WO2008028684A2 - Peptides présentant une affinité élevée avec le récepteur de la prolactine - Google Patents

Peptides présentant une affinité élevée avec le récepteur de la prolactine Download PDF

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WO2008028684A2
WO2008028684A2 PCT/EP2007/007863 EP2007007863W WO2008028684A2 WO 2008028684 A2 WO2008028684 A2 WO 2008028684A2 EP 2007007863 W EP2007007863 W EP 2007007863W WO 2008028684 A2 WO2008028684 A2 WO 2008028684A2
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amino acid
peptide according
isolated peptide
acid residue
seq
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PCT/EP2007/007863
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English (en)
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WO2008028684A3 (fr
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Kasper Rand
Mette Dahl Andersen
Ole Hvilsted Olsen
Jens Breinholt
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Novo Nordisk A/S
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Priority to PCT/EP2008/052784 priority Critical patent/WO2009003732A2/fr
Priority to EP08717531A priority patent/EP2167116A2/fr
Publication of WO2008028684A2 publication Critical patent/WO2008028684A2/fr
Publication of WO2008028684A3 publication Critical patent/WO2008028684A3/fr
Priority to PCT/EP2008/058593 priority patent/WO2009004058A2/fr
Priority to EP08802942A priority patent/EP2167059A2/fr
Priority to PCT/EP2008/058589 priority patent/WO2009004057A2/fr

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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
    • C07K14/575—Hormones
    • C07K14/57554—Prolactin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention related to variants of prolactin, which variants are binds to the prolactin receptor with higher affinity as well as method for producing such variants.
  • Such prolactin variant mutations may be useful for producing prolactin antagonists for use in the treatment of for instance breast cancer.
  • Prolactin is a cytokine with a variety of biological functions, mainly related to lactation, reproduction, osmoregulation and immunoregulation.
  • PRL is a four-helix bundle protein of 199 residues (Somers et al., Nature 372, 478-481 (1994)). The four antiparallel ⁇ - helices of the helix bundle are numbered 1 -4 as they occur from the N-terminus of the primary sequence i.e.
  • Helix 1 (residues 15-43), Helix 2 (residues 78-103), Helix 3 (residues 111-137) and Helix 4 (residues 161-193)
  • PRL furthermore comprises two minor helices denoted Helix 1 1 (residues 59-63) and Helix 1 " (residues 69-74), which are present in the loop connecting Helix 1 and Helix 2 (Teilum et al. J. MoI. Biol. 35J., 810-823 (2005)), see also Figure 1.
  • PRL is a potent growth factor for mammary epithelium and PRL has been associated with the development and growth of breast tumours. Furthermore, breast cancer cell lines often over-express the PRL receptor (PRL-R). Inhibiting pituitary secretion of PRL by dopamine agonists has no effect on breast tumours and it has been established that the tumour is bypassing the effect of the dopamine agonists by its own autocrine production of PRL. Thus for treatment of breast cancer it is not sufficient to inhibit the regular pituitary PRL production, but a PRL antagonist is necessary in order to prevent binding of autocrine PRL to the PRL-R on the tumour.
  • PRL-R PRL receptor
  • PRL binds two molecules of PRL-R through two regions on PRL referred to as binding site 1 (BS1 ) and binding site 2 (BS2).
  • the resulting dimerization of the receptor in a 1 :2 PRL:PRL-R complex is necessary for activation of the receptor and further signal transduction.
  • variants of PRL solely able to bind via BS1 will have antagonistic properties (see for instance Clevenger et al. Endocr Rev 24, 1 (2003); Goffin et al. Endocr Rev 26, 26 (2005).
  • PRL does not bind to the growth hormone receptor (GH-R); however growth hormone (GH) is able to bind both GH-R and PRL-R via with different, but overlapping, sites on GH (Cunningham and Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991 )).
  • PRL antagonists may be created by interfering with binding of PRL-R to PRL via BS2 for instance by mutating one or more small hydrophobic residues in BS2 to for instance large polar residues (e.g G129R, see for instance Goffin et al. Endocr Rev 26, 26 (2005)) or otherwise sterically interfere with binding of PRL-R to BS2.
  • Such a variant PRL can subsequently only bind PRL-R via BS1 and will thus have attained antagonistic properties.
  • prolactin G129R antagonists can inhibit tumor growth in vivo (Chen et al., Int. J. Oncology 20, 813-818 (2002)), it has also been stated that high level of prolactin receptor antagonists are necessary to obtain effects in vivo ( literature (Goffin et al., Endocrine Rev. 26, 400-422 (2005)). By improving pharmacokinetic parameters could lead to a compound which shows effect in vivo at a dose which are acceptable or desirable for a drug.
  • the binding affinity of the antagonist to BS1 should be retained, or even improved. Residues within BS1 of the PRL antagonist could for instance be mutated with the purpose of increasing favourable interactions or creating novel interactions in the binding interface with PRL-R at BS1.
  • BS1 has generally been described to comprise the region bordered by Helix 1 and Helix 4 specifically involving residues Val-23, His-30, Phe-37, Lys-69, Tyr-169, His-173, Arg- 176, Arg-177, His-180, Lys-181 , Tyr-185, and Lys-187 (Teilum et al. J. MoI. Biol. 351, 810- 823 (2005)),These results have been obtained by random mutagenesis of all PRL residues while screening for mutations that affect PRL-R binding. This is both a lengthy and potentially misleading approach due to, for instance, secondary effects of the mutations. Consequently, the creation of high affinity prolactin antagonists is problematic, since the PRL BS1 has not been precisely identified.
  • the present invention is concerned with peptides binding to the prolactin receptor, wherein said peptides have an improved binding via binding site 1 (BS1 ) to the prolactin receptor.
  • the present invention is concerned with an isolated peptide, which peptide is a variant of human prolactin or human growth hormone, and which binds to the prolactin receptor, said variant comprising
  • Figure 1 The primary sequence (using wt PRL numbering) and secondary structure of vPRL is displayed above the HX analyzed peptides (shown as horizontal bars).
  • ECD-PRL-R are colored in grey.
  • vPRL peptides Deuterium incorporation of vPRL peptides is plotted against time on a logarithmic scale in the presence (triangles) and the absence (circles) of ECD-PRL-R. Apart from peptide 101-1 13, the peptides shown are a part of BS1 in PRL.
  • FIG. 4 (A) Binding site 1 mapped onto the NMR structure of PRL by identifying peptides (shown in black) with reduced deuterium incorporation after 1000s hydrogen exchange in the presence of ECD-PRL-R. (B) A 90 c rotation along the vertical axis of the structure shown in (A). The figure was prepared using the NMR structure of human PRL (pdb entry: 1 RW5) using the software program PyMoI.
  • FIG. 1 Sequence alignment of prolactin and human growth hormone.
  • Asterisk (*) denotes identical amino acids
  • colon (:) denotes structurally and chemically similar amino acids
  • point (.) denotes aminoacids belonging to the same class (in casu hydrophobic or hydrophilic).
  • the sequence listed as "prolactin” is SEQ ID No. 1
  • the sequence listed as hGH is SEQ ID No. 2.
  • Negative bars represent residues for which backbone amide assignments are missing with shading according to Table 1.
  • FIG. 8 2D- 1 H 1 15 N-TROSY spectra of the complex between [ 2 H, 15 N]PRL-G129R and PRLR recorded in a buffer containing 95% 2 H 2 O.
  • Panel A blue
  • panel B red
  • panel C the two spectra from panel A and B are overlayed.
  • Panel D shows an expansion including the peaks assigned to K181 and L188, exhibiting strong attenuation.
  • Figure 9 The NH-signal intensity ratio observed in the cross-saturation experiment is for each amino acid residue shown by bars with most strongly attenuated positions (intensity ratio ⁇ 0.6) represented by colour coding. Positions for which the intensity ratio could not be calculated due to missing assignments or spectral overlap are represented by negative bars.
  • Figure 12 Ba/F3-PRLR proliferation assay result.
  • Figure 13 An example of Ba/F3-PRLR competition assay result.
  • the present invention is concerned with peptides binding to the prolactin receptor, wherein said peptides have an improved binding via binding site 1 (BS1 ) to the prolactin receptor.
  • the present invention is concerned with an isolated peptide, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, said variant comprising
  • the present invention is concerned with an isolated peptide, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, said variant having one or more amino acid mutations in the region corresponding to amino acid residue 66 to 83 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • a peptide of the invention has an amino acid sequence having at least 80% identity to SEQ ID No. 1 including one or more of the amino acid mutations according to the invention. In one embodiment, a peptide of the invention has an amino acid sequence having at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% identity to SEQ ID No. 1 including one or more of the amino acid mutations according to the invention.
  • a peptide of the invention has an amino acid sequence, which sequence is at least 80% similar to SEQ ID No. 1 including one or more of the amino acid mutations according to the invention. In one embodiment, a peptide of the invention has an amino acid sequence, which sequence is at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% similar to SEQ ID No. 1 including one or more of the amino acid mutations according to the invention. In one embodiment, the present invention is concerned with an isolated peptide, which peptide is a variant of human growth hormone, and which binds to the growth hormone receptor, said variant comprising
  • the present invention is concerned with an isolated peptide, which peptide is a variant of human growth hormone, and which binds to the prolactin receptor, said variant having one or more amino acid mutations in the region corresponding to amino acid residue 66 to 83 and/or in the region corresponding to amino acid residues 189 to 199 Of SEQ ID No. 1.
  • the present invention is concerned with an isolated peptide, which peptide is a variant of human growth hormone, and which binds to the growth hormone receptor, said variant having one or more amino acid mutations in the region corresponding to amino acid residue 66 to 83 and/or in the region corresponding to amino acid residues 189 to 199 Of SEQ ID No. 1.
  • a peptide of the invention has an amino acid sequence having at least 80% identity to SEQ ID No. 2 including one or more of the amino acid mutations according to the invention. In one embodiment, a peptide of the invention has an amino acid sequence having at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% identity to SEQ ID No. 2 including one or more of the amino acid mutations according to the invention.
  • a peptide of the invention has an amino acid sequence, which sequence is at least 80% similar to SEQ ID No. 2 including one or more of the amino acid mutations according to the invention. In one embodiment, a peptide of the invention has an amino acid sequence, which sequence is at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% similar to SEQ ID No. 2 including one or more of the amino acid mutations according to the invention.
  • Figure 6 shows an alignment of growth hormone to prolactin and shows which positions in growth hormone (SEQ ID No. 2) corresponds to which positions in prolactin (SEQ ID No. 1).
  • peptide is intended to indicate a sequence of two or more amino acids joined by peptide bonds, wherein said amino acids may be natural or unnatural.
  • the term encompasses the terms polypeptides and proteins, which may consists of two or more polypeptides held together by covalent interactions, such as for instance cysteine bridges, or non-covalent interactions. It is to be understood that the term is also intended to include peptides, which have been derivatized, for instance by the attachment of lipophilic groups, PEG or prosthetic groups.
  • peptide includes any suitable peptide and may be used synonymously with the terms polypeptide and protein, unless otherwise stated or contradicted by context; provided that the reader recognize that each type of respective amino acid polymer-containing molecule may be associated with significant differences and thereby form individual embodiments of the present invention (for example, a peptide such as an antibody, which is composed of multiple polypeptide chains, is significantly different from, for example, a single chain antibody, a peptide immunoadhesin, or single chain immunogenic peptide). Therefore, the term peptide herein should generally be understood as referring to any suitable peptide of any suitable size and composition (with respect to the number of amino acids and number of associated chains in a protein molecule). Moreover, peptides in the context of the inventive methods and compositions described herein may comprise non-naturally occurring and/or non-L amino acid residues, unless otherwise stated or contradicted by context.
  • a derivative is a peptide in which one or more of the amino acid residues of the peptide have been chemically modified (for instance by alkylation, acylation, ester formation, or amide formation) or associated with one or more non-amino acid organic and/or inorganic atomic or molecular substituents (for instance a polyethylene glycol (PEG) group, a lipophilic substituent (which optionally may be linked to the amino acid sequence of the peptide by a spacer residue or group such as ⁇ -alanine, ⁇ -aminobutyric acid (GABA), L/D-glutamic acid, succinic acid, and the like), a fluorophore, biotin, a radionuclide, etc.) and
  • Non-limiting examples of such amino acid residues include for instance 2-aminoadipic acid, 3-amino- adipic acid, ⁇ -alanine, ⁇ -aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-di- aminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methyl- isoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine
  • identity refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues.
  • Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms”).
  • Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
  • Preferred computer program methods to determine identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410 (1990)).
  • the BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra).
  • the well known Smith Waterman algorithm may also be used to determine identity. For example, using the computer algorithm GAP (Genetics Computer Group,
  • a gap opening penalty (which is calculated as 3. times, the average diagonal; the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually ⁇ fraction (1/10) ⁇ times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix see Dayhoff et al., Atlas of Protein Sequence and Structure, vol.
  • Preferred parameters for a peptide sequence comparison include the following: Algorithm: Needleman et al., J. MoI. Biol. 48, 443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., PNAS USA 89, 10915-10919 (1992); Gap Penalty: 12, Gap Length Penalty: 4, Threshold of Similarity: 0.
  • the GAP program is useful with the above parameters.
  • the aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps) using the GAP algorithm.
  • the term "similarity" is a concept related to identity, but in contrast to "identity”, refers to a sequence relationship that includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, (fraction (10/20)) identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If, in the same example, there are 5 more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% ((fraction (15/20))). Therefore, in cases where there are conservative substitutions, the degree of similarity between two polypeptides will be higher than the percent identity between those two polypeptides.
  • a peptide comprising an amino acid sequence of SEQ ID No. 1 (or SEQ ID No. 2) (and the corresponding modifications to the encoding nucleic acids) will produce peptides having functional and chemical characteristics similar to those of a peptide comprising an amino acid sequence of SEQ ID No. 1 (or SEQ ID No. 2).
  • substitutions in the amino acid sequence may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • a "conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis” (see, for example, MacLennan et al., Acta
  • Desired amino acid substitutions may be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of the peptides according to the invention, or to increase or decrease the affinity of the peptides described herein for the receptor in addition to the already described mutations.
  • Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, VaI, Leu, lie;
  • the hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine ('3.O); aspartate (+3.0 ⁇ 1 ); glutamate (+3.0 ⁇ 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • Peptides of the present invention may also include non-naturally occurring amino acids.
  • the present invention is concerned with an isolated peptide, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, and which peptide is a variant of the peptide having the amino acid sequence of SEQ ID No. 1 , said variant comprising (i) one or more amino acid mutations in the region corresponding to amino acid residue
  • a peptide according to the invention has an amino acid sequence comprising SEQ ID No. 1 having one or more amino acid mutations in amino acid residues 66 to 83 and/or in amino acid residues 189 to 199 of SEQ ID No. 1. In one embodiment, a peptide according to the invention has an amino acid sequence comprising SEQ ID No. 2 having one or more amino acid mutations in amino acid residues 66 to 83 and/or in amino acid residues 189 to 199 of SEQ ID No. 1.
  • HX-MS hydrogen exchange
  • MS mass spectrometry
  • Protein-protein complex formation may be detected by HX-MS simply by measuring the total amount of deuterium incorporated in either protein members in the presence and absence of the respective binding partner as a function of time.
  • the deuterium labels can be sub-localized to specific regions of either protein by proteolytic fragmentation of the deuterated protein sample into short peptides and analysis of the deuteron content of each peptide.
  • Peptides that display altered deuterium levels in the presence of binding partner either constitute or are structurally linked to the binding interface (for a recent review on the HX-MS technology see Wales and Engen, Mass Spectrom. Rev. 25, 158 (2006)).
  • a relevant example of application of the HX-MS technology may be found in Horn et al., Biochemistry 45, 8488-8498 (2006).
  • the HX-MS technology used provides information about which surface exposed amide hydrogens in PRL or variants thereof that become shielded from exchange with solvent upon PRL-R binding thereby facilitating a mapping of the binding interface.
  • the methodology can also reveal more indirect structural effects in PRL or variants thereof that give rise to altered HX upon binding. Examples of raw data and the resulting HX-time course plots of peptides from a variant of PRL (vPRL) are shown in Fig. 1 and Fig. 3.
  • BS1 is larger than previously known and that BS1 includes residues from helix 1 " and the second half of the loop between Helix 1 and Helix 2 (residues 66-83) and the S-S bonded C-terminus (residues 189-199).
  • BS1 is said to comprise the segments of PRL consisting of amino acid residues 20-36, 40-63, 66-83, 173-185 and 189-199 (Fig. 2 and Fig. 4).
  • residues in these regions are readily substituted/modified to increase binding affinity of PRL-R to BS1.
  • candidates for modifications may be substituted by residues of the same group of amino acid residues as the native residue or a closely related group so as not to cause large perturbations of the structural integrity of the respective segments of BS1.
  • Mutations in one region of BS1 may be performed alone or in combination with mutations in other regions of BS1 , or for instance mutations, which gives the variant antagonistic properties, such as antagonistic mutations in BS2.
  • prolactin receptor antagonists are currently known, namely G129R-hPRL, S179D-hPRL, G129R-hPRL( ⁇ 1-9), and G129R-hPRL ( ⁇ 1-14), see Goffin et al. Endocrine Rev. 26, 400-422 (2005)).
  • the decreased HX observed for amides hydrogens in Helix 2 and Helix 3 are an example of indirect structural effects propagated through PRL structure upon binding of PRL- R ( Figure 5).
  • a reduction in HX rates of Helix 2 and Helix 3, which are located on the opposite side of the PRL molecule and distal to BS1 shows that these regions are stabilized indirectly through changes in the structure of PRL during binding of PRL-R at BS1.
  • the stabilization observed in Helix 2 and Helix 3 indicate that the transient unfolding of these helices and possibly the entire four-helix bundle in PRL is significantly stabilized by PRL-R binding at BS1.
  • ECD-PRL-R Ectoplasmic domain of PRL-R 1 ie soluble form of the receptor, residues 25-2344 binding
  • Nuclear Magnetic Resonance (NMR) spectroscopy is a well established technique for characterizing binding interfaces of protein-protein complexes in solution.
  • the chemical shifts perturbation and H-D exchange methods do not distinguish between primary effects originating from direct molecular contacts at the protein-protein interface, and indirect effects attributable to changes in structure and/or dynamics induced by complex formation.
  • the cross saturation method relies on transfer of magnetization from one molecule to the other via short-range proton-proton contacts, and secondary effects do not interfere. The cross saturation method then (ideally) uniquely identifies back bone amide groups situated within a distance shorter than 5-7 A of the interface.
  • Mutations that would increase stabilization of the four-helix bundle structure in PRL include stabilization of the terminal part of any of the four helices of PRL (so-called helix capping) (including mutations such as E162D, A111 D, A111 N, A111 S, and A111 T), introducing new saltbridges in solvent exposed helical segments of PRL (including mutations such as E162D, A111 D, A111 N, A111S, and A111T), introducing new saltbridges in solvent exposed helical segments of prolactin (including mutations such as N92D), introduction of new S-S disulfide bonds (including mutations such as L81C/V134C, L88C/L127C, V102/L113C, L95C/E120C, S90C/Y147C, L32C/I119C, D160C/S193C, L81C/V134C, M105C/A108C, M36C/K115C, S33C/L175C, A
  • V102C/L113C S57C/N170C, R89C/Y147C, S82C/E143C, H195C/N198C, K190C/N198C, S33C/R176C, H138C/T141C, M158C/R164C, E93C/W150C, S86C/I146C, V85C/N144C, S82C/N144C, K78C/K142C, K78C/H138C, Q77C/V137C, L63C/S86C, T45C/I51C, L1C/S135C).
  • PRL in conjunction, one might also increase the stability of PRL by replacing solvent exposed hydrophobic residues by polar residues (including mutations such as I146S and V149S). Similarly, one might also increase the stability of PRL by improving the packing interactions at the hydrophobic core of the 4-helix bundle structure (including mutations such as L95V/H 19V/L175P).
  • the mutations according to the present invention are substitutions in one or more of the position corresponding to amino acid residues 25, 28, 31 , 33, 68, 73, 75, 76, 80, 179, and 190 of SEQ ID No. 1.
  • a peptide according to the present invention is a variant of a polypeptide having the amino acid sequence of SEQ ID No. 1 carrying substitution mutation in one or more of amino acid residues 25, 28, 31 , 33, 68, 73, 75, 76, 80, 179, and 190.
  • a peptide according to the present invention is a variant of a polypeptide having the amino acid sequence of SEQ ID No.
  • a peptide according to the present invention is a variant of a polypeptide having the amino acid sequence of SEQ ID No. 1 carrying the substitution mutations: Q73L, M75T, N76S, F80L and G129R.
  • a peptide according to the present invention is a variant of a polypeptide having the amino acid sequence of SEQ ID No. 1 carrying the substitution mutations: S33A, Q73L, G129R, and K190R.
  • Peptides and pharmaceutical compositions according to the present invention may be used in the treatment of diseases treatable by administration of prolactin antagonists, such as breast cancer.
  • treatment means the management and care of a patient for the purpose of combating a condition, such as a disease or a disorder.
  • the term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active peptides to prevent the onset of the symptoms or complications.
  • the patient to be treated is preferably a mammal, in particular a human being, but it may also include animals, such as dogs, cats, cows, sheep and pigs. It is to be understood, that therapeutic and prophylactic (preventive) regimes represent separate aspects of the present invention.
  • a “therapeutically effective amount” of a peptide as used herein means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications. An amount adequate to accomplish this is defined as “therapeutically effective amount”. Effective amounts for each purpose will depend on the type and severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary.
  • nucleic acid construct is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin.
  • construct is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a peptide of interest.
  • the construct may optionally contain other nucleic acid segments.
  • a nucleic acid construct of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. J. Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York) and by introducing the relevant mutations as it is known in the art.
  • a nucleic acid construct of the invention may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22, 1859-1869 (1981 ), or the method described by Matthes et al., EMBO Journal 3, 801-805 (1984).
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.
  • the nucleic acid construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239, 487-491 (1988).
  • the nucleic acid construct of the invention is a DNA construct which term will be used exclusively in the following for convenience. The statements in the following may also read on other nucleic acid constructs of the invention with appropriate adaptions as it will be clear for a person skilled in the art.
  • the present invention relates to a recombinant vector comprising a DNA construct of the invention.
  • the recombinant vector into which the DNA construct of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector may be an expression vector in which the DNA sequence encoding the peptide of the invention is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the peptide.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255, 12073-12080 (1980); Alber and Kawasaki, J. MoI. Appl. Gen.
  • suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4, 2093 - 2099 (1985)) or the tpiA promoter.
  • suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ - amylase, A. niger acid stable ⁇ -amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae those phosphate isomerase or A. nidulans acetamidase.
  • the promoter of a vector according to the invention is selected from the TAKA-amylase or the gluA promoters.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters or the E. coli lac, trp or tac promoters.
  • the DNA sequence encoding the peptide of the invention may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPH (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • suitable sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • DNA polymerase III complex encoding genes and origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P. R. Russell, Gene 40, 125-130 (1985)), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • selectable markers include amdS. pyrG. argB, niaD and sC.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5 1 to the DNA sequence encoding the peptide.
  • the secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.
  • the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed peptide into the secretory pathway of the cell.
  • the signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the ⁇ -factor signal peptide (cf. US 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 643-646 (1981)), a modified carboxypeptidase signal peptide (cf. L.A. VaIIs et al., Cell 48, 887-897 (1987)), the yeast BAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 127-137 (1990)).
  • a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and uptream of the DNA sequence encoding the peptide.
  • the function of the leader peptide is to allow the expressed peptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the peptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell).
  • the leader peptide may be the yeast ⁇ -factor leader (the use of which is described in e.g.
  • the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.
  • the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.
  • the signal peptide may be derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral ⁇ -amylase, A. niger acid-stable amylase, or A niger glucoamylase.
  • the procedures used to ligate the DNA sequences coding for the present peptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.).
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • Examples of bacterial host cells which, on cultivation, are capable of producing the peptide of the invention are grampositive bacteria such as strains of Bacillus, such as strains of 8. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or S. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as Echerichia coli.
  • Bacillus such as strains of 8. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or S. th
  • the transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • suitable hosts include S. mobaraense, S. lividans, and C. glutamicum (Appl. Microbiol. Biotechnol. 64, 447-454 (2004)).
  • the peptide When expressing the peptide in bacteria such as E. coli, the peptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the peptide is refolded by diluting the denaturing agent. In the latter case, the peptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the peptide.
  • sonication or osmotic shock to release the contents of the periplasmic space and recovering the peptide.
  • yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces reteyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous proteins therefrom are described, e.g. in US 4,599,311 , US 4,931 ,373, US 4,870,008, 5,037,743, and US 4,845,075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • a selectable marker commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • yeast An example of a vector for use in yeast is the POT1 vector disclosed in US 4,931 ,373.
  • the DNA sequence encoding the peptide of the invention may be preceded by a signal sequence and optionally a leader sequence , e.g. as described above.
  • suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula, e.g. H. polymo ⁇ ha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132. 3459-3465 (1986); US 4,882,279).
  • Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277 and EP 230 023.
  • the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al. Gene 78, 147-156 (1989).
  • a filamentous fungus When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell. This will make it more likely that the DNA sequence will be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.
  • the transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the present peptide, after which the resulting peptide is recovered from the culture.
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g.
  • the present invention provides a pharmaceutical formulation comprising a peptide of the present invention which is present in a concentration from 10 '15 mg/ml to 200 mg/ml, such as 10 '10 mg/ml - 5 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0.
  • said formulation may comprise one or more further cancer agents as described above.
  • the formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants.
  • the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension.
  • the pharmaceutical formulation is an aqueous solution.
  • aqueous formulation is defined as a formulation comprising at least 50 %w/w water.
  • aqueous solution is defined as a solution comprising at least 50 %w/w water, and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.
  • the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.
  • the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.
  • the invention relates to a pharmaceutical formulation comprising an aqueous solution of a peptide of the present invention, and a buffer, wherein said OGP protein is present in a concentration from 0.1-100 mg/ml, and wherein said formulation has a pH from about 2.0 to about 10.0.
  • the pH of the formulation is selected from the list consisting of 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
  • the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.
  • Each one of these specific buffers constitutes an alternative embodiment of the invention.
  • the formulation further comprises a pharmaceutically acceptable preservative.
  • the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p- hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol) or mixtures thereof.
  • the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention.
  • the use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the formulation further comprises an isotonic agent.
  • the isotonic agent is selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. L- glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol, 1 ,3- butanediol) polyethyleneglycol (e.g.
  • Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.
  • the sugar additive is sucrose.
  • Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol additive is mannitol.
  • the sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention.
  • the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml.
  • the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention.
  • the use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the formulation further comprises a chelating agent.
  • the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof.
  • the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml.
  • the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml.
  • the chelating agent is present in a concentration from 2mg/ml to 5mg/ml.
  • Each one of these specific chelating agents constitutes an alternative embodiment of the invention.
  • the use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the formulation further comprises a stabilizer.
  • a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceutical formulations.
  • aggregate formation is intended a physical interaction between the polypeptide molecules that results in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution.
  • a liquid pharmaceutical composition or formulation once prepared is not immediately administered to a subject. Rather, following preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject.
  • dried form is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J. Parenteral Sci. Technol.
  • compositions of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition.
  • amino acid base is intended an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms.
  • amino acids to use in preparing the compositions of the invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid.
  • Any stereoisomer i.e., L, D, or mixtures thereof
  • a particular amino acid e.g. glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof
  • a particular amino acid e.g. glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof
  • Compositions of the invention may also be formulated with analogues of these amino acids.
  • amino acid analogue is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the invention.
  • Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine
  • suitable methionine analogues include ethionine and buthionine
  • suitable cysteine analogues include S- methyl-L cysteine.
  • the amino acid analogues are incorporated into the compositions in either their free base form or their salt form.
  • the amino acids or amino acid analogues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.
  • methionine (or other sulphuric amino acids or amino acid analogous) may be added to inhibit oxidation of methionine residues to methionine sulfoxide when the polypeptide acting as the therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation.
  • inhibitor is intended minimal accumulation of methionine oxidized species over time. Inhibiting methionine oxidation results in greater retention of the polypeptide in its proper molecular form. Any stereoisomer of methionine (L, D, or mixtures thereof) or combinations thereof can be used.
  • the amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine such that the ratio of methionine added to methionine residues ranges from about 1 :1 to about 1000:1 , such as 10:1 to about 100:1.
  • the formulation further comprises a stabilizer selected from the group of high molecular weight polymers or low molecular compounds.
  • the stabilizer is selected from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (e.g. sodium chloride).
  • PEG 3350 polyethylene glycol
  • PVA polyvinyl alcohol
  • PVpyrrolidone polyvinylpyrrolidone
  • carboxy-/hydroxycellulose or derivates thereof e.g. HPC, HPC-SL, HPC-L and HPMC
  • cyclodextrins e.g. sulphur-containing substances as monothioglycerol,
  • compositions may also comprise additional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein.
  • Stabilizing agents of particular interest to the present invention include, but are not limited to, methionine and EDTA, which protect the polypeptide against methionine oxidation, and a nonionic surfactant, which protects the polypeptide against aggregation associated with freeze-thawing or mechanical shearing.
  • the formulation further comprises a surfactant.
  • the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such as Pluronic ® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g.
  • Tween-20, Tween-40, Tween-80 and Brij-35 monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (eg. phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) and lysophospholipids (eg.
  • phospholipids eg. dipalmitoyl phosphatidic acid
  • lysophospholipids eg.
  • ceramides e.g. sodium tauro-dihydrofusidate etc.
  • C6-C12 e.g.
  • acylcarnitines and derivatives N ⁇ -acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N ⁇ -acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N ⁇ -acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-11 -7]), docusate calcium, CAS registry no [128-49- 4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl sulphate or sodium lauryl sulphate), sodium caprylate, cholic acid or derivatives thereof, bile acids and salts thereof and glycine or taurine
  • N-alkyl-N,N-dimethylammonio-1-propanesulfonates 3-cholamido-i-propyldimethylammonio-i-propanesulfonate
  • cationic surfactants quaternary ammonium bases
  • cetyl-trimethylammonium bromide cetylpyridinium chloride
  • non- ionic surfactants eg. Dodecyl ⁇ -D-glucopyranoside
  • poloxamines eg.
  • Tetronic's which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the invention.
  • the use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine).
  • additional ingredients should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.
  • compositions containing a peptide of the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.
  • topical sites for example, skin and mucosal sites
  • sites which bypass absorption for example, administration in an artery, in a vein, in the heart
  • sites which involve absorption for example, administration in the skin, under the skin, in a muscle or in the abdomen.
  • Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.
  • routes of administration for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.
  • compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.
  • solutions for example, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses,
  • compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the peptide of the present invention, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof.
  • carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.
  • polymers for example cellulose and derivatives, polysaccharides, for example dextran and derivatives
  • compositions of the current invention are useful in the formulation of solids, semisolids, powder and solutions for pulmonary administration of a peptide of the present invention, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.
  • compositions of the current invention are specifically useful in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in formulation of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. Even more preferably, are controlled release and sustained release systems administered subcutaneous.
  • examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles,
  • Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, encapsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
  • General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).
  • Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • a further option is a composition which may be a solution or suspension for the administration of the peptide of the present inventionin the form of a nasal or pulmonal spray.
  • the pharmaceutical compositions containing the peptide of the present invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.
  • stabilized formulation refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.
  • physical stability of the protein formulation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
  • Physical stability of the aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods. Visual inspection of the formulations is performed in a sharp focused light with a dark background.
  • the turbidity of the formulation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a formulation showing no turbidity corresponds to a visual score 0, and a formulation showing visual turbidity in daylight corresponds to visual score 3).
  • a formulation is classified physical unstable with respect to protein aggregation, when it shows visual turbidity in daylight.
  • the turbidity of the formulation can be evaluated by simple turbidity measurements well-known to the skilled person.
  • Physical stability of the aqueous protein formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein.
  • the probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein.
  • Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
  • hydrophobic patch probes that bind preferentially to exposed hydrophobic patches of a protein.
  • the hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature.
  • these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like.
  • spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.
  • chemical stability refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure.
  • chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided and increasing amounts of chemical degradation products is often seen during storage and use of the protein formulation as well- known by the person skilled in the art.
  • the chemical stability of the protein formulation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by for instance increasing temperature).
  • the amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).
  • a “stabilized formulation” refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.
  • a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.
  • the pharmaceutical formulation comprising the peptide of the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage. In one embodiment of the invention the pharmaceutical formulation comprising the peptide of the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage.
  • the pharmaceutical formulation comprising the peptide of the present invention is stable for more than 4 weeks of usage and for more than two years of storage.
  • the pharmaceutical formulation comprising the peptide of the present invention is stable for more than 2 weeks of usage and for more than two years of storage.
  • Embodiment 1 An isolated peptide, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, said variant comprising (i) one or more amino acid mutations in the region corresponding to amino acid residue
  • Embodiment 2 An isolated peptide according to embodiment 1 , wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 25 Of SEQ ID No. 1.
  • Embodiment 3 An isolated peptide according to embodiment 2, wherein the amino acid residue in the position corresponding to amino acid residue 25 of SEQ ID No. 1 is substitued with a GIn.
  • Embodiment 4 An isolated peptide according to any of embodiments 1 to 3, wherein the mutation(s) described under (i) is in the region corresponding to amino acid residue 26 to 33 Of SEQ ID No. 1.
  • Embodiment 5. An isolated peptide according to any of embodiments 1 to 4, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 28 of SEQ ID No. 1.
  • Embodiment 6 An isolated peptide according to embodiment 5, wherein the amino acid residue in the position corresponding to amino acid residue 28 of SEQ ID No. 1 is substitued with an Asn.
  • Embodiment 7 An isolated peptide according to any of embodiments 1 to 6, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 31 of SEQ ID No. 1.
  • Embodiment 8 An isolated peptide according to embodiment 7, wherein the amino acid residue in the position corresponding to amino acid residue 31 of SEQ ID No. 1 is substitued with a Ser.
  • Embodiment 9 An isolated peptide according to any of embodiments 1 to 8, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 33 of SEQ ID No. 1.
  • Embodiment 10 An isolated peptide according to embodiment 9, wherein the amino acid residue in the position corresponding to amino acid residue 33 of SEQ ID No. 1 is substitued with an Ala.
  • Embodiment 11 An isolated peptide according to any of embodiments 1 to 10, wherein the mutation(s) described under (i) is not in the amino acid residue corresponding to amino acid residue 30 of SEQ ID No. 1.
  • Embodiment 12 An isolated peptide according to any of embodiments 1 to 11 , wherein at least one of the mutation(s) described under (ii) is in the position corresponding to amino acid residue 55 of SEQ ID No. 1.
  • Embodiment 13 An isolated peptide according to any of embodiments 1 to 12, wherein at least one of the mutation(s) described under (ii) is in the position corresponding to amino acid residue 56 of SEQ ID No. 1.
  • Embodiment 14 An isolated peptide according to any of embodiments 1 to 13, wherein the mutation(s) described under (ii) is not in the amino acid residue corresponding to amino acid residue 66 of SEQ ID No. 1.
  • Embodiment 15 An isolated peptide according to any of embodiments 1 to 14, wherein the mutation(s) described under (ii) is not in the amino acid residue corresponding to amino acid residue 69 of SEQ ID No. 1.
  • Embodiment 16 An isolated peptide according to any of embodiments 1 to 15, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 73 of SEQ ID No. 1.
  • Embodiment 17 An isolated peptide according to embodiment 16, wherein the amino acid residue in the position corresponding to amino acid residue 73 of SEQ ID No. 1 is substitued with a Leu.
  • Embodiment 18 An isolated peptide according to any of embodiments 1 to 17, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 75 of SEQ ID No. 1.
  • Embodiment 19 An isolated peptide according to embodiment 18, wherein the amino acid residue in the position corresponding to amino acid residue 75 of SEQ ID No. 1 is substitued with a Thr.
  • Embodiment 20 An isolated peptide according to any of embodiments 1 to 19, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 76 of SEQ ID No. 1.
  • Embodiment 21 An isolated peptide according to embodiment 20, wherein the amino acid residue in the position corresponding to amino acid residue 76 of SEQ ID No. 1 is substitued with a Ser.
  • Embodiment 22 An isolated peptide according to any of embodiments 1 to 21 , wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 80 of SEQ ID No. 1.
  • Embodiment 23 An isolated peptide according to embodiment 22, wherein the amino acid residue in the position corresponding to amino acid residue 80 of SEQ ID No. 1 is substitued with a Leu.
  • Embodiment 24 An isolated peptide according to any of embodiments 1 to 23, wherein at least one of the mutation(s) described under (iii) is in the region corresponding to amino acid residue 67 to 70 of SEQ ID No. 1.
  • Embodiment 25 An isolated peptide according to any of embodiments 1 to 24, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 68 of SEQ ID No. 1.
  • Embodiment 26 An isolated peptide according to embodiment 25, wherein the amino acid residue in the position corresponding to amino acid residue 68 of SEQ ID No. 1 is substitued with an Asn.
  • Embodiment 27 An isolated peptide according to any of embodiments 1 to 26, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 176 of SEQ ID No. 1.
  • Embodiment 28 An isolated peptide according to any of embodiments 1 to 27, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 177 of SEQ ID No. 1.
  • Embodiment 29 An isolated peptide according to any of embodiments 1 to 28, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 180 of SEQ ID No. 1.
  • Embodiment 30 An isolated peptide according to any of embodiments 1 to 29, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 181 of SEQ ID No. 1.
  • Embodiment 31 An isolated peptide according to any of embodiments 1 to 30, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 185 of SEQ ID No. 1.
  • Embodiment 32 An isolated peptide according to any of embodiments 1 to 31 , wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 187 of SEQ ID No. 1.
  • Embodiment 33 An isolated peptide according to any of embodiments 1 to 32, wherein at least one of the mutation(s) described under (iv) is in the position corresponding to amino acid residue 179 of SEQ I D No. 1.
  • Embodiment 34 An isolated peptide according to embodiment 33, wherein the amino acid residue in the position corresponding to amino acid residue 179 of SEQ ID No. 1 is substitued with a Thr.
  • Embodiment 35 An isolated peptide according to any of embodiments 1 to 34, wherein at least one of the mutation(s) described under (iv) is in the region corresponding to amino acid residue 188 to 199 of SEQ ID No. 1.
  • Embodiment 36 An isolated peptide according to any of embodiments 1 to 35, wherein at least one of the mutation(s) described under (iv) is in the position corresponding to amino acid residue 190 of SEQ ID No. 1.
  • Embodiment 37 An isolated peptide according to embodiment 36, wherein the amino acid residue in the position corresponding to amino acid residue 190 of SEQ ID No. 1 is substitued with an Arg.
  • Embodiment 38 An isolated peptide according to any of embodiments 1 to 37, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, said variant having one or more amino acid mutations in the region corresponding to amino acid residue 66 to 83 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • Embodiment 39 Embodiment 39.
  • An isolated peptide according to embodiment 38 which peptide is a variant of human prolactin, and which binds to the prolactin receptor, said variant having one or more amino acid mutations in the region corresponding to amino acid residue 67 to 70 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • Embodiment 40 An isolated peptide, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, and which peptide is a variant of the peptide having the amino acid sequence of SEQ ID No. 1 , said variant comprising (i) one or more amino acid mutations in the region corresponding to amino acid residue
  • Embodiment 41 An isolated peptide according to embodiment 40, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 25 of SEQ ID No. 1.
  • Embodiment 42 An isolated peptide according to embodiment 41 , wherein the amino acid residue in the position corresponding to amino acid residue 25 of SEQ ID No. 1 is substitued with a GIn.
  • Embodiment 43 An isolated peptide according to any of embodiments 40 to 42, wherein the mutation(s) described under (i) is in the region corresponding to amino acid residue 26 to 33 of SEQ ID No. 1.
  • Embodiment 44 An isolated peptide according to any of embodiments 40 to 43, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 28 of SEQ ID No. 1.
  • Embodiment 45 An isolated peptide according to embodiment 44, wherein the amino acid residue in the position corresponding to amino acid residue 28 of SEQ ID No. 1 is substitued with an Asn.
  • Embodiment 46 An isolated peptide according to any of embodiments 40 to 45, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 31 of SEQ ID No. 1.
  • Embodiment 47 An isolated peptide according to embodiment 46, wherein the amino acid residue in the position corresponding to amino acid residue 31 of SEQ ID No. 1 is substitued with a Ser.
  • Embodiment 48 An isolated peptide according to any of embodiments 40 to 47, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 33 of SEQ ID No. 1.
  • Embodiment 49 An isolated peptide according to embodiment 48, wherein the amino acid residue in the position corresponding to amino acid residue 33 of SEQ ID No. 1 is substitued with an Ala.
  • Embodiment 50 An isolated peptide according to any of embodiments 40 to 49, wherein the mutation(s) described under (i) is not in the amino acid residue corresponding to amino acid residue 30 of SEQ ID No. 1.
  • Embodiment 51 An isolated peptide according to any of embodiments 40 to 50, wherein at least one of the mutation(s) described under (ii) is in the position corresponding to amino acid residue 55 of SEQ ID No. 1.
  • Embodiment 52 An isolated peptide according to any of embodiments 40 to 51 , wherein at least one of the mutation(s) described under (ii) is in the position corresponding to amino acid residue 56 of SEQ ID No. 1.
  • Embodiment 53 An isolated peptide according to any of embodiments 40 to 52, wherein the mutation(s) described under (ii) is not in the position corresponding to amino acid residue 66 of SEQ ID No. 1.
  • Embodiment 54 An isolated peptide according to any of embodiments 40 to 53, wherein the mutation(s) described under (ii) is not in the position corresponding to amino acid residue 69 of SEQ ID No. 1.
  • Embodiment 55 An isolated peptide according to any of embodiments 40 to 54, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 73 of SEQ ID No. 1.
  • Embodiment 56 An isolated peptide according to embodiment 55, wherein the amino acid residue in the position corresponding to amino acid residue 73 of SEQ ID No. 1 is substitued with a Leu.
  • Embodiment 57 An isolated peptide according to any of embodiments 40 to 56, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 75 of SEQ ID No. 1.
  • Embodiment 58 An isolated peptide according to embodiment 57, wherein the amino acid residue in the position corresponding to amino acid residue 75 of SEQ ID No. 1 is substitued with a Thr.
  • Embodiment 59 An isolated peptide according to any of embodiments 40 to 58, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 76 of SEQ ID No. 1.
  • Embodiment 60 An isolated peptide according to embodiment 59, wherein the amino acid residue in the position corresponding to amino acid residue 76 of SEQ ID No. 1 is substitued with a Ser.
  • Embodiment 61 An isolated peptide according to any of embodiments 40 to 60, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 80 of SEQ ID No. 1.
  • Embodiment 62 An isolated peptide according to embodiment 61 , wherein the amino acid residue in the position corresponding to amino acid residue 80 of SEQ ID No. 1 is substitued with a Leu.
  • Embodiment 63 An isolated peptide according to any of embodiments 40 to 62, wherein at least one of the mutation(s) described under (iii) is in the region corresponding to amino acid residue 67 to 70 of SEQ ID No. 1.
  • Embodiment 64 An isolated peptide according to any of embodiments 40 to 63, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 68 of SEQ ID No. 1.
  • Embodiment 65 An isolated peptide according to embodiment 64, wherein the amino acid residue in the position corresponding to amino acid residue 68 of SEQ ID No. 1 is substitued with an Asn.
  • Embodiment 66 An isolated peptide according to any of embodiments 40 to 65, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 176 of SEQ ID No. 1.
  • Embodiment 67 An isolated peptide according to any of embodiments 40 to 66, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 177 of SEQ ID No. 1.
  • Embodiment 68 An isolated peptide according to any of embodiments 40 to 67, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 180 of SEQ ID No. 1.
  • Embodiment 69 An isolated peptide according to any of embodiments 40 to 68, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 181 of SEQ ID No. 1.
  • Embodiment 70 An isolated peptide according to any of embodiments 40 to 69, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 185 of SEQ ID No. 1.
  • Embodiment 71 An isolated peptide according to any of embodiments 40 to 70, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 187 of SEQ ID No. 1.
  • Embodiment 72 An isolated peptide according to any of embodiments 40 to 71 , wherein at least one of the mutation(s) described under (iv) is in the position corresponding to amino acid residue 179 of SEQ ID No. 1.
  • Embodiment 73 An isolated peptide according to embodiment 72, wherein the amino acid residue in the position corresponding to amino acid residue 179 of SEQ ID No. 1 is substitued with a Thr.
  • Embodiment 74 An isolated peptide according to any of embodiments 40 to 73, wherein the mutation(s) described under (iv) is in the region corresponding to amino acid residue 188 to 199 of SEQ ID No. 1.
  • Embodiment 75 An isolated peptide according to any of embodiments 40 to 74, wherein at least one of the mutation(s) described under (iv) is in the position corresponding to amino acid residue 190 of SEQ ID No. 1.
  • Embodiment 76 An isolated peptide according to embodiment 75, wherein the amino acid residue in the position corresponding to amino acid residue 190 of SEQ ID No. 1 is substitued with an Arg.
  • Embodiment 77 An isolated peptide according to any of embodiments 40 to 76, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, and which peptide is a variant of the peptide having the amino acid sequence of SEQ ID No. 1 , said variant having one or more amino acid mutations in the region corresponding to amino acid residue 66 to 83 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • Embodiment 78 An isolated peptide according to embodiment 77, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, said variant having one or more amino acid mutations in the region corresponding to amino acid residue 67 to 70 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • Embodiment 79 An isolated peptide, which peptide is a variant of human prolactin, and which binds to the prolactin receptor, wherein said variant comprises one or more amino acid mutations, which stabilizes the structure of the prolactin molecule.
  • Embodiment 80 An isolated peptide according to any of embodiments 1 to 79, wherein said variant comprises one or more amino acid mutations, which stabilizes the secondary structure of the prolactin molecule.
  • Embodiment 81 An isolated peptide according to embodiment 79 or embodiment 80, wherein the stabilization of PRL is detemined by use of HX-MS technology as described in Example 1.
  • Embodiment 82 An isolated peptide according to any of embodiments 79 to 81 , wherein one or more of said amino acid mutation(s) stabilizes the 4-helix bundle structure in prolactin.
  • Embodiment 83 An isolated peptide according to any of embodiments 79 to 82, wherein one or more of said amino acid mutation(s) improves the helix capping in helix 1 , helix 2, helix 3 and/or helix 4 of PRL.
  • Embodiment 84 An isolated peptide according to any of embodiments 79 to 83, wherein one or more of said amino acid mutations are selected from mutations in the amino acid residues corresponding to Ala-111 and Glu-162.
  • Embodiment 85 An isolated peptide according to embodiment 84, wherein the amino acid residue corresponding to Ala-111 is substituted with Asp, Asn, Ser or Thr.
  • Embodiment 86 An isolated peptide according to embodiment 84 or embodiment 85, wherein the amino acid residue corresponding to Glu-162 is substituted with Asp.
  • Embodiment 87 An isolated peptide according to any of embodiments 79 to 86, wherein one or more of said amino acid mutation(s) introduces salt bridges in helical segments exposed to solvent.
  • Embodiment 88 An isolated peptide according to any of embodiments 79 to 87, wherein one of said amino acid mutations is a mutation in the amino acid residue corresponding to Asn-92.
  • Embodiment 89 An isolated peptide according to embodiment 88, wherein the amino acid residue corresponding to Asn-92 is substituted with Asp.
  • Embodiment 90 An isolated peptide according to any of embodiments 79 to 89, wherein two or more of said amino acid mutation(s) introduces non-native disulfide bonds into prolactin.
  • Embodiment 91 An isolated peptide according to embodiment 90, wherein said two amino acid mutations are selected from mutations in the positions corresponding to L1C/S135C, A22C/G129C, V23C/L186C, S26C/D183C, L32C/I119C, S33C/L175C, S33C/R176C, S33C/S179C, M36C/K115C, F37C/L172C, T45C/I51 C, S57C/N170C, H59C/P148C, L63C/S86C, P66C/Q71C, P66C/A72C, Q77C/V137C, K78C/K142C, K78C/H138C, L81C/V134C, S82C/E143C, S82C/N144C, V85C/N144C, S86C/I146C, L88C/L127C, R89C/Y147C, S90C/Y147C,
  • Embodiment 92 An isolated peptide according to any of embodiments 1 to 91 , wherein one or more of said amino acid mutation(s) is a substitution of a solvent exposed hydrophobic residue with a polar residue.
  • Embodiment 93 An isolated peptide according to embodiment 92, wherein one or more of said amino acid mutations are selected from mutations in the amino acid residues corresponding to lle-146 and Val-149.
  • Embodiment 94 An isolated peptide according to embodiment 93, wherein the amino acid residue corresponding to lle-146 is substituted with serine or threonine.
  • Embodiment 95 An isolated peptide according to embodiment 93 or embodiment 94, wherein the amino acid residue corresponding to Val-149 is substituted with serine or threonine.
  • Embodiment 96 An isolated peptide according to any of embodiments 1 to 95, wherein one or more of said amino acid mutation(s) improves the packing interactions at the hydrophobic core of the 4-helix bundle structure.
  • Embodiment 97 An isolated peptide according to embodiment 96, wherein one or more of said amino acid mutations are selected from mutations in the amino acid residues corresponding to Leu-95, lle-1 19 and Leu-175.
  • Embodiment 98 An isolated peptide according to embodiment 97, wherein the amino acid residue corresponding to Leu-95 is substituted with VaI.
  • Embodiment 99 An isolated peptide according to embodiment 97 or embodiment 98, wherein the amino acid residue corresponding to lle-119 is substituted with VaI.
  • Embodiment 100 An isolated peptide according to any of embodiments 97 to 99, wherein the amino acid residue corresponding to Leu-175 is substituted with Pro.
  • Embodiment 101 An isolated peptide according to any of embodiments 1 to 100, wherein said peptide is also mutated in one or more positions corresponding to amino acid residues 20 to 36 and/or 40 to 63 and/or 173 to 185 of SEQ ID No. 1.
  • Embodiment 102 An isolated peptide according to any of embodiments 1 to 101 , wherein said peptide has an increased affinity to the prolactin receptor as compared to human prolactin.
  • Embodiment 103 An isolated peptide according to embodiment 102, wherein the affinity to the prolactin receptor is determined according to Assay (I) as described herein.
  • Embodiment 104 An isolated peptide according to any of embodiments 1 to 103, wherein the binding of said peptide for the prolactin receptor has a dissociation konstant (K d ) at least three times less than that of wildtype human PRL binding to the prolactin receptor.
  • Embodiment 105 An isolated peptide according to any of embodiments 1 to 104, wherein said peptide is capable of binding to the human growth hormone receptor.
  • Embodiment 106 An isolated peptide according to embodiment 105, wherein the binding to the human growth hormone receptor is determined by use of the assay as described as Assay (I) herein.
  • Embodiment 107 An isolated peptide according to any of embodiments 1 to 106, which is an antagonist of the prolactin receptor.
  • Embodiment 108 An isolated peptide according to embodiment 107, wherein said antagonism is determined using Assay (II) as described herein.
  • Embodiment 109 An isolated peptide according to embodiment 107 or embodiment 108, wherein said antagonism is achieved by introducing one or more mutations into BS-2 to prevent or reduce interaction of BS2 with PRL-R.
  • Embodiment 110 An isolated peptide according to any of embodiments 107 to 109, wherein at least one or more of said antagonistic mutations are selected from mutations in the amino acid residues corresponding to Gly-129 and SeM 79.
  • Embodiment 111 An isolated peptide according to embodiment 1 10, wherein at least one or more of said antagonistic mutations are selected from mutations corresponding to G129R and S179D.
  • Embodiment 112 An isolated peptide according to embodiment 111 , wherein at least one or more of said antagonistic mutations are selected from a mutation corresponding to G129R.
  • Embodiment 113 An isolated peptide according to embodiment 1 12, wherein the amino acid residues corresponding to positions 1 to 9 in PRL have been deleted.
  • Embodiment 114 An isolated peptide according to embodiment 1 13, wherein the amino acid residues corresponding to positions 1 to 14 in PRL have been deleted.
  • Embodiment 115 An isolated peptide, which peptide is a variant of human growth hormone, and which binds to the growth hormone receptor, said variant comprising
  • Embodiment 116 An isolated peptide according to embodiment 115, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 25 of SEQ ID No. 1.
  • Embodiment 117 An isolated peptide according to embodiment 116, wherein the amino acid residue in the position corresponding to amino acid residue 25 of SEQ ID No. 1 is substitued with a GIn.
  • Embodiment 118 An isolated peptide according to any of embodiments 115 to 117, wherein the mutation(s) described under (i) is in the region corresponding to amino acid residue 26 to 33 of SEQ ID No. 1.
  • Embodiment 119 An isolated peptide according to any of embodiments 115 to 118, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 28 of SEQ ID No. 1.
  • Embodiment 120 An isolated peptide according to embodiment 119, wherein the amino acid residue in the position corresponding to amino acid residue 28 of SEQ ID No. 1 is substitued with an Asn.
  • Embodiment 121 An isolated peptide according to any of embodiments 115 to 120, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 31 of SEQ ID No. 1.
  • Embodiment 122 An isolated peptide according to embodiment 121 , wherein the amino acid residue in the position corresponding to amino acid residue 31 of SEQ ID No. 1 is substitued with a Ser.
  • Embodiment 123 An isolated peptide according to any of embodiments 40 to 122, wherein at least one of the mutation(s) described under (i) is in the position corresponding to amino acid residue 33 of SEQ ID No. 1.
  • Embodiment 124 An isolated peptide according to embodiment 123, wherein the amino acid residue in the position corresponding to amino acid residue 33 of SEQ ID No. 1 is substitued with an Ala.
  • Embodiment 125 An isolated peptide according to any of embodiments 115 to 124, wherein the mutation(s) described under (i) is not in the amino acid residue corresponding to amino acid residue 30 of SEQ ID No. 1.
  • Embodiment 126 An isolated peptide according to any of embodiments 115 to 125, wherein at least one of the mutation(s) described under (ii) is in the position corresponding to amino acid residue 55 of SEQ ID No. 1.
  • Embodiment 127 An isolated peptide according to any of embodiments 115 to 126, wherein at least one of the mutation(s) described under (ii) is in the position corresponding to amino acid residue 56 of SEQ ID No. 1.
  • Embodiment 128 An isolated peptide according to any of embodiments 115 to 127, wherein the mutation(s) described under (ii) is not in the amino acid residue corresponding to amino acid residue 66 of SEQ ID No. 1.
  • Embodiment 129 An isolated peptide according to any of embodiments 115 to 128, wherein the mutation(s) described under (ii) is not in the amino acid residue corresponding to amino acid residue 69 of SEQ ID No. 1.
  • Embodiment 130 An isolated peptide according to any of embodiments 115 to 129, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 73 of SEQ ID No. 1.
  • Embodiment 131 An isolated peptide according to embodiment 130, wherein the amino acid residue in the position corresponding to amino acid residue 73 of SEQ ID No. 1 is substitued with a Leu.
  • Embodiment 132 An isolated peptide according to any of embodiments 115 to 131 , wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 75 of SEQ ID No. 1.
  • Embodiment 133 An isolated peptide according to embodiment 132, wherein the amino acid residue in the position corresponding to amino acid residue 75 of SEQ ID No. 1 is substitued with a Thr.
  • Embodiment 134 An isolated peptide according to any of embodiments 115 to 133, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 76 of SEQ ID No. 1.
  • Embodiment 135. An isolated peptide according to embodiment 134, wherein the amino acid residue in the position corresponding to amino acid residue 76 of SEQ ID No. 1 is substitued with a Ser.
  • Embodiment 136 An isolated peptide according to any of embodiments 115 to 135, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 80 of SEQ ID No. 1.
  • Embodiment 137 An isolated peptide according to embodiment 136, wherein the amino acid residue in the position corresponding to amino acid residue 80 of SEQ ID No. 1 is substitued with a Leu.
  • Embodiment 138 An isolated peptide according to any of embodiments 115 to 137, wherein at least one of the mutation(s) described under (iii) is in the region corresponding to amino acid residue 67 to 70 of SEQ ID No. 1.
  • Embodiment 139 An isolated peptide according to any of embodiments 115 to 138, wherein at least one of the mutation(s) described under (iii) is in the position corresponding to amino acid residue 68 of SEQ ID No. 1.
  • Embodiment 140 An isolated peptide according to embodiment 139, wherein the amino acid residue in the position corresponding to amino acid residue 68 of SEQ ID No. 1 is substitued with an Asn.
  • Embodiment 141 An isolated peptide according to any of embodiments 115 to 140, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 176 of SEQ ID No. 1.
  • Embodiment 142 An isolated peptide according to any of embodiments 115 to 141 , wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 177 of SEQ ID No. 1.
  • Embodiment 143 An isolated peptide according to any of embodiments 115 to 142, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 180 of SEQ ID No. 1.
  • Embodiment 144 An isolated peptide according to any of embodiments 115 to 143, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 181 of SEQ ID No. 1.
  • Embodiment 145 An isolated peptide according to any of embodiments 115 to 144, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 185 of SEQ ID No. 1.
  • Embodiment 146 An isolated peptide according to any of embodiments 115 to 145, wherein the mutation(s) described under (iv) is not in the amino acid residue corresponding to amino acid residue 187 of SEQ ID No. 1.
  • Embodiment 147 An isolated peptide according to any of embodiments 115 to 146, wherein at least one of the mutation(s) described under (iv) is in the position corresponding to amino acid residue 179 of SEQ ID No. 1.
  • Embodiment 148 An isolated peptide according to embodiment 147, wherein the amino acid residue in the position corresponding to amino acid residue 179 of SEQ ID No. 1 is substitued with a Thr.
  • Embodiment 149 An isolated peptide according to any of embodiments 115 to 148, wherein at least one of the mutation(s) described under (iv) is in the region corresponding to amino acid residue 188 to 199 of SEQ ID No. 1.
  • Embodiment 150 An isolated peptide according to any of embodiments 115 to 149, wherein at least one of the mutation(s) described under (iv) is in the position corresponding to amino acid residue 190 of SEQ ID No. 1.
  • Embodiment 151 An isolated peptide according to embodiment 150, wherein the amino acid residue in the position corresponding to amino acid residue 190 of SEQ ID No. 1 is substitued with an Arg.
  • Embodiment 152 An isolated peptide according to any of embodiments 115 to 151 , which peptide is a variant of human growth hormone, and which binds to the growth hormone receptor, said variant comprises one or more amino acid mutations in the region corresponding to amino acid residue 66 to 83 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • Embodiment 153 An isolated peptide according to embodiment 152, which peptide is a variant of human growth hormone, and which binds to the growth hormone receptor, said variant comprises one or more amino acid mutations in the region corresponding to amino acid residue 67 to 70 and/or in the region corresponding to amino acid residues 189 to 199 of SEQ ID No. 1.
  • Embodiment 154 An isolated peptide, which peptide is a variant of human growth hormone, and which binds to the growth hormone receptor, wherein said variant has one or more amino acid mutations, which stabilizes the secondary structure of the growth hormone molecule.
  • Embodiment 155 An isolated peptide according to any of embodiments 115 to 154, wherein said variant comprises one or more amino acid mutations, which stabilizes the secondary structure of the growth hormone molecule.
  • Embodiment 156 An isolated peptide according to embodiment 154 or embodiment 155, wherein the stabilization of the growth hormone molecule is detemined by use of HX- MS technology as described in Example 1.
  • Embodiment 157 An isolated peptide according to any of embodiments 154 to 156, wherein one or more of said amino acid mutation(s) stabilizes the 4-helix bundle structure in growth hormone.
  • Embodiment 158 An isolated peptide according to any of embodiments 154 to 157, wherein one or more of said amino acid mutation(s) improves the helix capping in helix 1 , helix 2, helix 3 and/or helix 4 of growth hormone.
  • Embodiment 159 An isolated peptide according to any of embodiments 154 to 158, wherein one or more of said amino acid mutations are selected from mutations in the amino acid residues corresponding to Ala-111 and Glu-162 of SEQ ID No. 1.
  • Embodiment 160 An isolated peptide according to embodiment 159, wherein the amino acid residue corresponding to Ala-111 is substituted with Asp, Asn, Ser or Thr.
  • Embodiment 161. An isolated peptide according to embodiment 159 or embodiment
  • Embodiment 162 An isolated peptide according to any of embodiments 154 to 161 , wherein one or more of said amino acid mutation(s) introduces salt bridges in helical segments exposed to solvent.
  • Embodiment 163. An isolated peptide according to any of embodiments 154 to 162, wherein one of said amino acid mutations is a mutation in the amino acid residue corresponding to Asn-92 of SEQ ID No. 1.
  • Embodiment 164 An isolated peptide according to embodiment 163, wherein the amino acid residue corresponding to Asn-92 is substituted with Asp.
  • Embodiment 165 An isolated peptide according to any of embodiments 154 to 164, wherein two or more of said amino acid mutation(s) introduces non-native disulfide bonds into growth hormone.
  • Embodiment 166 An isolated peptide according to embodiment 165, wherein said two amino acid mutations are selected from mutations in the positions corresponding to L1C/S135C, A22C/G129C, V23C/L186C, S26C/D183C, L32C/I119C, S33C/L175C, S33C/R176C, S33C/S179C, M36C/K115C, F37C/L172C, T45C/I51C, S57C/N170C, H59C/P148C, L63C/S86C, P66C/Q71C, P66C/A72C, Q77C/V137C, K78C/K142C, K78C/H138C, L81C/V134C, S82C/E143C, S82C/N144C, V85C/N144C, S86C/I146C, L88C/L127C, R89C/Y147C, S90C/Y147C
  • V102C/L113C M105C/A108C, H138C/T141C, M158C/R164C, and D160C/S193C of SEQ ID No. 1.
  • Embodiment 167 An isolated peptide according to any of embodiments 115 to 166, wherein one or more of said amino acid mutation(s) is a substitution of a solvent exposed hydrophobic residue with a polar residue.
  • Embodiment 168 An isolated peptide according to embodiment 167, wherein one or more of said amino acid mutations are selected from mutations in the amino acid residues corresponding to lle-146 and Val-149 of SEQ ID No. 1.
  • Embodiment 169 An isolated peptide according to embodiment 168, wherein the amino acid residue corresponding to lle-146 is substituted with serine or threonine.
  • Embodiment 170 An isolated peptide according to embodiment 168 or embodiment 169, wherein the amino acid residue corresponding to Val-149 is substituted with serine or threonine.
  • Embodiment 171 An isolated peptide according to any of embodiments 154 to 170, wherein one or more of said amino acid mutation(s) improves the packing interactions at the hydrophobic core of the 4-helix bundle structure.
  • Embodiment 172 An isolated peptide according to embodiment 171 , wherein one or more of said amino acid mutations are selected from mutations in the amino acid residues corresponding to Leu-95, lle-119 and Leu-175 of SEQ ID No. 1.
  • Embodiment 173. An isolated peptide according to embodiment 172, wherein the amino acid residue corresponding to Leu-95 is substituted with VaI.
  • Embodiment 174 An isolated peptide according to embodiment 172 or embodiment 173, wherein the amino acid residue corresponding to lle-119 is substituted with VaI.
  • Embodiment 175. An isolated peptide according to any of embodiments 172 to 174, wherein the amino acid residue corresponding to Leu-175 is substituted with Pro.
  • Embodiment 176 An isolated peptide according to any of embodiments 152 to 175, wherein said peptide is also mutated in one or more positions corresponding to amino acid residues 20 to 36 and/or 40 to 63 and/or 173 to 185 of SEQ ID No. 1.
  • Embodiment 177 An isolated peptide according to any of embodiments 115 to 176, wherein said peptide has an increased affinity to the prolactin receptor as compared to human growth hormone.
  • Embodiment 178 An isolated peptide according to embodiment 177, wherein the affinity to the prolactin receptor is determined according to Assay (I) as described herein.
  • Embodiment 179 An isolated peptide according to any of embodiments 115 to 178, wherein the binding of said peptide for the prolactin receptor has a dissociation konstant (Kd) at least three times less than that of wildtype human growth hormone binding to the prolactin receptor.
  • Kd dissociation konstant
  • Embodiment 180 An isolated peptide according to any of embodiments 115 to 179, wherein said peptide has an increased affinity to the growth hormone receptor as compared to human growth hormone.
  • Embodiment 181 An isolated peptide according to embodiment 180, wherein the affinity to the growth hormone is determined according to Assay (I) as described herein.
  • Embodiment 182 An isolated peptide according to any of embodiments 115 to 181 , wherein the binding of said peptide for the growth hormone receptor has a dissociation konstant (Kd) at least three times less than that of wildtype human growth hormone binding to the growth hormone receptor.
  • Kd dissociation konstant
  • Embodiment 183 An isolated peptide according to any of embodiments 115 to 182, which is an antagonist of the prolactin receptor.
  • Embodiment 184 An isolated peptide according to embodiment 183, wherein said antagonism is determined using Assay (II) as described herein.
  • Embodiment 185 An isolated peptide according to embodiment 183 or embodiment 184, wherein said antagonism is achieved by introducing one or more mutations into BS-2 to prevent or reduce interaction of BS2 with PRL-R.
  • Embodiment 186 An isolated peptide according to any of embodiments 183 to 185, wherein at least one or more of said antagonistic mutations are selected from mutations in the amino acid residues corresponding to Gly120 in SEQ ID No. 2.
  • Embodiment 187 An isolated peptide according to embodiment 186, wherein at least one or more of said antagonistic mutations are selected from G120R or G120K.
  • Embodiment 188 An isolated nucleic acid encoding a peptide according to any of embodiments 1 to 187.
  • Embodiment 189 A vector comprising a nucleic acid construct according to embodiment 188.
  • Embodiment 190 A host cell comprising a nucleic acid construct of embodiment
  • Embodiment 191 An antibody that specifically binds a peptide according to any of embodiments 1 to 187.
  • Embodiment 192 An antibody according to embodiment 191 , which antibody does not bind to a peptide comprising the amino acid sequence of SEQ ID No. 1.
  • Embodiment 193 An antibody according to embodiment 191 or embodiment 192, which antibody does not bind to a peptide comprising the amino acid sequence of SEQ ID No. 2.
  • Embodiment 194 A pharmaceutical composition comprising a peptide according to any of embodiments 1 to 187.
  • Embodiment 195 A method for treating breast cancer, which method comprising administering a peptide according to any of embodiments 1 to 187 or a formulation according to embodiment 194 to a patient in need thereof.
  • Embodiment 196 Use of a peptide according to any of embodiments 1 to 187 for the preparation of a medicament for treatment of breast cancer.
  • Embodiment 197 Use of a peptide according to any of embodiments 1 to 187 for generating prolactin antagonists for the treatment of breast and prostate cancers.
  • the prolactin molecule used in this example is a variant of PRL, wherein amino acid residues 1-11 has been deleted and which contains the mutations Q12S and G129R.
  • the G129R mutation and 1-11 deletion disrupts BS2, whereas the Q12S mutation has been introduced in order to ensure optimal activity of MetAP leading to a more homogeneous product.
  • Methionine aminopeptidases are the enzymes responsible for the removal of the initiator NH 2 -terminal methionine from newly synthesized proteins.
  • this variant will be named vPRL.
  • the pET32-a(+) expression vector (Novagen, Madison Wl) was used for expression of all proteins.
  • Recombinant hPRL and mutated vPRL were produced in Escherichia coli as inclusion bodies. Solubilization in 8M Urea, 0.1 M Tris, 2-20 mM DTT 1 pH 8.5 buffer and following refolding by dilution into a 20 mM Tris, 0.05 % Tween 20, pH 8.0. Protein purification was performed using Source30Q ion exchange columns (Amersham Biosciences) followed by a macro-prep Caramic Hydroxyapatite column (BioRad) and a final size-exclusion chromatography on a Sephadex G25 column.
  • ECD-PRL-R was refolded in two dilution steps, first in 0.4M Arginine, pH 8.5 and then diluted further in 20 mM Tris, 0.05 % Tween 20, pH 8.0.
  • HX Amide hydrogen/deuterium exchange
  • Peptic peptides were identified in separate experiments using standard MS/MS methods. Average masses of peptide isotopic envelopes were determined from lockmass- corrected centroided data (processed using MassLynx software, Waters Inc.) using an Excel spreadsheet. Complete deuteration of control samples was achieved by incubation for 6 hrs at 90 0 C. Average back-exchange (i.e. deuterium loss) was measured to be approx. 15-20% for the analyzed peptides.
  • BS1 is said to comprise the segments of PRL consisting of amino acid residues 20-36, 40-63, 66-83, 173-185 and 189-199.
  • Samples of [ 2 H, 15 N]PRL-G129R and [ 2 H, 13 C, 15 N]PRL-G129R at concentrations ranging between 0.2-0.5 mM were prepared in 2 mM NH 4 HCO 3 , 1 mM NaN 3 and 10% (v/v) 2 H 2 O (pH 8.0) (denoted NMR buffer).
  • the complex between [ 2 H 1 15 N]PRL-GI 29R (or [ 2 H, 13 C 1 15 N]PRL-GI 29R) and PRLR (the soluble extracellular domain of the human prolactin receptor) was prepared by mixing [ 2 H, 15 N]PRL-G129R and PRLR in a ratio 1 :1.2.
  • the binary complex was purified by gel filtration in 2 mM NH 4 HCO 3 and 50 mM NaCI (pH 8.0) using a Superdex 75 Prep 26/60 column. Finally the complex was concentrated and exchanged into the NMR buffer.
  • the buffer contained 10% 2 H 2 O.
  • the buffer contained 90-95% 2 H 2 O in order to quench potential amide proton mediated spin diffusion in [ 2 H 1 15 N]PRL-GI 29R. NMR experiments
  • Back-bone amide resonances in PRL-G129R in complex with PRLR were assigned using a set of TROSY based 3D triple resonance experiments, including HNCO, HNCA, NHCOCA, HNCACB and HNCOCACB 1 supplemented by a 3D 15 N-edited NOESY-HSQC experiment.
  • the back-bone assignments are shown in Table 1. Mapping of binding interface using the chemical shift perturbation method
  • a 1 H 1 15 N-TROSY spectrum was recorded for the one-to-one complex between [ 2 H 1 15 N]PRL-GI 29R and PRLR at 800 MHz. Only signals corresponding to the PRL part of the complex is observed as PRLR is unlabeled. A reference spectrum was acquired for [ 2 H 1 15 N]PRL-GI 29R under identical conditions.
  • Peak intensities in the two spectra were measured and the ratio of the peak intensity in the spectrum with saturation relative to the intensity of the corresponding peak in the reference spectrum was calculated. Signals displaying strong attenuation in the saturated spectrum are attributable to the residues in PRL-G129R for which the corresponding amide proton is in close proximity ( ⁇ 7A distance) of protons in the receptor chain, and therefore are likely to be important for receptor interaction.
  • the cross-saturation data serve to identify residues in PRL-G 129R making direct contact with the receptor.
  • the results from the cross-saturation experiment are mapped on the 3D PRL structure in Figure 10.
  • the overall site 1 binding interface determined by the NMR methods is generally in accordance with results from mutation experiments aimed at identifying residues important for receptor binding (Goffin, V. et al., MoI. Endocrinol. 6, 1381-1392 (1992) and Kinet, S. et al., J. Biol. Chem. 271, 14353-14360 (1996).
  • additional important interactions in the PRL-G129R/PRLR complex are indicated by the NMR data.
  • C- terminal Cystine C199
  • Two hotspot libraries were generated with Error-prone PCR using PRL G129R as the template.
  • the libraries were screened with Scintillation Proximity Assay (SPA). About 1 % of the hits were cherry picked and confirmed with SPA. About 10% of the hits identified by confirmation SPA were purified and analyzed with Biacore assay and cell-based bioassay.
  • Two hits, [PRL Q73L, M75T, N76S, F80L, G129R] and [PRL S33A, Q73L, G129R, K190R] were identified to have higher affinity than wt PRL and 6 to 8 fold higher antagonist activity compared with PRL G129R.
  • the library LibMixNew was generated based on EZclone strategy(Genemorphll EZclone Domain Mutagenesis Kit, stratagene catalog# 200552). Mixture of primer Lib23-83 and Lib173-199 which generated by error prone PCR were used as mega primer for round the world PCR by pfu polymerase. After Dpn ⁇ digestion, 6 separate reactions were performed as 3 ⁇ l of PCR product were transformed into 50 ⁇ l DH5 competent cell, recover 20 mins at 37°C, Plate on LBA plate for O/N at room temperature. Collect around 50,000 colonies from all the plates for plamid purification, 10 ⁇ g plamid can be recovered.
  • the pET39b-BirATag-Ser-PRLR(1-210)/E. coli BL21(DE3) was cultivated at 37°C in LB medium supplemented with 25 ⁇ g/ml of Kanamycin and 10 ⁇ g/ml of chloramphenicol to an optical density of 0.8, and the cells were induced with 0.5 mM IPTG and 100 ⁇ M biotin for 6 hours (37°C, 250 rpm).
  • the cell pellet was harvested by centrifugation, resuspended in the buffer (20 mM Tris, pH 8.0, 5 mM EDTA, 2 mM DTT, 0.05% Tween 20) and disrupted with the cell disruptor (Z-plus, Constant Systems).
  • the inclusion bodies were pelleted and solubilised with 100 mM Tris, pH 8.0, 8 M urea, 5 mM DTT.
  • the solubilised material was clarified by centrifugation, then diluted 20-fold into the refolding buffer (20 mM Tris, pH 8.0, 0.05% Tween 20, 0.5 mM GSSH, 0.1 mM GSSG) and stirred at 16°C for 65 hours.
  • the refolded protein was purified with QHP sepharose (GE), followed by affinity purification with SoftLinkTM Soft Release Avidin Resin (Promega).
  • SPA assay :
  • the cells in 96-well plates were harvested by centrifugation.
  • the cell pellet was resuspended with the lysis buffer (CelLytic Express, Sigma) and stayed at RT for 1 hr for complete lysis.
  • the cell lysate was diluted with pure water 3 times. 15 ⁇ l of the lysate was added into 85 ⁇ l of the assay buffer (50 mM Tris, pH 8.0, 0.05% Triton X-100, 0.2%BSA) containing 0.3 mg streptavidin SPA beads (RPNQ0066V, GE), 0.1 ⁇ Ci of tritium labelled wt PRL and 150 nM BirA-Ser-PRLR (1-210). Stay at room temperature for 3 hours and count with the luminescence counter (MicroBeta TriLux, PerkinElmer). The pipetting was performed with the liquid handler (Biomek FX 1 Beckman). Purification of the hits:
  • the pET32_PRL mutant (hits)/E. coli Origami was cultivated at 37°C in LB medium supplemented with 100 ⁇ g/ml Ampicillin to an optical density of 0.8-1.0, and the cells were induced with 50 ⁇ M IPTG overnight.
  • the cell pellet was harvested by centrifugation, and then lysed with the lysis buffer (CelLytic Express, Sigma).
  • the cell lysate was clarified by centrifugation and purified with Ser-PRLR (1-210) coupled sepharose 4 FF (NHS-activated sepharose 4 FF, GE).
  • Biacore assay :
  • Biotinylated prolactin receptor BirATag-Ser-PRLR (1-210) was diluted to 20 ⁇ g/ml in 10 mM sodium acetate pH 4.0 (Biacore BR-1003-49) and immobilized on the CM5 chip (Biacore BR-1006-68) with the immobilization reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 1.0 M ethanolamine- HCI pH 8.5 (Biacore BR1000-50).
  • the immobilization level was 1500 RU.
  • the PRL analogues were diluted to a series of concentrations as following: 1.6/3.13/6.25/12.5/25 nM and run through the PRLR immobilized chip using HBS-EP (10 mM HEPES pH 7.4; 150 mM NaCI; 3 mM EDTA; 0.005% v/v Tween-20) as the running buffer under the following conditions:
  • Ba/F3 cells were transfected with PRLR gene containing plasmid. Cells could survived under 1 ng/ml wtPRL stimulation were subcloned and 48 clones with fast growing were chosen for further dose-response study under different wtPRL stimulation. About 50% of these 48 clones can survive at 0.4 ng/ml wtPRL but only 2 of them kept proliferation at 0.1 ng/ml wtPRL.
  • Figure 11 shows the Biacore assay results of some prolactin analogs.
  • [PRL S61 A, Q71A, Q73A, G129R] was a rational designed mutant.
  • [PRL Q73L, M75T, N76S, F80L, G129R] and [PRL S33A, Q73L, G129R, K190R] were two hits identified by SPA assay. The result indicated that the affinity of the two hits was almost 2-fold higher than wt PRL and [PRL G129R].
  • Figure 12 shows a Ba/F3-PRLR proliferation assay result.
  • the wtPRL reached highest stimulation activity around 1 nM and the EC50 is 1.02E-10M.
  • the PRL-G129R reached highest stimulation activity around 110 nM and the EC50 is 3.2E-09M.
  • the highest proliferation rate under PRL-G129R stimulation is only 12% that of wtPRL.
  • very weak agonist activity could be detected.
  • FIG. 13 shows an example of Ba/F3-PRLR competition assay result.
  • HTPN-62 is the mutant [PRL Q73L, M75T, N76S, F80L, G129R], one of the hits identified by SPA assay and Biacore as-say. The result indicated that the antagonist activity of the mutant was about 4 fold higher than that of [PRL G129R].
  • Test compound in this case ECD-PRL-R (25 ⁇ g/ml in 10 mM sodium acetate, pH 3.0), was injected into a Biacore 3000 instrument at a flow rate of 5 ⁇ l/min and coupled to a CM5 sensor chip by amine coupling chemistry.
  • Prolactin and variants thereof 500 nM in buffer; 20 mM Hepes, pH 7.4, containing 0.1 M NaCI, 2 mM CaCI 2 and 0.005% P20 were then injected over the immobilized receptor for 5 minutes at the same flow rate, followed by a 10-min dissociation period during which buffer was injected, to assess receptor binding affinity.
  • Data evaluation was performed in BiaEvaluation 4.1. Regeneration was accomplished with 4.5 M MgCI 2 between runs.
  • AU 565 cells were cultured for 2 days in 6-well dishes. Cells was starved for 18 hours in medium with ⁇ 1 % FCS after which they were stimulated for 15 min with PRL or variants thereof. Cell lysate was prepared and analyzed for STAT5 tyrosine phosphorylation by western blotting.

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Abstract

L'invention concerne des variants de la prolactine présentant une affinité élevée avec le récepteur de la prolactine.
PCT/EP2007/007863 2006-09-08 2007-09-10 Peptides présentant une affinité élevée avec le récepteur de la prolactine WO2008028684A2 (fr)

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PCT/EP2008/052784 WO2009003732A2 (fr) 2007-07-05 2008-03-07 Peptides dotés d'une haute affinité pour le récepteur de la prolactine
EP08717531A EP2167116A2 (fr) 2007-07-05 2008-03-07 Peptides ayant une forte affinite pour le recepteut de la prolactine
PCT/EP2008/058593 WO2009004058A2 (fr) 2007-07-05 2008-07-03 Nouveaux composés de prolactine
EP08802942A EP2167059A2 (fr) 2007-07-05 2008-07-03 Nouveaux composés de prolactine
PCT/EP2008/058589 WO2009004057A2 (fr) 2007-07-05 2008-07-03 Ligands du récepteur de prolactine dimère muté

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058142A1 (fr) * 1998-05-12 1999-11-18 Chen Wen Y Utilisation d'agents anti-prolactine pour le traitement d'etats proliferatifs
US6780613B1 (en) * 1988-10-28 2004-08-24 Genentech, Inc. Growth hormone variants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6780613B1 (en) * 1988-10-28 2004-08-24 Genentech, Inc. Growth hormone variants
WO1999058142A1 (fr) * 1998-05-12 1999-11-18 Chen Wen Y Utilisation d'agents anti-prolactine pour le traitement d'etats proliferatifs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLEVENGER C V ET AL: "The role of prolactin in mammary carcinoma" ENDOCRINE REVIEWS, BALTIMORE, MD, US, vol. 24, no. 1, February 2003 (2003-02), pages 1-27, XP002408798 *

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