WO2001092298A9 - MUTEINE EINER KETTE EINES PROTEINS AUS DER SUPERFAMILIE DES WACHSTUMSFAKTORS TGF-$g(b) - Google Patents
MUTEINE EINER KETTE EINES PROTEINS AUS DER SUPERFAMILIE DES WACHSTUMSFAKTORS TGF-$g(b)Info
- Publication number
- WO2001092298A9 WO2001092298A9 PCT/EP2001/006166 EP0106166W WO0192298A9 WO 2001092298 A9 WO2001092298 A9 WO 2001092298A9 EP 0106166 W EP0106166 W EP 0106166W WO 0192298 A9 WO0192298 A9 WO 0192298A9
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- bmp
- mutein
- tgf
- muteins
- protein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/495—Transforming growth factor [TGF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
Definitions
- the present invention relates to muteins of a chain of a protein from the superfamily of growth factor TGF- ⁇ with antagonistic and / or partially agonistic activity, derivatives of a protein from the TGF-ß superfamily, pharmaceutical compositions which comprise the muteins and / or derivatives according to the invention and the for the nucleic acids encoding the muteins or derivatives thereof.
- TGF- (transforming growth factor) -ß comprises a large number of structurally related polypeptide growth factors, each of which regulates a beautiful range of cellular processes, including cell proliferation, cell line determination, differentiation, mobility, adhesion and cell death.
- the factors are expressed in accordance with complex temporal and tissue-specific patterns and play an important role in the development, homeostasis and repair of almost all tissues in eukaryotic organisms, from the fruit fly to humans. Overall, these factors are responsible for a substantial part of the intracellular signals that determine cell fate.
- TGF-ß signal transduction pathway involves receptor serine kinases on the cell surface, the substrates of which, the SMAD proteins, migrate into the nucleus after phosphorylation, where they activate the transcription of the target gene in cooperation with DNA-binding partners.
- the multifunctional nature of TGF-ß and the other factors belonging to the TGF-ß superfamily seem to be based on the interaction of different receptors, SMAD proteins and DNA-binding proteins. Disorders of this signal transduction pathway are the cause of various forms of human carcinoma and developmental disorders.
- the TGF-ß superfamily comprises different subfamilies, each with two to four members. A detailed overview of the different subfamilies and their properties is given, for example, in Massague (1998).
- Table I gives an overview of some of the most important approximately 20 members of the TGF-ß superfamily known to date which, as “bone morphogenetic proteins” and “growth and differentiation factors”, control the formation and regeneration of tissue in the adult organism and in early ones and late stages of embryonic development are significantly involved.
- the amino acid primary sequences of the members of the TGF- ⁇ superfamily sometimes have relatively little correspondence with one another, there are structural features common to all proteins of the different subfamilies. For example, all proteins of the superfamily are dimers, which are made up of two mostly identical monomers. Another common feature is the signal transduction pathway: all TGF-ß-like protein factors signal via cellular receptors that are composed of two different types of serine kinase receptor chains.
- the type I chain has a cytoplasmic GS box and a serine kinase that activates the cellular SMAD1 and -5 signaling proteins.
- the type I I chain activates a type I receptor serine kinase by transphosphorylation of the GS box segment.
- the small receptor ectodomains of type I or type II chains each 120 to 150 amino acids long, show very little similarity to one another, and different chains of the same type are also relatively little preserved.
- a common feature of all known receptor chains of the TGF-ß superfamily are four conserved disulfide bridges; additional disulfide bridges and the positions of a few amino acid residues appear to be characteristic of either type I or type II receptor proteins.
- the members of the TGF-ß superfamily break down into two groups with regard to their binding mechanism, that of the TGF-ß / activin-like proteins and that of the BMP (bone-morphogenetic protein) -2-like proteins.
- TGF-ß For the namesake of the superfamily, ie TGF-ß, an ordered sequential mechanism of binding to its cellular receptors has been described. Accordingly, an external ligand first 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 receptor that is highly affinity for TGF- ⁇ . There seems to be a comparable mechanism for the interaction of activins with their receptors.
- the members of the TGF-ß superfamily, the binding mechanism of which corresponds to that described for TGF-ß, were therefore referred to collectively as TGF-ß / activin-like proteins. These include all members of the TGF-ß superfamily, the N-terminal a 4th disulfide bridge exhibit. According to current knowledge, these are TGF-ß1, TGF-ß2, TGF-ß3, all activins and inhibins as well as BMP-11 and GDF-8.
- BMP-2 binding of BMP-2 to its cellular receptors follows a mechanism that differs from that established for the TGF-ß / activin-like proteins.
- the high affinity receptors for BMP-2 are the type I chains BMPR-IA, BMPR-IB and possibly also ActR-1.
- Type II chains themselves can also bind dissolved BMP-2, but with much less affinity. From this it was concluded that the order of type I and type II receptor interaction with BMP-2 is reversed compared to the order established for TGF-ß.
- the knowledge gained for the TGF-ß / -activin-like proteins can therefore not 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) are transmitted.
- TGF-ß superfamily All factors of the TGF-ß superfamily, including the closely related representatives of a subgroup, have their own specific function in the organism, which is reflected in the cell and stage-specific expression of the genes and in the effects of mutations.
- the inactivation of BMP or GDF genes can lead to death in mammals in various embryonic stages (BMP-2, BMP-4) or in the perinatal period (BMP-7); Furthermore, specific changes in the development of skeletal elements (GDF-5, BMP-5) have been observed.
- 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 the regeneration of ischemic kidney damage (BMP-7).
- GDF-8 myostatin
- the object of the invention is therefore to provide means with which the pathophysiological effects of members of the TGF- ⁇ superfamily can be reduced.
- this object is achieved by a mutein of a chain of a protein from the superfamily of the growth factor TGF- ⁇ , the mutein having antagonistic and / or partially agonistic activity after formation of a homodimer, the mutein being changed at one or more positions which is / are involved in a low-affinity binding to its receptor in the unchanged protein.
- an antagonist is understood to mean proteins which bind to the receptors for the natural proteins of the super family of the growth factor TGF- ⁇ , but which do not trigger the normal biological subsequent reactions with their binding. No signal transduction takes place after binding of an antagonist.
- “partially agonistic activity” is understood to mean an activity which, to a certain extent, triggers the normal biological subsequent reactions, but the extent of these subsequent reactions remains far behind that of the subsequent reaction triggered by a natural protein.
- a partial agonistic activity is therefore understood in connection with the present invention to be an activity which has less than 80%, preferably less than 50% and particularly preferably less than 25% of the activity of the corresponding natural protein. Test that is described below in the chapter "Material and Methods”.
- a “low-affinity bond” is understood to mean a bond which only reaches a half-maximum saturation of the at a concentration of the ligand of 10 nM or more, often even more than 100 nM or 1 ⁇ M
- a “high-affinity binding” is understood to mean a binding which, at a ligand concentration of less than 10 nM, often even less than 1 nM, leads to a half-maximum saturation of a receptor protein. The saturation as a function of the ligand concentration can be measured using a biosensor system as described in Example 4.
- a "high affinity” binding enables the ligand to bind to receptor type II even in the absence of receptor type I, as can be demonstrated in whole cells by chemical crosslinking with radiolabelled ligands
- the "low affinity” binding of the ligand to the receptor chain I in the absence of the type II chain is possible with very little efficiency and is enhanced in the presence of the type II chain. This can also be checked by cross-linking with radiolabelled ligands (see e.g. Massague 1998; Wuytens et al., 1999).
- an “unchanged protein” is understood to mean a growth factor from the TGF- ⁇ superfamily in the form in which it is found naturally in a mammal and exerts biological activity.
- the expression “in one or more position (s)” means that only a single amino acid is changed in mutein, but that, e.g. B. where more extensive deletions have been carried out, a larger number of amino acids can be changed. In preferred embodiments, between 1 to 50, particularly preferably 1 to 25 or 1 to 10, very particularly preferably between 1 to 5 amino acids are changed.
- the muteins can have a deletion of one or more amino acids, wherein the deletion of several amino acids can relate to several positions of the protein chain.
- muteins in which one or more amino acids are substituted by other amino acids are preferred, it being possible for the several amino acids to be adjacent or not adjacent.
- the group of basic amino acids includes arginine, lysine and histidine.
- the group of acidic amino acids includes glutamic acid and aspartic acid.
- the uncharged / polar amino acids include glutamine, asparagine, serine, threonine and tyrosine.
- the non-polar amino acids include methionine, phenylalanine, tryptophan, cysteine, glycine, alanine, valine and proline, leucine and isoleucine.
- a non-conservative substitution means the replacement of a given amino acid by an amino acid from another physicochemical group. It is particularly preferred to replace an amino acid of a first group with an amino acid of a second group, the amino acids of the second group having a different charge than the amino acids of the first group.
- a large amino acid with one of the small amino acids glycine, alanine or serine. It is also preferred to replace one of the small amino acids with one of the large amino acids tryptophan, tyrosine, phenylalanine, leucine, isoleucine or glutamine.
- one or more amino acids are inserted. Multiple amino acid insertions can occur at one or more positions in the chain.
- one or more of the specified amino acid residues are chemically modified.
- the modification can be e.g. B. is the covalent compound with one or more radicals selected from the following group: carboxylic acids, amines, polyethylene glycol, biotin and sugar (DeSantis et al., 1999).
- the mutein is derived from a chain of a BMP-2-like protein.
- the family of BMP-2-like proteins includes the BMP-2 subfamily, BMF-5 subfamily and GDF-5 subfamily (see classification of these Families of Massague (1998)).
- 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%, based on BMP-2 exhibit.
- 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.
- the inventors have determined the binding epitopes for all receptors involved.
- the amino acid positions of BMP-2, which determine the binding affinity for the BMPR-IA or BMPR-II receptor chains, were found to form two non-overlapping sets.
- FIG. 1 shows that these determinants are distributed over the entire BMP-2 sequence.
- the spatial model in Figure 5 shows that the functional residues form two separate epitopes on the surface of the homodimeric BMP-2 molecule.
- the determinants for the BMPR-IA interactions are arranged. 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 are identified by normal letters.
- the epitope is highly discontinuous and contains residues from the ß1-sheet, the loop in front of the helix ⁇ 3 and the helix ⁇ 3 from one monomer as well as parts of the large ⁇ -loop between the sheets ß2 and ß3 as well as the sheet ß8 of the other monomer.
- One of the monomers thus contributes the residues V26, D30 and W31 from the ⁇ -sheet areas ß-2 and ß3 and the residues K101 and Y103 from the ß-sheet area ß8.
- the other monomer carries the residues I62, L66, N68 and S69 from Helix 3 and the residues F49, P50, A52 and H54 from the Area in front of the helix ⁇ 3 at.
- this epitope is referred to as the “Wris epitope: the monomers are compared with an open hand, in which the central helix ⁇ 3 represents the wrist and 2 ß-leaflets 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.
- the first epitope located around the central a-helix is on the 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 located on the back of the hand near the outer finger segments, is responsible for the low affinity binding of BMP-2 to the BMPR-II. It is composed only of amino acid residues of a subunit and is also referred to as the "Knuckle" epitope.
- the amino acid residues A34 and H39 are arranged in the ⁇ -leaflets ß-3 and ß-4, the amino acid residues S88 and L90 in the leaflet ß- 7 and L100 in leaflet ß-8.
- amino acid residue E109 is another amino acid residue that is important for contact
- the second epitope seems to be much smaller than the first epitope, since there are many amino acid residues at the borders of the second Epitopes can be modified without any visible effects on receptor binding or biological activity, however it cannot be ruled out at the present time that the epitope contains further functional amino acid residues.
- the two epitopes are functionally and spatially separated. All binding determinants were found to be specific for either BMPR-I (Type I) or BMPR-II / ActR-II (Type II). In the case of BMP-2, antagonistic muteins could only be found for the Knuckle epitope.
- the different epitopes are defined by binding determinants and delimited by neutral residues. They do not form overlapping areas on the surface of the established 3-dimensional structure of BMP-2. However, it cannot be excluded that cooperative effects occur during type I and type II receptor binding.
- the wrist epitope and the knuckle epitope are separated from one another only by the thickness of a ⁇ -sheet, which can change the conformation after binding to the ectodomain and on this way can convey cooperative effects.
- the spatial separation of the epitopes further suggests that each of the symmetry-related parts of the dimeric BMP-2 molecule contains a pair of functional epitopes and that two independent wrist epitopes and two independent Knuckle epitopes can bind a total of 4 receptor chains.
- a complex between a BMP-2 and two BMPR-IA ectodomains has already been identified (Kirsch et al., 2000 (c)).
- 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 5 o 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 determine for BMP-2.
- BMP-2-like proteins activate their corresponding receptors according to the same mechanism as that shown for BMP-2, ie via one High affinity wrist epitope and a low affinity knuckle epitope
- antagonistic muteins of these proteins can also be generated by amino acid substitutions in the knuckle epitope.
- amino acid residues which form the surface-exposed regions from the ⁇ -sheet structures ⁇ -3, ⁇ -4, ⁇ -7, ⁇ -8 or ⁇ -9 are changed. These residues exposed to the surface are as follows:
- ß-3 V33, A34 between ß-3 and ß-4: P35, P36; ⁇ -4: G37, Y38, H39; after ⁇ -4: F41, Y42; ⁇ -6: T82, E83, L84, S85; ⁇ -7: A86, I87, S88, L90; ß-8: K97, V98. V99, L100; ß-9: V107, E109, G110.
- one or more of the specified amino acid residues are deleted individually or in groups of up to 5 amino acids.
- Amino acids for which an interaction with the BMP receptor II is proven or whose deletion has effects on the conformation of the “knuckle” epitope are preferably deleted.
- 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 :
- the BMP-2 position V33 in BMP-7 corresponds to an isoleucine
- A34 is also alanine
- P35 is also alanine
- P36 is also alanine
- P36 is also alanine
- P35 is also alanine
- P36 is also alanine
- P35 is also alanine
- P36 is also alanine
- P35 is also alanine
- P36 a glutamate
- H39 an alanine
- S88 a serine amino acid
- L90 a leucine V98 a valine
- L100 a leucine
- E109 an arginine
- FIG. 6 shows an alignment of the sequences of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, GDF-5, GDF-6, GDF-7 carried out with the "Multalin” program , 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-ß1, TGF-ß2, TGF-ß3, Inh-a, MIS and GDNF, from which the amino acids corresponding to a particular BMP-2 amino acid can be found in other members of the TGF-ß superfamily
- the positions determined by comparison with BMP-2 can also be changed by substitution, deletion or chemical modification, and the epitopes can also lose binding affinity by insertion, insertions being preferred immediately before or after the positions indicated.
- the invention further relates to muteins which have at least 50% identity at the amino acid level with a growth factor from the TGF- ⁇ superfamily and also have antagonistic and / or partially agonistic activity.
- This also includes muteins whose amino acid sequence differs from the amino acid sequence of the corresponding natural protein chains even in areas which are suitable for a antagonistic or partially agonistic activity are not decisive.
- identity known to the person skilled in the art denotes the degree of relationship between two or more DNA molecules or two or more polypeptide molecules, which is determined by the agreement between the sequences. The percentage of “identity” results from the percentage of identical regions in two or more sequences, taking into account gaps or other sequence peculiarities.
- 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., NucleicAcids 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 from other sources (BLAST Handbuch, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul, S., et al., J. Mol. 215: 403/410 (1990)).
- NCBI National Center for Biotechnology Information
- the well-known Smith Waterman algorithm can also be used to determine identity.
- Preferred parameters for sequence comparison include the following:
- the GAP program is also suitable for use with the above parameters.
- the above parameters are the default parameters for amino acid sequence comparisons, with gaps at the ends not reducing the homology value. In the case of very small sequences compared to the reference sequence, it may also be necessary to increase the expected value to up to 100,000 and, if necessary, to reduce the word length to up to 2.
- gap opening penalties can be used.
- the selection will depend on the comparison to be performed 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.
- 50% identity A match of 50% determined using the above-mentioned algorithm is referred to as 50% identity. The same applies to higher degrees of identity.
- the muteins according to the invention have an identity of 60% or more, e.g. B. more than 70% or 80%, with the sequence of a 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 more than 90, 95 or 97% identity are even more preferred.
- the epitope that binds low-affinity is the "knuckle” epitope
- the epitope that binds high-affinity to the receptor is the "Wrisf” epitope
- Muteins may still be derived from a protein of the TGF- ⁇ / activin family, but in this case it has surprisingly been shown that changes in the "Knuckle” epitope, but changes in the "Wris epitope lead to muteins with antagonistic and / or partially agonistic activity. Accordingly, there are one or more in muteins according to the invention which are derived from a protein of the TGF- ⁇ / activin family Amino acids from the “Wrisf epitope changed.
- the TGF-ß / Aktivin family includes TGF-ß 1, TGF-ß2, TGF-ß3, all activins, inhibins, BMP-11 and GDF-8.
- Previously known activins include, for example, activin ⁇ A, activin ⁇ B, activin ⁇ C and activin ⁇ E.
- the inhibins include ßA, ßB and ßC.
- antagonists or partial agonists are primarily caused by changes in amino acids in the areas exposed to the surface, i.e. H. those areas involved in binding to the receptor. These are essentially the helix in front of the leaflet structure ⁇ 1, the leaflet structure ⁇ 1, the long loop between the leaflet structures ⁇ 2 and ⁇ 3, the loop in front of the helix ⁇ 3, the helix ⁇ 3 and the leaflet structure ⁇ 8.
- changes can be brought about by deletions, substitutions or modifications, 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 here accordingly.
- At least one of the following amino acids is changed, i.e. H. deleted, substituted and / or modified and / or one or more amino acids inserted, the position information relating to BMP-2:
- this position information in TGF-ß1 corresponds to: Y6, N14, L28, G29, W30, K31, missing, W32, P49, Y50, 151, S53, missing, missing, Q57, K60, V61, L64, N66, Q67, H68, E99, L101.
- the muteins according to the invention can additionally be changed in the regions which are not essential for binding to the receptor.
- Muteins with an identity of at least 50% with a chain of a TGF- ⁇ / activin-like growth factor with an antagonistic and / or partially agonistic activity are also included.
- Such muteins can e.g. B. also come from other mammals, for example mouse, rat, rabbit, guinea pig, cattle, pig or sheep.
- Such muteins have an antagonistic and / or partially agonistic activity in the C2C12 cell test, they are also the subject of the invention.
- muteins according to the invention by making major changes to the underlying molecules, e.g. B. by inserting an insertion in addition to a substitution.
- Conceivable muteins of both subfamilies of the TGF- ⁇ superfamily also contain an insertion in addition to a deletion, or a deletion in addition to a substitution, or at least one substitution, deletion or insertion in connection with a chemical modification.
- 2 changes of one type e.g. B. a substitution in two different places, alone or in combination with a change of a second type, e.g. B. an insertion at another location.
- more than 2 types of changes can be combined.
- both a deletion at one point and an insertion at another point can be present.
- the person skilled in the art knows how to produce the muteins.
- the Merrifield synthesis are particularly suitable for substitution, deletion and insertion recombinant methods.
- mutations can be inserted in a targeted manner, e.g. B. by oligonucleotide-dependent site-specific mutagenesis. Fragments can be deleted or used.
- DNA sequences coding for the muteins can be synthesized de novo.
- the mutein described above is covalently linked to a target-specific molecule.
- a target-specific molecule This can e.g. be a heparin-binding epitope which effects an increased binding to the glycosaminoglycans of the extracellular matrix or the cell surface (see, for example, PCT / EP00 / 00637).
- this target-specific molecule is an antibody, e.g. selectively prevent signal transduction in cells that have a surface protein that is recognized by the antibody.
- target specificity can be imparted to the mutein not only by covalent binding to an antibody, but possibly also by covalent binding to a ligand that is specific for a receptor that only occurs on the target cell.
- muteins with a target-specific molecule covalently bound thereto are produced by recombinant expression of a fusion protein which optionally contains a spacer between the mutein-coding sequence and the target-specific molecule.
- the invention further relates to derivatives of proteins from the TGF- ⁇ superfamily which contain, as an essential component, a mutein according to the present invention and, to form a dimer, a further chain of a protein from the group of the TGF- ⁇ superfamily or a further mutein according to the invention ,
- the derivatives can therefore both homodimers and heterodimers Form muteins according to the invention.
- a derivative can comprise a mutein and a natural chain of a protein from the TGF- ⁇ super family.
- the derivative is a heterodimer consisting of a mutein with at least one mutation in the “wrisf epitope and a natural chain of a protein from the TGF-beta super family.
- the natural chain of the heterodimer BMP-2 is particularly preferred.
- the mutein of the heterodimer BMP-2 with the double substitution F49A and P50A is also particularly preferred.
- the heterodimer particularly preferably consists of BMP-2 and a BMP-2 mutein with the double substitution F49A and P50A.
- heterodimer consisting of a mutein with at least one mutation in the “wrisf epitope and a natural chain of a protein from the TGF-beta super family is a partial agonist works (see Example 8).
- the heterodimer has about 75% less biological activity than a homodimer, which consists of two natural chains.
- the derivative is a heterodimer which consists of a mutein with at least one mutation in the “knuckle” epitope and a natural chain of a protein from the TGF-beta super family.
- the natural chain of the heterodimer is particularly preferred BMP-2.
- the mutein of the heterodimer BMP-2 with the double substitution A34D and D53A is particularly preferred.
- the particularly preferred heterodimer consists of BMP-2 and a BMP-2 mutein with the double substitution A34D and D53A.
- a heterodimer consisting of a mutein with at least one mutation in the “knuckle” epitope and a natural chain of a protein from the TGF-beta super family acts as a complete high-affinity antagonist, as by the inhibition of Induction of the ALP activity in C2C12 cells was determined (cf. Example 9). In the absence of an agonist, the heterodimer has no ALP-inducing activity of its own (cf. Example 8).
- heterodimer Due to the mutation of a monomer, such a heterodimer should only have an intact “knuckle” epitope, which would be expected to show a reduced but present ALP activity compared to a homodimer consisting of two natural chains. However, the remaining “knuckle” epitope in the heterodimer is evidently unable to compensate for the loss of the other epitope so that the heterodimer acts as a complete antagonist.
- a further advantage here is the fact that the antagonist is highly affine and thus effectively displaces the natural ligand from the receptor even at a low concentration. The efficiency at low concentration makes the heterodimer particularly interesting for therapeutic use.
- the invention further relates to pharmaceutical compositions which contain at least one protein according to the invention and / or a derivative according to the invention.
- the invention also includes pharmaceutically acceptable salts thereof.
- the pharmaceutical compositions can be provided in the form of ointments, creams, lotions for topical application, in the form of solutions or lyophilisates for intramuscular or subcutaneous injections.
- the formulation and packaging of the pharmaceutical compositions is carried out according to the state of the art and includes u. a. the stabilization.
- a mutein according to the invention and / or a derivative according to the invention for producing pharmaceutical compositions is claimed.
- These can be used for the prophylaxis and / or for the treatment of diseases which are mediated by a protein from the superfamily of the TGF- ⁇ growth factor. Examples of such diseases are ectopic bone formation, psoriasis and muscle wasting, scarring, fibrosis and cirrhosis.
- a mutein of one of the growth factors BMP-2 or BMP-4 is preferably used, while z. B.
- a mutein of one or more of the growth factors TGF- ⁇ 1, -ß2 or -ß3 or a derivative thereof is preferably used.
- antibodies against a mutein according to the invention or a derivative according to the invention are provided. Since the muteins are characterized by a change in areas of the molecule exposed to the surface, this also has an effect on the antibody populations which react specifically with the molecule.
- Antibodies can be produced in a conventional manner either by immunizing animals (e.g. rabbits, mice or rats) to produce polyclonal antibodies or by immunizing and then immortalizing antibody-producing cells in the case of monoclonal antibodies.
- the invention further relates to the nucleic acids coding for the muteins according to the invention.
- These contain a nucleic acid sequence that codes for a desired mutein.
- the nucleic acid sequence for BMP-2 is e.g. B. from Wozney et al. (1988) known for TGF- ⁇ 2 from Madisen et al. (1988).
- the nucleic acid sequence coding for a mutein differs therefrom primarily by the triplets coding for the modified amino acids, ie by the absence, exchange or insertion of one or more codons.
- the mutein is a mutein with an identity of 50% or more at the amino acid level
- the corresponding nucleic acids also have a reduced identity compared to the nucleic acid sequence used for a natural mature protein chain. Nucleic acids deviating from this because of the degeneration of the genetic code are also included.
- the nucleic acid sequences encoding the muteins are complementary and nucleic acids which hybridize with these complementary sequences under stringent conditions and encode a mutein which, after the formation of a homodimer, has antagonistic or partially agonistic BMP-2 activity.
- Stringent conditions include hybridization at 68 ° C in 0.5 x SSC.
- the nucleic acid according to the invention can be a genomic DNA, a cDNA, a synthetic DNA or an RNA.
- Genomic DNAs or cDNAs can be isolated from the corresponding gDNA or cDNA banks by methods known in the art.
- tissue or cell line-specific banks e.g. B. from U-2 OS osteosarcoma banks or prostate adenocarcinoma banks, preferred.
- Synthetic DNA can be produced by known methods
- RNA can be isolated either by means of RNA vectors or from mRNA.
- a genomic DNA or cDNA will be preferred for the recombinant production of muteins according to the invention, although expression by means of RNA vectors is not excluded.
- codon coding for the original amino acid can be replaced by methods known in the prior art.
- codons coding for one or more amino acids are removed, while in the case of insertions codon triplets which code for the desired amino acids are used.
- codons in the case of substitution or insertion the person skilled in the art will endeavor to take the codon use of the intended host organism into account. The corresponding information is available in the prior art.
- nucleic acids are also provided which contain a promoter suitable for expression control, the nucleic acid sequence coding for a mutein according to the invention being under the control of this promoter.
- a suitable promoter is in turn dependent on the choice of the expression system. The person skilled in the art can choose between a large number of known, inducible or constitutive promoters for a wide variety of host organisms.
- the invention further relates to a vector which contains a nucleic acid according to the invention and to host organisms which contain a Nucleic acid sequence coding for a mutein, either integrated directly into the genome or containing in the form of an autonomously replicating vector.
- a vector which contains a nucleic acid according to the invention and to host organisms which contain a Nucleic acid sequence coding for a mutein, either integrated directly into the genome or containing in the form of an autonomously replicating vector.
- Numerous prokaryotic and eukaryotic expression systems are known in the prior art, the host cells being 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.
- certain CHO production lines are known in the prior art whose glycosylation patterns have changed compared to CHO cells.
- the polypeptides obtained by using glycosylation-efficient or glycosylation-reduced host cells have an altered spatial structure, which may be associated with an altered biological activity.
- the invention also relates to a method for producing a mutein according to the invention, the method comprising culturing a host cell under the conditions suitable for expression and, if appropriate, purifying the expressed mutein according to methods known in the art.
- FIG. 1 shows the sequences for BMP-2, BMP-7, TGF-ß2 and TGF-ß3, the corresponding amino acids being arranged one below the 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. Show simple vertical lines indicated that no measurable changes in the function of the corresponding muteins could be found
- the numbering refers to the BMP-2 sequence.
- Figure 2 provides information about the biological activity and the inhibitory properties of BMP-2 muteins.
- 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.
- Muteins with filled symbols are changed with regard to their BMPR-II interaction. Plus or cross symbols indicate muteins with modified ones Association or dissociation constants for binding to the BMPR-IA receptor.
- FIG. 3 shows the biosensor analysis of the binding of BMP-2 and BMP-2 muteins to (A) type I or (B) type II BMP receptor chains.
- FIG. 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 in order 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 , ot r and EQ45 values from BMP-2 as standard.
- FIG. 5 is a spatial model of BMP-2 (Scheufler et al., 1999) in which the residues of the “wrisf epitope determining the type I receptor binding and the residues of the“ knuckle ”epitope determining the type I I receptor binding are designated are.
- the dimeric protein is rotated by 90 degrees around the long axis in the paper plane.
- FIG. 7 shows the dose-dependent induction of the activity of alkaline phosphatase (ALP activity) in C2C12 cells by BMP-2 muteins.
- the ALP activity values were determined after subtracting the background (approx. 70 arbitrary ALP units) and related to the maximum response of 100% (approx. 2300 arbitrary ALP units).
- ALP activity induced by BMP-2 muteins in C2C12 cells was determined in the presence of 250 nM BMP protein (ALP250). The values show an average of 12 measurements +/- standard deviation (SD). The response in the presence of medium alone was determined as a control value.
- FIG. 8 shows the antagonistic activity of the heterodimer B2ell- / B2m-, which carries mutations in the “knuckle” epitope.
- the heterodimer B2ell- / B2m- is a complete, high-affinity antagonist with regard to the induction of ALP activity.
- 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, pH 8.3, 300 mM NaCI, 300 mM imidazole) and thoroughly against high salt HBS buffer (10 mM HEPES, pH 7.4, 500 mM NaCI, 3, 4 mM EDTA) dialyzed.
- 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 N-biotinylated by incubation with equimolar concentrations of sulfo-NHS-LC-Biotin (Pierce) as described (Shen et al., 1996). Production of BMP-2 muteins
- a BMP-2 cDNA encoding residues 283-396 of the mature BMP-2 protein plus the two N-terminal amino acids MA was subjected to an in vitro cassette mutagenesis (Wang et al. , 1997), for which synthetic double-stranded oligonucleotides were used.
- the BMP-2 muteins were expressed in E. coli, isolated as inclusion bodies, renatured and purified as described in Ruppert et al., See above.
- B2m- denotes a BMP-2 molecule with an altered N-terminal segment (referred to as EHBMP-2 in Ruppert et al., 1996).
- the heterodimer B2el- / B2m- consists of a monomer B2m- and a BMP-2 mutein with the amino acid substitutions F49A and P50A, which are in the wrist epitope.
- the homodimer B2el- / B2el- is the corresponding homodimer, which is deficient in two wrist epitopes.
- the heterodimer B2ell- / B2m- consists of a monomer B2m- and a BMP-2 mutein, which has the amino acid substitutions A34D and D53A. These amino acid substitutions relate to the "knuckle" epitope.
- the homodimer B2ell- / B2ell- represents the corresponding homodimer which has the mutations in both monomers of the BMP molecule.
- 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 ⁇ l DMEM medium stimulated with 2% calf serum and antibiotics (100 U / ml penicillin G and 100 ⁇ g / ml streptomycin) 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 ul 1% NP40 in ALP buffer (0.1 M glycine, pH 9.6, 1mM MgCl 2 , 1mM ZnCl 2 ).
- ALP activity was determined by incubating the lysed cells with 100 ⁇ l ALP buffer plus 1 mg / ml p-nitrophenyl phosphate for 15 minutes and measuring the absorbance at 405 nm.
- An A 05 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.
- 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 ⁇ l / min at 25 ° C.
- 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, and the basic sensogram recorded for flow cell 1 (streptavidin control) was derived from the sensograms for flow cells 2 (BMPR-II), 3 (ActR-II) and 4 (BMPR-IA) were subtracted.
- the differential sensograms were evaluated in accordance with the "fitting routine 2" in accordance with the BIA evaluation software 2.2.4 (Biacore).
- the equilibrium binding of BMP-2 muteins at a concentration of 45 nM was measured twice twice with a maximum standard deviation (SD) of +/- 20%.
- the given rate constants k on for the association rate and k 0ff 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.
- 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 a dose-dependent high alkaline phosphatase (ALP) activity in starving C2C12 cells with an ED 50 of 20 +/- 10 nM ( Figure 2 B).
- ALP alkaline phosphatase
- 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 they were examined at a concentration of 250 nM on C2C12 cells (FIG. 2A). Muteins A34D and L 90A do not induce any significant response at all.
- Some other mutant proteins showed reduced activity in the range of 2% to 30% of BMP-2 activity.
- the symbols indicating the activity of the individual proteins at a concentration of 250 nM are consistent with the results of a receptor interaction analysis (see below) colored. Red symbols indicate a reduced affinity for the BMPR-II ectodomain, while blue symbols indicate a changed interaction with the BMPR-IA ectodomain.
- muteins were able to inhibit BMP-2 activity at a concentration of 10 to 250 nM.
- 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 which was induced by BMP-2 in the absence of mutant proteins (FIG. 2 c).
- the inhibitory properties of these antagonists / partial agonists were confirmed by determining the dose / inhibition curves shown in Figure 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 the respective amino acid substitutions have caused special changes that affect the potency of the BMP-2 protein affect, but broadly unaffected by receptor binding affinity.
- Sensograms as shown in FIG. 3 were recorded and evaluated.
- differences in the rate constants for complex formation (k o ⁇ ) 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. 4 A) 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.
- One set of muteins had 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. 4 C, 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 reduced rate constants for association with the BMPR-IA ectomain with K on values that were 5 to 10 times lower than those of the BMP-2 (FIG. 4 C, dark blue symbols).
- the sensograms shown in Figure 3B show that the muteins show clear differences in the 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 ;ur> 10 "2 s " 1 ). This prevented a reliable evaluation of the k on or k off values.
- 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 Figure 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 ß-2 and ß-3, as well as the weak determinants K101 and Y103 that occur in sheet ß-8.
- the other monomer contributes residues I62, L66 and N68 from helix ⁇ 3 and residues F49, P50 and H54 from the long loop before helix ⁇ 3.
- the muteins with substitutions in this latter loop have remarkably reduced association rate constants. This may be related to the observation that the remnants 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 Knuckle epitope of BMP-2 which is involved in the binding of BMPR-II, is composed of the residues of only one subunit.
- the residues A34 and H39 occur in the leaflet ß3 and ß4, respectively, while S88 and L90 occur in ß7 and L100 in ß8.
- Residue E109 which, after substitution with arginine, was found to produce a mutein with higher affinity for BMPR-II, may be another contact residue.
- the Knuckle epitope appears to be smaller than the wrist epitope, since many residues at the border to the Knuckle epitope could be changed without having a detectable effect on receptor binding via biological activity. It cannot be 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.
- the similar biological activity of these 3 homo- or heterodimers is in agreement with the biosensor-based binding studies, which have shown similar affinities of these proteins for the BRIA or BRII ectodomains.
- the heterodimer B2el- / B2m- showed a reduced biological activity, measured by the determination of the ALP activity, which corresponded to approximately 25% of the activity of the heterodimer BMP-2 / B2m- at a concentration of 250 nM (FIG. 7B). From the shift to the right of the dose response curve of the heterodimer B2el- / B2m- an ED 50 value can be estimated which is approximately 10 times lower than that of the homodimer B2m- / B2m-.
- the homodimer B2ell- / B2ell- which is deficient in both "knuckle" epitopes, had no significant ALP activity (FIG. 7B).
- the heterodimer B2ell- / B2m- was not able to perform ALP activity in C2C12 cells (Figure 7B), although this heterodimer has one epitope for the BRII and two epitopes for the BRI binding.No significant activity was observed for the heterodimer B2ell / B2m ⁇ . Because the standard deviation 5 was arbitrary ALP units (FIG. 7 C), the activity of the heterodimer B2ell- / B2m- is less than 0.2% of the activity of the heterodimer BMP-2 / B2m-, which caused a maximum response of 2400 units (FIG. 7 C) ,
- Example 9 Antagonistic activity of the heterodimer B2ell- / B2m-
- the heterodimer B2ell- / B2m- has the property of a high affinity complete antagonist.
- the dose-dependent inhibition of the heterodimer B2ell- / B2m- shown in FIG. 8 shows a half-maximum inhibition at 20 nM in the presence of 10 nM BMP-2 / B2m-. Larger amounts are required if 20 nM BMP-2 / B2m- is present. Similar dose inhibition curves were obtained with the homodimer B2m- / B2m- and BMP-2.
- the IC 50 value is only twice as high as the ED 5 o-value of the agonistic BMP-2 protein.
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DE2000126713 DE10026713A1 (de) | 2000-05-30 | 2000-05-30 | Mutein einer Kette eines Proteins aus der Superfamilie des Wachstumsfaktors TGF-beta |
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EP1571159A1 (de) | 2004-03-04 | 2005-09-07 | Bayerische Julius-Maximilians-Universität Würzburg | Mutein von Knochenmorphogenetisches Protein und seine Verwendung |
JP2007530055A (ja) | 2004-03-26 | 2007-11-01 | アクセルロン ファーマ インコーポレーテッド | Bmp−3プロペプチドおよび関連する方法 |
WO2005113590A2 (en) | 2004-05-12 | 2005-12-01 | Acceleron Pharma Inc. | Bmp10 propeptides and related methods |
EP1751185A2 (de) * | 2004-05-27 | 2007-02-14 | Acceleron Pharma Inc. | Tgf derepressoren und deren verwendungen |
US7465706B2 (en) | 2004-06-24 | 2008-12-16 | Acceleron Pharma Inc. | GDF3 propeptides and related methods |
US7795389B2 (en) | 2004-09-28 | 2010-09-14 | The Board Of Regents Of The University Of Texas System | Antagonizing TGF-beta activity with various ectodomains TGF-beta receptors used in combination or as fusion proteins |
EP1721909A1 (de) * | 2005-05-10 | 2006-11-15 | BIOPHARM GESELLSCHAFT ZUR BIOTECHNOLOGISCHEN ENTWICKLUNG VON PHARMAKA mbH | Wachstumsfaktoren mit geänderten biologischen Eigenschaften |
GB0604966D0 (en) | 2006-03-11 | 2006-04-19 | Renovo Ltd | Medicaments and proteins |
GB0604964D0 (en) | 2006-03-11 | 2006-04-19 | Renovo Ltd | Protein folding |
GB0604938D0 (en) * | 2006-03-11 | 2006-04-19 | Renovo Ltd | Proteins, nucleic acids and medicaments |
CA2708549C (en) * | 2007-12-21 | 2014-04-01 | Stryker Corporation | Bmp mutants with decreased susceptibility to noggin |
CN102369212B (zh) * | 2009-03-12 | 2015-12-16 | 哈瑟投资公司 | 具有降低的bmp拮抗剂敏感性的骨形成蛋白2(bmp2)变体 |
US9688735B2 (en) | 2010-08-20 | 2017-06-27 | Wyeth Llc | Designer osteogenic proteins |
EP2949338B1 (de) | 2010-08-20 | 2017-10-25 | Wyeth LLC | Osteogene designerproteine |
EP2602264A1 (de) | 2011-12-05 | 2013-06-12 | Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka mbH | GDF-5-Mutant zur Induzierung der Knorpelbildung |
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US5284763A (en) * | 1985-03-22 | 1994-02-08 | Genentech, Inc. | Nucleic acid encoding TGF-β and its uses |
US5631142A (en) * | 1986-07-01 | 1997-05-20 | Genetics Institute, Inc. | Compositions comprising bone morphogenetic protein-2 (BMP-2) |
DE01201555T1 (de) * | 1988-04-08 | 2004-07-08 | Stryker Corp., Kalamazoo | Biosynthetische osteogene Proteine und solche enthaltende osteogene Einrichtungen |
GB8927546D0 (en) * | 1989-12-06 | 1990-02-07 | Ciba Geigy | Process for the production of biologically active tgf-beta |
US5399677A (en) * | 1993-12-07 | 1995-03-21 | Genetics Institute, Inc. | Mutants of bone morphogenetic proteins |
EP0691349A3 (de) * | 1994-07-04 | 1997-09-10 | Hoechst Japan | Dimere Knochenmorphogeneseproteine und ihre Fragmente und Analoge und sie enthaltende pharmazeutische Zubereitungen |
TW517059B (en) * | 1994-07-25 | 2003-01-11 | Ciba Geigy Ag | New process for the production of biologically active protein |
WO2000001410A1 (en) * | 1998-07-06 | 2000-01-13 | Beth Israel Deaconess Medical Center | Methods of inhibiting proliferative diseases by inhibiting tgf-beta mediated angiogenesis |
US6677432B1 (en) * | 1998-10-07 | 2004-01-13 | Stryker Corporation | Mutations of the C-terminal portion of TGF-β superfamily proteins |
JP2000119192A (ja) * | 1998-10-09 | 2000-04-25 | Aventis Pharma Ltd | 骨誘導因子アンタゴニスト活性を有する成熟型蛋白質 |
ES2226467T5 (es) * | 1998-11-13 | 2009-08-27 | Stryker Corporation | Aliviamiento de los sintomas del cancer de prostata. |
EP1292330A1 (de) * | 2000-03-31 | 2003-03-19 | Vaccine Chip Technology APS | Immunostimulierende eigenschaften eines tgf-beta-fragmentes |
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