific molecule.

The invention further relates to derivatives of proteins from TGF-β Superfamily, which is an essential component of a mutein according to the present Invention and the formation of a dimer another chain of a protein from the Group of the TGF-β superfamily or contain another mutein according to the invention. The derivatives can therefore both homodimers and heterodimers  Form muteins according to the invention. In addition, a derivative can be a mutein and comprise a natural chain of a protein from the TGF-β super family.

The invention further relates to pharmaceutical compositions which at least one protein according to the invention and / or one derivative according to the invention contain. The invention also includes pharmaceutically acceptable salts from that. The pharmaceutical compositions can, depending on the Nature of the mutein contained therein and depending on the one to be treated pathological condition in the form of ointments, creams, lotions for the topical Application can be provided in the form of solutions or lyophilisates for intramuscular or subcutaneous injections. The formulation and packaging of the Pharmaceutical compositions are made according to the prior art known requirements and includes u. a. the stabilization.

According to a further embodiment, the use of an inventive Muteines and / or a derivative according to the invention for producing claimed pharmaceutical compositions. These can be used for prophylaxis and / or used to treat diseases caused by a protein from the superfamily of the TGF-β growth factor. Examples of such Diseases include ectopic bone formation, psoriasis and muscle wasting, Scarring, fibrosis and cirrhosis. In the case of ectopic bone formation preferably a mutein of one of the growth factors BMF-2 or BMP-4 is used, while e.g. B. in the case of cirrhosis, a mutein one or more of the preferred Growth factors TGF-β1, -β2 or -β3 or a derivative containing them is used.

In further embodiments of the present invention, antibodies against a mutein according to the invention or a derivative according to the invention is provided. There the muteins are characterized by a change in areas of the Characterize molecule, this also affects those specific to the molecule reacting antibody populations. Antibodies can be prepared in a conventional manner Manner either by immunizing animals (e.g. rabbits, mice or rats) for the production of polyclonal antibodies or by immunization and the following  Immortalize antibody-producing cells in the case of monoclonal Antibodies are produced. The procedures required for this are the expert now very familiar, but due to the high phylogenetic invariance of the superfamily, be sure to use one for the generation of antibodies To choose host whose growth factors differ from that against the antibody to be generated largely differentiate.

The invention further relates to those coding for the muteins according to the invention Nucleic acids. These contain a nucleic acid sequence that is required for a desired Mutein encoded. The nucleic acid sequence for BMP-2 is e.g. B. from Wozney et al. (1988) known for TGF-β2 from Madisen et al. (1988). The one coding for a mutein Nucleic acid sequence differs primarily from that for the modified ones Triplets encoding amino acids, i. H. through the absence, the exchange or the Insertion of one or more codons. As far as the mutein is a mutein with one Identity of 50% or more at the amino acid level is appropriate identity reduced for a natural mature protein chain. From this because of the Degeneracy of the genetic code are also different nucleic acids includes. Furthermore, the nucleic acid sequences coding for the muteins complementary sequences as well as with these complementary sequences below stringent conditions hybridizing nucleic acids that code for a mutein, that after the formation of a homodimer, antagonistic or partially agonistic BMP-2 Has activity includes. Stringent conditions are one example Hybridization at 68 ° C in 0.5 × SSC. These and other stringent ones Hybridization conditions can be found in the manual by Maniatis et al., 1989, be looked up.

The nucleic acid according to the invention can be a genomic DNA, a cDNA, a be synthetic DNA or an RNA. Genomic DNAs or cDNAs can be im Methods known from the prior art from the corresponding gDNA or cDNA Banks are isolated. When isolating nucleic acids from cDNA banks tissue or cell line specific banks, e.g. B. from U-2 OS osteosarcoma banks or prostate adenocarcinoma banks. Synthetic DNA can be used  known methods can be produced from RNA either by means of RNA vectors or from mRNA can be isolated. For the recombinant production of the invention Depending on the expression system, muteins will become genomic DNA or cDNA prefer, but the expression by means of RNA vectors is not excluded is.

In the case of substitution changes can be made by known in the art Process to replace the codon coding for the original amino acid. in the Case of deletions are codons coding for one or more amino acids removed while in the case of insertions codon triplets that are required for the Encode amino acids. When choosing codons in the case of a The person skilled in the art will endeavor to substitute or insert the codon use of to take into account the intended host organism. The corresponding Information is available in the prior art.

According to the invention, nucleic acids are also made available which are used for Expression control suitable promoter, the for a Mutein coding nucleic acid sequence according to the invention under the control of this Promoter stands. The choice of a suitable promoter is in turn dependent on the choice of Expression system dependent. The expert has the choice between one A large number of known, inducible or constitutive promoters for the most varied Host organisms.

This is used for the recombinant expression of the nucleic acid according to the invention preferably used in a vector. The invention further relates to a vector which contains a nucleic acid according to the invention, and host organisms which a Nucleic acid sequence coding for a mutein, either integrated directly into the genome or contains in the form of an autonomously replicating vector. In the state of the art numerous prokaryotic and eukaryotic expression systems are known, the host cells are selected, for example, from prokaryotic cells, e.g. B. Bacteria such as E. coli or B. subtilis, from eukaryotic cells such as yeast cells, Plant cells, insect cells and mammalian cells, e.g. B. CHO cells, COS cells or HeLa cells and derivatives thereof. For example, in the prior art  certain CHO production lines known, their glycosylation patterns compared changed to CHO cells. The more efficient by using glycosylation or glycosylation-reduced host cells obtained have a changed spatial structure, possibly with a changed biological Activity.

The invention also relates to a method for producing an invention Muteines, the method of culturing a host cell under the Expression suitable conditions and, if necessary, purification of the expressed mutein according to methods known in the art.

The following examples and figures illustrate the invention without, however, pointing to it restrict.

Figure descriptions Fig. 1

Fig. 1 shows the sequences for BMP-2, BMP-7, TGF-β2 and TGF-β3, and corresponding amino acids are arranged among each other. The amino acid residues altered by substitution are indicated above the BMP-2 sequence. BMP-2 muteins with reduced binding affinity for the type II receptor BMPR-II are identified by a double vertical line in. Altered binding affinities for the type I receptor BMPR-IA, which are based on a decreased association or an increased dissociation rate constant, are marked with a plus (+) or cross (x) at the corresponding positions. Simple vertical lines indicate that no measurable changes in the function of the corresponding muteins could be found
The numbering refers to the BMP-2 sequence.

Fig. 2

Fig. 2 provides information on the biological activity and the inhibitory properties of BMP-2 muteins.

• A) After incubation of BMP-2 or a BMP-2 mutein (250 nM), the alkaline phosphatase activity was measured. The response elicited by each mutein was expressed as a% of the BMP-2 response. The values represent the mean values (+/- standard deviation) of 4 measurements.
  Muteins with filled symbols are changed with regard to their BMPR-II interaction, as shown in FIG. 4. Plus or cross symbols indicate muteins with an altered association or dissociation constant for binding to the BMPR-IA receptor chain.
• B) The dose-dependent induction of the activity of the alkaline phosphatase in starved C2C12 cells is for BMP-2 (○) and for BMP-2 muteins A34D (), D30K (▱) and P50A (Δ) are shown. The background absorption at 405 nm from 0.080 +/- 0.020 was not subtracted from the signal / background ratio to represent.
• C) The inhibition of the induction of alkaline phosphatase activity in starved C2C12 cells was determined after incubation with 250 nM BMP-2 mutein in the presence of 10 nM (o) or 20 nM (Δ) BMP-2. The response obtained alone in the presence of BMP-2 is indicated by a dotted line and was set at 100%. The values represent an average +/- standard deviation of 4 measurements.
  Muteins with filled symbols are changed with regard to their BMPR-II interaction. Plus or cross symbols indicate muteins with altered association or dissociation constants for binding to the BMPR-IA receptor.
• D) Inhibition of BMP-2 activity (10 nM BMP-2) by increasing doses possibly antagonistic (partially agonistic) BMP-2 muteins in starved C2C12 cells. The dose-response curves of Muteine A34D (o) H39D (▱) S88A (Δ), L90A (∇) and L100A () in the presence of 10 nM BMP-2  were induced after incubation of the cells (3 days) and analysis of alkaline Obtained phosphatase activity.

Fig. 3

Fig. 3 shows the biosensor analysis of the binding of BMP-2 and BMP-2 muteins of (A) Type I or (B) type II BMP receptor chains.

Fig. 4

Figure 4 shows the interaction of BMP-2 muteins with type I (BMPR-IA) or type II (BMPR-II, ActR-II) receptorectodomains.

The rate constants for the association (k _on ) and dissociation (k _off ) of a BMP-2 mutein at a concentration of 15, 30 and 45 nM with immobilized BMPR-IA receptor ectodomain were derived from the sensograms shown in FIG. 3 (A). The sensograms shown in Fig. 3 (B) were evaluated to derive the equilibrium binding of 45 nM mutein (EQ45) to immobilized BMPR-II or ActR-II receptor ectodomains. All values were normalized by taking the k _on , k _off and EQ45 values from BMP-2 as standard.

• A) Equilibrium binding of increasing concentrations of BMP-2 to the type I receptors BMPR-IA and BMPR-IB and to the type II receptors BMPR-II and ActR-II. The sensograms shown in FIG. 3 were evaluated to determine the binding of equilibrium to the immobilized receptor domains.
• B) Differential binding affinity of BMP-2 muteins to BMPR-II or ActR-II Receptors. The equilibrium bond during the biosensor analysis of 45 nM Mutein (EQ45) on BMPR-II is plotted against binding to ActR-II. The values are through the equilibrium binding of BMP-2 to the corresponding Normalized receptors.  
• C) Graphical representation of the rate constants for the association (k _on ) and dissociation (k _off ) of a BMP-2 mutein with the BMPR-IA receptor. Muteins that are specifically changed in terms of their k _on are identified by plus symbols, those with a specifically modified k _off by cross symbols.
• D) Graphical representation of the association constant (k _on ) for the BMPR-IA binding and the equilibrium binding (EQ45) to BMPR-II for the same muteins as in (C). Because both the association constants and the equilibrium bond depend on the concentration of the BMP-2 mutein, specific (and concentration-independent) changes become visible. Muteins with a specific decrease in the binding equilibrium are marked by filled circles.

Fig. 5

Figure 5 is a spatial model of BMP-2 (Scheufler et al., 1999) in which the type I receptor binding residues of the "wrist" epitope and the type II receptor binding residues of the "knuckle" epitope are designated. The assignment is shown in Fig. 1, as well as from the tables and lists on pages 12/13 and 17. Remains of one subunit are bold and italic, remainders of the other with simple capital letters.

On the small inserted schematic drawing, the dimeric protein is in the paper plane rotated 90 degrees around the long axis.

Fig. 6

Sequence assignment of factors of the TGF-β superfamily. The numbering follows Amino acid sequence of the mature human BMP-2.  

EXAMPLES material and methods Production of recombinant receptor ectodomains

An extracellular domain of human BMPR-IA comprising residues 24-142 (ten Dijke et al., 1993) including an N-terminal extension (GSGAMA) was expressed as a soluble thioredoxin fusion protein in E. coli. After thrombin cleavage, the protein was purified by affinity chromatography on BMP-2 Sepharose as described by Kirsch et al., (2000 (a)).

The extracellular domains of ActR-II (amino acid residues 19-126) (Matzuk and Bradley, 1992), BMPR-II (amino acid residues 27-151) (Rosenzweig et al., 1995) and BMPR-IB (amino acid residues 14-126) (Astrom et al., 1999) were expressed with a C-terminal thrombin cleavage site (LVPRGS) together with a 6 × His tag in SF-9 insect cells Pharmingen in accordance with the manufacturer's instructions. The corresponding DNA sequences were inserted into the BamHI site of the baculovirus transfer vector pAcGP67B (Pharmingen). The culture medium, which had been incubated for four days after infection of the SF9 cells with a MOI (multiplicity of infection) of 3, was applied to Ni-NTA-Agarose Qiagen in a washing buffer (50 mM NaH ₂ PO ₄ , pH 8.3 , 300 mM NaCl, 10 mM imidazole) at 4 ° C. The recombinant proteins were eluted with elution buffer (50 mM NaH ₂ PO ₄ , pH 8.3, 300 mM NaCl, 300 mM imidazole) and thoroughly against high salt HBS buffer (10 mM HEPES, pH 7.4, 500 mM NaCl, 3 , 4 mM EDTA) dialyzed. Finally, the ectodomains were adsorbed onto a BMP-2-Sepharose affinity matrix (Kirsch et al., 2000 (a)), washed and eluted with 4 M MgCl ₂ . The purified proteins were transferred into low salt HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3, 4 mM EDTA), concentrated using YM 10 ultrafiltration membranes and stored at -80 ° C.

The purified receptor proteins were incubated with equimolar Concentrations of sulfo-NHS-LC-biotin (Pierce) as described N-biotinylated (Shen et al., 1996).  

Production of BMP-2 muteins

A BMP-2 cDNA covering residues 283-396 of the mature BMP-2 protein plus the two N-terminal amino acids MA encoded (Ruppert et al., 1996) was one in vitro Cassette mutagenesis (Wang et al., 1997) for which synthetic double-stranded oligonucleotides were used. The BMP-2 muteins were in E. coli expressed, isolated as inclusion body, renatured and as in Ruppert et al., see. O., described, cleaned.

C2C12 [Alkaline Phosphatase (ALP)] test

The promyoblast cells C2C12 (ATCC CRL-1772, Blau et al., 1983) were at a density of 3 × 10 ⁴ cells per well in a microtiter plate with 96 wells for 3 days with 1 to 250 nM of each BMP-2 variant in 100 µl DMEM medium with 2% calf serum and antibiotics (100 U / ml penicillin G and 100 µg / ml streptomycin) stimulated at 37 ° C in a humidified atmosphere with 5% CO ₂ . The cells were washed with PBS and then lysed for 1 hour with 100 µl 1% NP40 in ALP buffer (0.1 M glycine, pH 9.6, 1 mM MgCl ₂ , 1 mM ZnCl ₂ ). The ALP activity was determined by incubating the lysed lines with 100 μl ALP buffer plus 1 mg / ml p-nitrophenyl phosphate for 15 minutes and measuring the absorbance at 405 nm. An A ₄₀₅ absorbance unit corresponds to 1.5 nmol p-nitrophenolate production per minute per 3 × 10 ⁴ cells. The results were expressed as means obtained in four independent experiments with a standard deviation (SD) of +/- 39%. Inhibition experiments carried out in the presence of 10 or 20 nM BMP-2 showed larger standard deviations, which are shown in the corresponding figures.

Biosensor interaction analysis

The BIA2000 system (Biacore) was used to record the binding of BMP-2 muteins to immobilized receptor ectodomains. The biotinylated proteins were separately fixed to a streptavidin-coated matrix of biosensor CM5 in flow cells 2 , 3 and 4 at a density of approximately 200 resonance units (RU), which corresponds to 200 pg protein (approximately 15 fmol receptor) per mm ² . BMP-2 muteins in concentrations of 15 to 30 nM in HBS buffer (10 mM Hepes, pH 7.4, 500 mM NaCl, 3.4 mM EDTA, 0.005% P20 (Biacore) via the flow cells 1 , 2 , 3 and 4 were perfused in series at a flow rate of 10 µl / min at 25 ° C. and the sensograms were recorded at a data sampling rate of 2.5 Hz, the association period was 20 minutes and the dissociation period was set to 6 minutes Free receptors were regenerated by perfusion with 0.1 M acetic acid, 1 M NaCl for 2 minutes The basic sensogram recorded for flow cell 1 (streptavidin control) was obtained from the sensograms used for flow cell 2 (BMPR -II), 3 (ActR-II) and 4 (BMPR-IA) were subtracted The differential sensograms were evaluated in accordance with the "fitting routine 2" according to the BIA evaluation software 2.2.4 (Biacore). The equilibrium binding of BMP-2 muteins at a concentration of 45 nM (EQ ₄₅ ) was done twice ppelt measured with a maximum standard deviation (SD) of +/- 20%. The given rate constants k _on for the association rate and K _off for the dissociation rate for the interaction between BMPR-IA and BMP-2 muteins are mean values which have been obtained in at least 12 measurements which were carried out with at least three different concentrations of the ligands . The standard deviations were 13% for k _on and 19% for k _off . Because the real stoichiometry of the complex formation is not yet certain, all sensograms were evaluated on the basis of an unsecured 1: 1 association model, and therefore only apparent but no absolute constants are given.

example 1 Selection of BMP-2 muteins

In order to identify functionally important amino acid side chains and receptor-binding epitopes in the mature portion of human BMP-2, 57 amino acid residues were individually substituted by in vitro mutagenesis (Kirsch et al., 2000 (b)). The substituted residues are shown in Fig. 1 above the BMP-2 sequence. The muteins were expressed in E. coli. A set of 42 muteins substituted at 40 different positions was obtained. Expression in E. coli resulted in dimeric proteins which could be obtained with a purity of better than 95% and in yields which were sufficient for the subsequent analysis of the biological activity and receptor binding.

In a first round of mutagenesis, 20 residues were found on the surface of the molecule exposed side chains selected covering the entire surface of the BMP-2 span like a net. After muteins with promising phenotypes had been obtained, the juxtapositioned surface residues became systematic exchanged. First, the residues were replaced by alanine to contribute to the to be able to estimate the replaced side chain for binding energy. Later that Amino acid residues replaced by charged residues to change the phenotypic Properties to observe after a charge has been introduced.

Example 2 Biological activity of BMP-2 muteins

The C2C12 cell test, which was used for the quantitative determination of the biological activity of the BMP-2 muteins, makes it possible to detect relatively small changes reproducibly. The mouse promyoblast cells rapidly differentiate into multinuclear myotubes under hunger conditions. BMP-2 inhibits this myogenic pathway and induces the formation of osteoblast-like cells that are positive for alkaline phosphatase (ALP). BMP-2 induced dose-dependent high alkaline phosphatase (ALP) activity in starving C2C12 cells with an ED ₅₀ of 20 +/- 10 nM ( Fig. 2B). The functional importance of BMPR-IA for the osteoinductive effects of BMP-2 in C2C12 cells is already known. The BMPR-IB receptor is only detected in negligible amounts and therefore probably does not play a functional role in these cells. The type II receptors BMPR-II and ActR-II are present in C2C12 cells and can be cross-linked with the BMP-2 ligand in the absence, more efficiently but in the presence of BMPR-IA. To date it has not been clearly established whether both type II receptors mediate the BMP-2 responses in host cells. Some BMP-2 muteins showed a clearly reduced activity when examined at a concentration of 250 nM on C2C12 cells ( Fig. 2A). Muteins A34D and L90A do not induce a significant response at all. Some other mutant proteins showed reduced activity in the range of 2% to 30% of BMP-2 activity. The symbols, which indicate the activity of the individual proteins at a concentration of 250 nM, are colored in accordance with the results of a receptor interaction analysis (see below). Red symbols indicate a reduced affinity for the BMPR-II ectodomain, while blue symbols indicate a changed interaction with the BMPR-IA ectodomain.

Representative examples of muteins with approximately 50% (D30K), less than 10% (P50A) and less than 1% (A34D) residual activity are shown in Figure 2c by activity curves. Because the BMP-2 proteins precipitated in the culture medium at concentrations above 500 nM, doses above 250 nM were not analyzed.

The changes in the biological activity of the muteins described can be caused by significant changes in the structure, stability or solubility of the protein due to amino acid substitutions. Alternatively, functional ones Side chains involved in the binding of type I or type II BMP-2 receptors, have been affected. The latter possibility, namely special changes in the muteins has been described in the following Experiments checked.

Example 3 Antagonist activity

Surprisingly, some of the muteins were able to inhibit BMP-2 activity at a concentration of 10 to 250 nM. When C2C12 cells were stimulated with a constant amount of BMP-2 in the presence of 250 nM mutein, the induction of ALP activity by the mutein A34D was reduced to less than 1%, by L90A to approximately 3%, by L100A to approximately 20% and by S88A to 80% of the value induced by BMP-2 in the absence of mutant proteins ( Fig. 2c).

The inhibitory properties of these antagonists / partial agonists were confirmed by determining the dose / inhibition curves shown in Fig. 2D. The mutant proteins A34D, L90A and L100A inhibited half-maximally at concentrations of 20 to 40 nM. This IC50 value is similar to a concentration of 10 nM BMP-2 during the test. Accordingly, the inhibitory muteins work at concentrations similar to BMP-2, most likely competing with BMP-2 for a common receptor binding site.

The detection of the BMP-2 muteins with antagonistic or partially agonistic Properties shows that special amino acid substitutions Changes have been made to the potency of the BMP-2 protein affect, but on the whole, receptor binding affinity leave undisturbed.

Example 4 Interaction of BMP-2 muteins with receptor ectodomains

It is already known that it is in BMC-2 receptors BMPR-IA and type C2C12 the type II receptors BMPR-II and ActR-II are there that mediate the BMP-2 responses seem to be. There was therefore a remote possibility that the functional Changes that were observed in some of the BMP-2 muteins, the result specific changes in BMP-2 epitopes for the binding of these receptor chains would. This hypothesis was substantiated by an interaction analysis investigated in which the recombinant ectodomains of BMPR-IA, BMPR-II and ActR-II were used. It would have been difficult in quantitative radioligand Binding experiments to bind BMP-2 muteins to whole cells investigate since the BMP-2 protein attaches to those in the extracellular matrix and to the Cell surface binds existing glycosaminoglycans. The interaction between the recombinant receptor and the ectodomains was able to Biosensor system can be recorded. It is already for other receptor systems that small changes in binding affinity or kinetics of Ligand binding with biosensor immobilized receptor domains are shown can. It turns out that the BMPR-II receptor proteins immobilized on the biosensor are very stable and without a few dozen cycles of ligand binding and dissociation Survive change in binding properties. The kinetics and the Equilibrium binding of all BMP-2 proteins were therefore among the same Conditions measured. Differences between BMP-2 and the muteins could be so  can be determined with certainty even if the values of the kinetics and the Equilibrium constants tend to be relative values.

Sensograms as shown in Fig. 3 were recorded and evaluated. Using immobilized BMPR-IA ectodomains, differences in the rate constants of complex formation (k _on ) and dissociation (k _off ) with the BMP-2 muteins could be easily analyzed, as shown in Fig. 3A for sensograms, all at 45 nM Concentration of muteins have been recorded. The concentration dependence of the equilibrium binding of BMP-2, as in ( Fig. 4A), leads to an apparent K _d of approximately 1 nM. This affinity is in the area of high affinity binding, such as e.g. B. between hGH and hGHbp or IL-4 and IL-4Rα, and is mainly due to the low dissociation rate of the ligand (apparente k _off ≅ 4 × 10 ^-4 s ^-1 ), which has a half-life for the complex of approximately 0 Means 5 hours. It could not be conclusively determined whether this exceptionally long half-life was caused by the simultaneous interaction of BMP-2 with 2 immobilized receptors or whether it resulted from a real 1: 1 interaction. The association rate (apparente k _on ≅ 7 × 10 ⁵ M ^-1 s ^-1 ) is comparable to that of other receptors. One set of muteins showed specifically increased dissociation rates for the complex with BMPR-IA, the K _off values being 2 to 5 times greater than those of BMP-2 (see FIG. 4C, light blue symbols). 2 muteins with a modification at position D30 (D30A, D30K) and 2 muteins at position W31 (W31A, W31C) belong to this subgroup. Another subgroup with 4 muteins had decreased rate constants for association to the BMPR-IA ectomain with K _on values that were 5 to 10 times lower than that of BMP-2 ( Fig. 4C, dark blue symbols). The reduced K _on values for V26A, F49A, P50A and H54D were only observed for BMPR-IA, but not for BMPR-II or ActR-II interactions, and therefore cannot be due to instability or impurity, ie lower effective concentrations of these muteins . Two muteins, K101E and Y103A, showed a 2-fold change in both K _on and K _off . The kinetics of the other BMP-2 muteins do not differ significantly from BMP-2 with regard to their interaction with BMPR-IA.

The binding of BMP-2 and BMP-2 muteins to the BMPR-II ectodomain could also be recorded despite the low affinity of this interaction ( FIG. 3 B). An apparent dissociation constant K _d of approximately 100 nM was derived from the concentration dependence of the equilibrium bond, as shown in Fig. 4A. The affinity of BMP-II for the ActR-II ectodomain was found to be slightly higher (K _d ≅ 50 nM). In contrast, the apparent K _d for the binding of activin to the ActR-II ectodomain was given as 2-7 nM (Donaldson et al., 1999). The sensograms shown in Figure 3B show that the muteins have clear differences in equilibrium binding to the type II receptor BMPR-II. Mutein A34D bound 5 times weaker than BMP-2 or muteins D30K and P40A. The kinetic constants for the interaction between BMP- 2 and the type II receptors are relatively large (k _on <10 ⁶ M ^-1 s ^-1 ; k _off <10 ^-2 s ^-1 ). This prevented a reliable evaluation of the K _on or k _off values. A specific subset of muteins that had substitutions at 5 different positions showed an equilibrium bond (EQ ₄₅ ) to the BMPR-II ectodomain that was 3 to 15 times lower than that of BMP-2 ( Fig. 4D; filled symbols) . These deviations were specific for the BMPR-II interaction. The k _on values of these muteins for BMPR-IA binding were normal.

Example 5 Binding determinants and antagonist / agonist activity of BMP-2 muteins

Muteins with a reduced affinity for BMPR-II were observed to behave as competitive inhibitors of BMP-2 during the C2C12 test. The determinants of the BMP-2 muteins for the ActR-II binding differ from those of the BMPR-II ( Fig. 4B). Mutein H39D showed no decrease, and muteins A34D and L90A showed only a 2-fold decrease in ActR-II affinity. The binding affinity of S88A and L100A was similarly changed for both type II receptor chains. Such differential effects of amino acid substitutions on binding to different receptors allows the construction of selective agonists that preferentially activate one or the other receptor chain.

In contrast to other receptor systems, none were used in the present system "hot spots" observed for binding. It could at most 5 to 30 times Change in kinetics or equilibrium constants in some muteins to be watched. However, there is a possibility that some of the The main determinants in the current collection of BMP-2 muteins are not are represented. These possibly missing determinants can be found under the Residues that after substitution led to proteins that were not expressed or could be won. Examples of this could e.g. B. G27 and W28. A however, another, more likely possibility is that hydrogen bonds bonds involving N or O atoms of the main peptide bonds with the Receptors interact and thus contribute to binding affinity.

It is also interesting that despite the small changes in binding affinity the biological activity of some of the muteins was significantly reduced. It cannot that some muteins are excluded by amino acid substitution are destabilized and during the 3-day incubation as part of the C2C12 test partially inactivated. However, this cannot be done for the antagonistic muteins apply, which in the case of A34D z. B. a 100-fold reduced biological activity exhibit. It is more likely, however, that the discrepancy to the extent changes in physical receptor binding and cellular activity in some of the muteins of avidity effects during the interaction of the multiple Binding epitopes of BMP-2 with multimeric cellular receptors in the membrane come from.

Example 6 Localization of binding epitopes

The amino acid positions of BMP-2, which determine binding affinity for either BMPR-IA or BMPR-II receptor chains, belong to 2 non-overlapping subgroups. As shown in Fig. 1, these determinants are distributed over the entire BMP-2 sequence. However, the space-filling model (Scheufler et al., 1999) of FIG. 5 shows that the functional residues form 2 separate epitopes on the surface of the homodimeric BMP-2 molecule.

The determinants for the BMPR-IA interaction colocalize in the wrist epitope that includes residues of subunit 1 (italic letters) and subunit 2 (normal letters). A monomer contributes residues V26, D30 and W31 from the long loop that connects sheets β-2 and β-3 and the weak determinants K101 and Y103 that appear in sheet β-8. The other monomer contributes residues I62, L66 and N68 from helix α3 and residues F49, P50 and H54 from the long loop before helix α3. The muteins with substitutions in this latter loop have remarkably reduced association rate constants. This may be related to the observation that the residues in this loop had the highest B factors of 40-90. The B factors are a measure of the order of the atoms in the crystal and the accuracy with which the atoms are defined in the protein model. The B factors are thus indirectly a measure of the mobility of the atoms in the protein crystal. An even higher mobility of this loop in the muteins could reduce the likelihood of a productive encounter with BMPR-IA and therefore slow down an association. The weak determinant H17 appears to be separate from the other functional wrist epitope residues. BMP-2 amino acid residues that have not yet been analyzed and are between H17 and H54 may represent additional contact points for BMPR-IA.

The BMP-2 knuckle epitope involved in BMPR-II binding settles from the remains of only one subunit. The residues A34 and H39 occur in the Leaflet β3 and β4 respectively, while S88 and L90 occur in β7 and L100 in β8. The Residue E109, which was found to be a by substitution with arginine Mutein with higher affinity for BMPR-II may be another contact residue. The Knuckle epitope appears to be smaller than the wrist epitope because there are many residues on the Border to the Knuckle epitope could be changed without a detectable To have an effect on receptor binding via biological activity. It cannot excluded that the Knuckle epitope includes other functional residues.  

The homodimeric BMP-2 protein has a double axis of symmetry, which lies in the paper plane in FIG. 5 and runs from top to bottom. There is therefore a second pair of wrist and knuckle epitopes on the back of the protein.

Example 7 Combination of amino acid substitutions in BMP-2 double mutants

The simultaneous substitution of 2 involved in the low affinity binding to BMPR-II Amino acids significantly increase the effects on biological activity. How The inhibitory activity was shown in Table III below of doubly substituted muteins increased considerably. Amazingly, it showed the combination protein A34D / D53A, in which both an amino acid from the Wrist epitope as well as an amino acid from the Knuckle epitope have been replaced at 20 nM BMP-2 the highest antagonistic activity of all muteins examined. This could be due to the fact that the D53A substitution (wrist epitope) is simple substituted mutein an increase in BMPR-IA affinity (and also in biological Activity) causes the interaction with the required for biological activity BMPR-II, however, is efficiently weakened by A34D at the same time.

Overall, however, it turns out that the combination of two mutations in the area of amino acids responsible for the low affinity binding a considerable antago has a nistic effect.  

Table III

Increased effects on the kinetic constants k _on and k _off for the association and dissociation of the complex with the BMPR-IA ectodomain; Equilibrium binding at 45 nM concentrations, EQ ₄₅ , to the BMPR-II and ActR-II ectodomains. Alkaline phosphatase activity in the presence of 250 nM mutein, ALP ( 250 ), was determined in the absence and presence of 10 nM or 20 nM BMP-2.

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SEQUENCE LOG

Claims (28)

1. Mutein a chain of a protein from the superfamily of growth factor TGF-β, the mutein being antagonistic after formation of a homodimer and / or partially agonistic activity and wherein the mutein on one or several position (s) is changed, which is in the unchanged protein at one low affinity binding to its receptor is / are. 2. Mutein according to claim 1, wherein the mutein by deletion and / or substitution and / or insertion and / or modification of one or more amino acids is changed. 3. Mutein according to claim 1 or 2, wherein the mutein from a chain of a BMP-2- similar protein is derived and one or more position (s) in the mutein from the "Knuckle" epitope is / are changed. 4. Mutein according to claim 3, wherein the BMP-2-like protein from the group is selected, the BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6 and GDF-7 includes. 5. Mutein according to one of claims 3 or 4, characterized in that a or more amino acid (s) selected from the group of amino acids which the surface-exposed areas from the β-sheet structures β3, β4, β7, β8 and / or form β9, is deleted and / or substituted and / or modified and / or that at least one of said surface-exposed areas is changed by insertion of one or more amino acids. 6. Mutein according to one of claims 3 or 4, characterized in that at least one of the amino acids connecting the β-sheets β3 and β4 deleted and / or substituted and / or modified and / or in which the β- Leaflets β3 and β4 connecting amino acid region one or more Amino acids are inserted.   7. Mutein according to one of claims 3 to 6, characterized in that at least one of the following amino acids is deleted, substituted and / or modified, the position information relating to BMP-2:
V33 A34 P35 P36 G37 Y38 H39 F41 Y42 T82 E83 L84 S85 A86 I87 S88 L90 K97 V98 V99 L100 V107 E109 and G110. 8. Mutein according to one of claims 1 to 7, characterized in that it at least 50% identity with a chain of a BMP-2-like Has growth factor from the TGF-β superfamily and antagonistic and / or shows partial agonistic activity. 9. Mutein according to one of claims 1 or 2, wherein the mutein of a protein is derived from the TGF-β / activin family and one or more in mutein Position (s) from the "Wrist" epitope is / are changed. 10. Mutein according to claim 9, wherein the protein of the TGF-β / activin family from the Group is selected, the TGF-β1, TGF-β2, TGF-β3, activins, inhibins, BMP-11 and GDF-8. 11. Mutein according to one of claims 9 or 10, characterized in that at least one amino acid selected from the group of amino acids which the surface-exposed areas from the helix in front of the sheet structure β1, the sheet structure β1, the long loop between the sheet structures β2 and β3, the loop in front of the helix α3, the helix α3 and the sheet structure β8 form, deleted and / or substituted and / or modified is / are and / or that at least one of the areas exposed to the surface Insertion of one or more amino acids is changed. 12. Mutein according to at least one of claims 9 to 11, characterized in that at least one of the following amino acids is deleted, substituted and / or modified, the position information relating to BMP-2:
K5, S13, V26, G27, W28, N29, D30, W31, P48, F49, P50, A52, D53, H54, N59, I62, V63, L66, N68, S69, V70, K101 and Y103. 13. Mutein according to one of claims 1 to 12, characterized in that it at least 50% identity with a chain of a TGF-β / activin-like Has growth factor from the TGF-β superfamily and antagonistic and / or shows partial agonistic activity. 14. Mutein according to one of claims 1 to 13, characterized in that it with is covalently linked to a target-specific molecule. 15. Mutein according to claim 14, characterized in that the target-specific Molecule with which mutein forms a fusion protein. 16. derivative of a protein from the TGF-β superfamily comprising a mutein according to one of claims 1 to 15 and a further chain of a protein the group of the TGF-β superfamily or another mutein according to one of the Claims 1 to 15. 17. A derivative according to claim 16, comprising a homodimer of muteins according to one of claims 1 to 15. 18. A derivative according to claim 16, comprising a heterodimer of muteins according to one of claims 1 to 15. 19. A pharmaceutical composition comprising at least one mutein according to one of claims 1 to 15 and / or a derivative according to any one of the claims 16 to 18 and / or a pharmaceutically acceptable salt thereof. 20. Use of a mutein according to any one of claims 1 to 15 and / or one Derivatives according to one of claims 16 to 18 for producing a Composition for the prophylaxis and / or treatment of diseases which  mediated by a protein from the superfamily of the growth factor TGF-β become. 21. Antibodies against a mutein according to any one of claims 1 to 15 or a derivative according to one of claims 16 to 18. 22. Nucleic acid containing a nucleic acid sequence selected from:

• a) a nucleic acid sequence coding for a mutein according to any one of claims 1-15;
• b) a nucleic acid sequence complementary to the nucleic acid sequence according to (i) and
• c) a nucleic acid sequence which hybridizes with a nucleic acid sequence according to (ii) and codes for a mutein which has antagonistic or partially agonistic activity after formation of a homodimer.

23. Nucleic acid according to claim 22, characterized in that the hybridi is carried out according to (iii) under stringent conditions. 24. Nucleic acid according to claim 19, characterized in that the nucleic acid genomic DNA, cDNA, synthetic DNA or RNA. 25. A nucleic acid according to any one of claims 22 to 24, further comprising one promoter suitable for expression control, the one for a mutein coding nucleic acid sequence is under the control of the promoter. 26. Vector containing a nucleic acid according to one of claims 22 to 28. 27. Host organism containing a nucleic acid according to one of claims 22 to 25 or a vector according to claim 26.   28. A method for producing a mutein according to any one of claims 1-15, comprising cultivating a host organism according to claim 27 below Expression suitable conditions and, if necessary, the purification of the expressed Muteins.