WO2005097994A1 - Muteines se fixant sur la digoxygenine de la proteine de pieris brassicae qui se fixe sur la biline - Google Patents

Muteines se fixant sur la digoxygenine de la proteine de pieris brassicae qui se fixe sur la biline Download PDF

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WO2005097994A1
WO2005097994A1 PCT/EP2004/003741 EP2004003741W WO2005097994A1 WO 2005097994 A1 WO2005097994 A1 WO 2005097994A1 EP 2004003741 W EP2004003741 W EP 2004003741W WO 2005097994 A1 WO2005097994 A1 WO 2005097994A1
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mutein
digoxigenin
digoxin
amino acid
binding
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PCT/EP2004/003741
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English (en)
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Arne Skerra
Evelyn Braungart
Steffen Schlehuber
Alexander Peim
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Pieris Proteolab Ag
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Priority to PCT/EP2004/003741 priority Critical patent/WO2005097994A1/fr
Publication of WO2005097994A1 publication Critical patent/WO2005097994A1/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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

  • the present invention relates to novel muteins of the bilin-binding protein of Pieris brassicae, which bind both digoxin and digoxigenin with the substantially same affinity.
  • V & affinity for digoxigenin of these muteins when expressed in terms of the dissociation constant Kj for the complex formed between digoxigenin and the mutein is 25 nM or less.
  • the invention also refers to the corresponding nucleic acid molecules encoding such muteins as well as to a method for their production by means of genetic engineering.
  • the invention is directed to a composition, preferably a pharmaceutical composition, composition and a kit comprising such a mutein as well as to the use of a muteina in various applications.
  • the bilin-binding protein of the butterfly Pieris brassicae is a prototypical member of the lipocalin family of polypeptides (Pervaiz, S., and Brew, K. (1987) FASEB J 1, 209-214).
  • the members of this family are typically small, secreted proteins, which bind various, principally hydrophobic molecules (such as retinoids, fatty acids, cholesterols, biliverdins, pheromones, tastants, and odorants).
  • lipocalins fulfill a variety of physiological -functions such as in olfaction, pheromone signaling, regulation of the immune response and mediation of cell homoeostasis (reviewed, e.g., in Flower, D.R. (1996) Biochem. J. 318, 1-14; Plower, D.R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).
  • lipocalin muteins named anticalins ® , i.e. polypeptides exhibiting specific binding characteristics for a given ligand (also reviewed, e.g., in Skerra, A. (2000) J Mol. Recognit. 13, 167-187; Weiss, G.A. and Lowman, H.B. (2000) Chem. Biol. 7, R177-R184). Due to their antibody-like functions in recognizing prescribed ligands combined with their small size and advantageous biophysical properties, engineered lipocalins have been regarded as attractive candidates for the design of "customized" binding modules as novel tools in diagnostic as well as therapeutic applications.
  • digoxin and to a much lesser extent digitoxin play a prominent role in the treatment of ventricular tachyarehythmias and congestive heart failure (reviewed, for example, in Kelly, R.A. and Smith, T.W. (1996) In: Goodman Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., McGraw-Hill, New York, NY, pp. 809-838; Rahimtoola, S.H. and Tak, T. (1996) Curr. Probl. Cardiol. 21, 787-853; Hauptman, P.J. and Kelly, R.A. (1999) Circulation 99, 1265-1270).
  • Digoxigenin-based labeling systems for the non-radioactive labeling of nucleic acids and proteins have become popular in recent years. Not only do they provide similar sensitivity than radioactively labeled probes, for example, but are also more convenient to use.
  • Digoxigenin can, for example, be easily coupled to nucleotides like UTP, dUTP or ddUTP and incorporated into nucleic acids using generally available polymerases such as Klenow DNA polymerase, Taq DNA polymerase, or RNA polymerases.
  • the probes thus generated can be used in standard blotting and hybridization procedures including in situ hybridization, gel shift assays or RNase protection assays and detected with anti-digoxigenin conjugates (reviewed in, for example, Holtke, H.J. et al. (1995) Cell. Mol. Biol. 41, 883-905; Hughes, J.R. et al. (1995) Non-isotopic methods in molecular biology. Oxford IRL Press at Oxford University Press, pp. 145-182; McCreery, T. (1997) Mol. Biotechnol. 7, 121-124).
  • a first promising molecule that might replace antibodies in digoxigenin-based applications is the digoxigenin-binding mutein IDigAl ⁇ .
  • This mutein was obtained in a two-step procedure.
  • the mutein DigA which binds digoxigenin with a C D of approximately 300 nM was isolated from a random BBP-library which was obtained by random mutagenesis of 16 amino acid residues (sequence positions 34 to 37, 58, 60, 69, 88, 90, 93, 95, 97, 114, 116, 125, and 127) of the bilin-binding protein.
  • affinity maturation was carried out by selectively randomizing only residues in the first loop of the lipocalin binding site (sequence positions 28, 31, 34, 35, 36, 37) and subjecting the collection of mutants again to screening against the digoxigenin group.
  • This approach yielded the mutant DigA 16 which binds digoxigenin with a K ⁇ of approximately 30 nM (WO 00/75308; Schlehuber et al., J Mol. Biol. 297 (2000) 1105- 1120).
  • Such a mutein is a digoxigenin binding mutein of the bilin-binding protein of Pieris brassicae, comprising: an amino acid replacement at the sequence position 56 and/or 86, and further comprising an amino acid replacement at at least one of any of the sequence positions 28, 31, 34,
  • the mutein of the invention binds digoxigenin with a K Q of 20 nM, and more preferred with a i-T D of 16 nM, 11 nM or even less.
  • a preferred mutein of the invention also binds both digitoxin and digitoxigenin with substantially mutually the same affinity, which renders such a mutein also a promising candidate for use in applications in which binding to either the digitoxin or digitoxigenin group or both is required.
  • Such a mutein preferably binds digitoxigenin with a R-rj of 2 nM, 1.5 nM, 1.0 nM or even less.
  • the complex formation between the respective mutein and its ligand is influenced by many different factors such as the concentrations of the respective binding partners, the presence of competitors, pH and the ionic strength of the buffer system used, and the experimental method used for determination of the dissociation constant K Q (for example fluorescence titration, competition ELISA or surface plasmon resonance, just to name a few) or even the mathematical algorithm which is used for evaluation of the experimental data.
  • the Rpo values (dissociation constant of the complex formed between the respective mutein and its ligand) given here may vary within a certain experimental range, for example ⁇ 1, 2 or 3 nM, depending on the method and experimental setup that is used for determining the affinity of a particular BBP mutein for digoxigenin or digitoxigenin. This means, there may be a slight deviation in the measured KQ values or a tolerance range depending, for example, on whether the K ⁇ value was determined by fluorescence titration or by competition ELISA.
  • the determination of the KQ value of a given mutein is preferably carried out by fluorescence titration using a suitable physiological buffer system that best resembles the conditions of a pharmaceutical in vivo use and allows measurements under conditions of the-modynamic equilibrium. More preferably, PBS (4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 , 115 mM NaCl, pH 7.4), optionally containing 1 mM EDTA (PBS/E), and the respective BBP mutein in a concentration of 1 ⁇ M or 0.1 ⁇ M is used as buffer for the fluorescence measurement (cf. Example 3). The so obtained data is then preferably normalized to an initial fluorescence intensity of 100 % and fitted by non-linear least squares regression according to the theory of bimolecular complex formation using the formula (I):
  • the term "substantially the same affinity for digoxigenin and digoxin” means that the measured dissociation constant K Q can vary within certain limits that axe caused, for example, a) by an actual slight intrinsic difference in the binding characteristics and b) by the experimental error in the determination of the dissociation constant.
  • a BBP mutein of the invention has a K Q for the binding of digoxigenin that is within the range of 25 to 10 nM
  • the term "substantially the same affinity for digoxigenin and digoxin” means that the dissociation constants of the complexes formed between the mutein (or fusion protein thereof, for example) and either digoxigenin or digoxin can differ in a range of up to about ⁇ 10 nM. This means, if digoxigenin and digoxin are bound by a respective mutein with a j of 11 nM and 21 nM, they are still bound with substantially the same affinity.
  • a BBP mutein of the invention binds digoxigenin with a K Q that is within the range of 10 to 1 nM
  • the term "substantially the same affinity for digoxigenin and digoxin” means that the dissociation constants of the complexes formed between the mutein (or a fusion protein thereof, for example) and either digoxigenin or digoxin can differ by up to about ⁇ 5 nM. This means, if digoxigenin and digoxin are bound by a respective mutein with a K Q of 3 nM and 8 nM, they are still bound with substantially the same affinity.
  • the term "substantially the same affinity for digoxigenin and digoxin" means that the dissociation constants of the complexes formed between the mutein and either digoxigenin or digoxin can differ by up to about ⁇ 500 pM.
  • a K Q of 800 pM for the binding of digoxigenin and 300 pM for the digoxin binding is considered to be substantially the same in the present invention. The same holds true, if the K Q value becomes still lower, for example 100 pM for digoxigenin and 600 pM for digoxin.
  • the term "substantially the same affinity for digoxigenin and digoxin” means that the dissociation constants of the complexes formed between the mutein protein and either digoxigenin or digoxin can differ by up to about ⁇ 50 pM.
  • the definition "substantially the same affinity” means that both, the dissociation constant K Q for the complex of the respective mutein with digoxigenin and digoxin is 25 nM or less, or preferably less than 20, 16 or 11 nM.
  • the term "substantially the same affinity for digitoxigenin and digitoxin” is used within the same meaning as explained with respect to digoxigenin and digoxin above, except that the dissociation constants K- Q for the complex of the respective mutein with digitoxigenin and the one with digitoxin are both preferably 2 nM, 1.5 nM, 1.0 nM or even less.
  • the muteins of the present invention show an improved binding specificity for digoxin and digoxigenin.
  • the ratio r Kn for the complex formed between mutein and Progesterone K ⁇ > for the complex formed between mutein and Digoxin
  • the invention is based on the surprising finding that an amino acid substitution at sequence position 56 and/or 86 is sufficient to significantly improve the affinity of known digoxigenin-binding BBP muteins for both digoxigenin (about 4-fold) and digitoxigenin (about 20-fold), while maintaining a superior or at least equal binding of the naturally occurring cardiac glycosides digoxin and digitoxin compared to their respective aglycons.
  • IVIuteins of the invention bind digoxigenin with K Q values of about 1 to 3 nM and digitoxigenin with K Q values of about 300 to 100 pM (cf. examples), which makes them highly versatile and valuable molecular tools not only in diagnostic applications but also - and more importantly - as an effective therapeutic in the treatment of digitalis intoxications such as digoxin intoxications.
  • valine residue and the histidine residue occurring at position 56 and position 86, respectively, in the wild type sequence of the bilin-binding protein of Pieris brassicae can be replaced by any other suitable amino acid as long as this replacement leads to a mutein having the substantially same binding affinity for both digoxin and digoxigenin and wherein said mutein binds digoxigenin with a K Q of 25 nM or less.
  • the His residue at sequence position 86 is replaced by a Ser, Asn, Gin, Gly, Ala, Thr, Cys, Leu, Asp or Glu residue.
  • the Nal residue at sequence position 56 can be replaced by a hydrophobic residue such as Leu, He or Met.
  • a Leu or Met residue is introduced at position 56 of the polypeptide sequence of a BBP.
  • the substitutions at sequence positions 56 and 86 can be carried out independently from each other. Accordingly, a mutein of the invention can be a "single mutant" (in which only the position 56 or the position 86 is mutated) or a "double mutant".
  • Examples of such "double mutants" are the muteins that comprise the following amino acid substitutions: 1) His86— >Ser, Nal56 ⁇ Leu; 2) His86->Ser, Val56 ⁇ Met; 3) E_is86 ⁇ Asn, Nal56 ⁇ Leu; 4) His86- Asn, Nal56 ⁇ Met; 5) His86 ⁇ Gln, Val56 ⁇ Leu; 6) His86 ⁇ Gln, Nal56 ⁇ Met; 7) His86 ⁇ Cys, Nal56 ⁇ Leu; 8) His86 ⁇ Cys, Val56 ⁇ Met; 9) His86 ⁇ Thr, Nal56 ⁇ Leu; 10) His86 ⁇ rhr, Nal56 ⁇ Met; 11) His86 ⁇ Ala, Val56 ⁇ Leu; 12) His86- Ala, Nal56 ⁇ Met; 13) His86 ⁇ Ser, Nal56 ⁇ fle; 14) His86 ⁇ Asn, Val56 ⁇ Ile; 15) His86 ⁇ Gln, Nal56 ⁇ Ile (cf. Examples).
  • the binding site of the BBP in general or digoxigenin-binding muteins such as DigA16 derived from the BBP can accommodate a rather large number of amino acid substitutions, both with respect to sequence position and to the side chain at a specific position, without losing its ability of binding digoxigenin.
  • the Glu residue at secjuence position 96 of wild type BBP can be replaced by a Nal or a Gly residue
  • the Phe residue at position 99 of wild type BBP can be substituted by a Ser, Nal or Gly residue
  • the Lys at position 116 of wild type BBP can be replaced by either Ser, Trp or Arg
  • the Phe residue at position 127 of wild type BBP can be substituted by Leu, Tyr, or Ala, to name a few of s ⁇ ch possibilities.
  • a BBP mutein of the invention can further comprise an amino acid mutation at at least one, more preferably at least any four, more preferably at least any eight, at least any 12, 17, 19 or at all 20 sequence positions 28, 31, 34, 35, 36, 37, 58, 60, 69, 88, 90, 95, 96, 97, 99, 114, 116, 119, 125, and 127.
  • muteins are preferred that comprise at least 12, 17 or 19 or all 20 amino acid mutations at any of these sequence positions of the bilin-binding protein, wherein the mutations are selected from Glu28— >Gln, Lys31— Ala, Asn34— >As ⁇ , Ser35— His, Nal36 ⁇ Ile, Glu37->Thr, Asn58 ⁇ Arg, His60 ⁇ Ser, Ile69 ⁇ Ser, Leu88->Tyr, Tyr90 ⁇ Ile, Tyr90 ⁇ Leu, Lys95 ⁇ Gln, Lys95 ⁇ Thr, Glu96-»Nal, Glu96 ⁇ Gly, Asn97 ⁇ Gly, Phe99 ⁇ Ser, Tyrll4 ⁇ Phe, Lysll ⁇ Ser, Lysll6 ⁇ Arg, Lysll ⁇ Trp, Glnll9 ⁇ - ys, Glnl25 ⁇ Met, Glnl25 ⁇ Leu, Phel27 ⁇ Leu, Phel27 ⁇ Tyr, and Phel27 ⁇ A
  • the inventive BBP mutein comprises or has the amino acid sequence that corresponds to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ -ED NO: 20.
  • amino acid replacement means that the amino acid naturally occurring at a given sequence position of the BBP of Pieris brassicae (SWISS PROT data bank accession code P09464) is substituted by at least one amino acid that is not present at this specific position in the natural (wild type) polypeptide sequence.
  • Such mutations can be introduced easily on the DNA level using established standard methods such as oligodeoxynucleotide-directed mutagenesis (cf., for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • amino acid replacement also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids.
  • one amino acid at a chosen sequence position is replaced/substituted by a stretch of three amino acids, leading to an insertion of two amino acid residues vith respect to the length of (the respective segment) of the wild-type protein.
  • a stretch of consecutive amino acids for example, of three or four amino acids, is replaced by a single amino acid residue.
  • amino acids other than the 20 naturally occurring amino acids such as selenocystein or pyrrolysine
  • "artificial" codons in order to introduce other unusual amino acids, for example o- methyl-L-tyrosine or p-aminophenylalanine (Wang, L. et al. (2001) Science 292, 498-500; Wang, L. and Schultz, P.G. (2002) Chem. Comm. 1, 1-11).
  • the BBP muteins of the invention may comprise the wild-type amino acid sequence at any non-mutated position.
  • the BBP muteins disclosed herein may also contain amino acid mutations apart from the sequence positions as defined in the claims and that participate in the ligand binding. Such mutations are often tolerated or can even prove to be advantageous, for example if they contribute to an improved folding efficiency, protein stability or ligand binding affinity of the mutein (cf. also the possible variations of amino acids in the binding site explained above).
  • possible alterations of the amino acid sequence include insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e. an amino acid residue is replaced with a chemically similar amino acid residue.
  • conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysirie; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan (see also Creighton, T.E. (1993) Proteins: structures and molecular properties, pp. 6-20, 2 nd ed., W.H. Freeman and Company, New York).
  • Creighton, T.E. (1993) Proteins: structures and molecular properties, pp. 6-20, 2 nd ed., W.H. Freeman and Company, New York One the other hand, it is also possible to introduce non- conservative alterations in the amino acid sequence.
  • th-e inventive BBP muteins may have amino acid substitutions, which prevent BBP oligo-merization, such as the Asnl ⁇ Asp substitution, or suppress proteolytic cleavage within ttie polypeptide chain that may occur during production in Escherichia coli (E. col ⁇ ), such as the Lys87— >Ser substitution.
  • modifications of the amino acid sequence include directed mutagenesis of single amino acid positions in order to simplify sub-cloning of the mutein gene or its parts by incorporating cleavage sites for certain restriction enzymes. Furthermore, mutations can specifically be introduced in order to improve certain characteristics of the mutein.
  • the inventive mutein is fused at its N-terminus or its C-terminus to a protein, a protein domain or a peptide such as a signal sequence, a toxin and or an affinity tag.
  • the coupling to the conjugated molecule can be realized by any suitable reactive group of the BBP mutein, for example, the terminal ⁇ -amino group of lysine residues or purposely introduced Cys residues.
  • the fusion partner may confer new characteristics to the inventive -mutein such as enzymatic activity or improved affinity.
  • suitable fusion proteins are alkaline phosphatase, horseradish peroxidase, glutathion-S-transferase, the albumin-binding domain of protein G, the Z domain of protein A, antibody fragments, oligomerization domains, and lipocalin muteins of same or different binding specificity (which results in the formation of "duocalins", cf. PCT application WO 99/16873 or Schlehuber, S. & Skerra, A. (2001) Biol. Chem. 382, 1335-1342).
  • Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T.G.M. et al. (1996) J Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag, the His 6 -tag, the X7-tag or the HA-tag or proteins that allow easy detection and/or purification of recombinant proteins or enhance their solubility are further examples of preferred fusion partners.
  • proteins with chromogenic or fluorescent properties such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP) are suitable fusion partners for a lipocalin mutein of the invention as well.
  • fusion protein also comprises BBP muteins according to the invention containing a C- or N-terminal signal sequence.
  • Signal sequences at the N-terminus of a polypeptide direct this polypeptide to a specific cellular compartment, for example the periplasm of E. coli or the endoplasmatic reticulum of eukaryotic cells. A large number of signal sequences is known in the art.
  • a preferred signal sequence for the secretion of a polypeptide into the periplasm of E. coli is the OmpA-signal sequence.
  • An example of a useful C-terminal signal sequence is the signal sequence of the hemolysin A transporter of E. coli.
  • the invention is also directed to muteins, which are conjugated to a label.
  • This label can be selected from enzyme labels, radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, biotin, metal complexes, metals, and colloidal gold, to name a few.
  • the mutein may also be conjugated- to an organic molecule.
  • organic molecule as used herein preferably denotes an organic molecule comprising at least two carbon atoms, but preferably not more than seven rotatable carbon bonds, having a molecular weight in the range between 100 and 2000 Dalton, preferably 1000
  • Dalton Dalton, and optionally including one or two metal atoms.
  • the lipocalin mutein with any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical or enzymatic reaction.
  • An example for a physical reaction is the emission of fluorescence or phosphorescence upon irradiation or excitation or the emission of X-rays when using a radioactive label.
  • Alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, and ⁇ -lactamase are examples of enzyme labels, which catalyze the formation of chromogenic reaction products.
  • all labels commonly used for antibodies can also be used for conjugation to the muteins of the present invention. Such conjugates can be produced by methods well known in the art.
  • the present invention also relates to nucleic acid molecules (DNA and RNA) comprising nucleotide sequences coding for BBP muteins as described herein. Since the degeneracy of the genetic code permits substitutions of certain codons by other codons specifying the sa-me amino acid, the invention is not limited to a specific nucleic acid molecule encoding a mutein of the invention but includes all nucleic acid molecules comprising nucleotide sequences encoding functional BBP muteins, which bind both digoxin and digitoxin with substantially the same affinity as described herein.
  • the nucleic acid sequences encode BBP muteins which bind digoxigenin with a K Q of 25 nM, and more preferred with a K Q of 20, 16 nM, 11 nM or less.
  • the nucleic acid sequences encode BBP muteins which bind digitoxigenin with a K Q of 2 nM, 1.5 nM, 1 nM, or less.
  • the nucleic acid sequence encoding: an inventive mutein comprises nucleotide mutations at a sequence position corresponding to amino acid position 56 and/or 86, and nucleotide mutations at at least any four, more preferably at least any eight, at least any 12, 17, 19 or at all 20 sequence positions 28, 31, 34, 35, 36, 37, 58, 60, 69, 88, 90, 95, 96, 97, 99, 114, 116, 119, 125, and 127 of the BBP polypeptide.
  • the inventive nucleic acid molecule comprises or has a nucleic acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • the invention as disclosed herein also includes nucleic acid molecules encoding BBP muteins which comprise additional nucleotide mutations at sequence positions other than those mentioned above. Such mutations are often tolerated or can even prove to be advantageous, for example, if they contribute to an improved folding efficiency, protein stability or ligand binding affinity of the mutein.
  • a nucleic acid molecule disclosed in this application may be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of this nucleic acid molecule.
  • a nucleic acid molecule such as DNA
  • capable of expressing genetic information giving rise to transcription and/or translation of an encoded protein
  • a nucleic acid molecule or capable “to allow expression of a nucleotide sequence” if it comprises sequence elements which contain information regarding to transcriptional and/or translational regulation, and such sequences are "operably linked” to the nucleotide sequence encoding the polypeptide.
  • An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression.
  • promoter regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e. DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation.
  • promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/-10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5 -capping elements in eukaryotes.
  • These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.
  • the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactorily functional in a particular host cell, they may be substituted with signals functional in that cell.
  • a nucleic acid molecule of the invention can include a regulatory sequence, preferably a promoter sequence.
  • a nucleic acid molecule of the invention comprises a promoter sequence and a transcriptional termination sequence.
  • Suitable prokaryotic promoters are, for example, the tet promoter, the Z ⁇ cUV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SN40 promoter or the CMN promoter.
  • the nucleic acid molecule of the invention can also be comprised in a vector or any other cloning vehicles, such as plasmids, phagemids, phages, baculoviruses, cosmids or artificial chromosomes.
  • cloning vehicles can include replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art, and are commercially available.
  • the D ⁇ A molecule encoding a BBP mutein of the invention can be transfected into a host cell capable of expressing the gene. Transformation can be performed using standard techniques (Sambrook, J. et al. (1989), supra). Thus, the invention is also directed to a host cell containing a nucleic acid molecule as disclosed herein.
  • the transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a fusion protein of the invention.
  • Suitable host cells can be prokaryotic, such as E. coli or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) or primary mammalian cells.
  • the invention also relates to a method for the production of a BBP mutein of the invention, wherein the mutein, a fragment of the mutein or a fusion protein of the mutein and another polypeptide is produced starting from the nucleic acid coding for the mutein by means of genetic engineering methods.
  • the method can be carried out in vivo, the mutein can for example be produced in a bacterial or eukaryotic host organism and then isolated from this host organism or its culture. It is also possible to produce a protein in vitro, for example by use of an in vitro translation system.
  • a nucleic acid encoding a mutein of the invention is introduced into a suitable bacterial- or eukaryotic host organism by means of recombinant D ⁇ A technology (as already outlined above).
  • the host cell is transformed with a cloning vector comprising a nucleic acid molecule encoding such a mutein using established standard methods (Sambrook, J. et al. (1989), supra).
  • the host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium.
  • nascent polypeptide can be direct to a cell compartment having an oxidizing redox milieu using an appropriate signal sequence.
  • an oxidizing environment is provided by the periplasm of Gram-negative bacteria such as E. coli or in the lumen of the endoplasmatic reticulum of eukaryotic cells and usually favors the correct formation of the disulfide bonds.
  • a mutein of the invention in the cytosol of a host cell, preferably E. coli.
  • the polypeptide can, for instance, be produced in form of inclusion bodies, followed by renaturation in vitro or as soluble material.
  • a further option is the use of specific host strains having an oxidizing intracellular milieu, which thus allow the production of the native protein in the cytosol.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one inventive mutein, a fusion protein or a conjugate thereof and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is used for the treatment of intoxications caused by digoxin, digoxigenin, digitoxin or digitoxigenin, or derivatives and structural or functional analogues thereof and in particular for such intoxications of the heart.
  • the terms “derivatives” and “structural or functional analogues” include all compounds to which a mutein of the invention binds with detectable affinity.
  • the terms further include such compounds in which the tridigitoxose, if present, is replaced by another sugar moiety, for example a monosaccharide (for example rhamnose or thevetose), a disaccharide (for example, didigitoxose) or different trisaccharide, or in which the free hydroxyl group at the 3-position of the steroid moiety is converted into an ester, an amide or is acetylated, for example.
  • a monosaccharide for example rhamnose or thevetose
  • a disaccharide for example, didigitoxose
  • different trisaccharide or in which the free hydroxyl group at the 3-position of the steroid moiety is converted into an ester, an amide or is acetylated, for example.
  • These terms also include compounds with modifications at the 12- or 16-position of the steroid moiety such as 12-acetyldigoxin or 16-acetylgitoxin.
  • Examples of such derivatives and structural or functional analogues include but are not hmited to digoxigenin bisdigitoxoside, digoxin monodigitoxoside, 3-epidigoxigenin, evomonoside, neriifolin, gitoxin, diginatin, 12-acetyldigoxin, digoxigenin-3,12-diacetate, dihydrodigoxigenin, dihydrodigoxin, gitoxigenin monodigitoxoside, strospeside, gitoxigenin, gitoxigenin-3-acetate, gitaloxin, gitaloxigenin, 16-acetylgitoxin, oleandrin, oleandrigenin monodigitoxoside, oleandrigenin, gitoxigenin-3,16-dia
  • intoxication includes the known suicidal or accidental consumption of fatal doses of the above-mentioned compounds (e.g. ingestion of 10 mg digoxin or more in previous healthy adults or 4 mg digoxin or more in previously healthy children), chronic ingestions causing digoxin blood serum concentrations greater than 5 ng/ml as well as (acute) intoxications due to digoxin or digitoxin overdoses during the treatment of cardiac diseases.
  • the BBP muteins according to the invention can be administered via any parenteral or non- parenteral (enteral) route that is therapeutically effective for proteinaceous drugs.
  • Parenteral application methods comprise, for example, intracutaneous, subcutaneous, intramuscular or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures, as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders.
  • Non-parenteral delivery modes are, for instance, orally, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or rectally, e.g. in the form of suppositories.
  • the muteins of the invention can be administered systemically or topically in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers , additives and vehicles as desired.
  • the pharmaceutical composition is administered parenterally, with intravenous infusion or injection being the most preferable application method.
  • the dosage of the mutein applied may vary within wide limits to achieve the desired therapeutic response for a particular patient. It will, for instance, depend on the affinity of the BBP mutein for digoxin or digitoxin as well as the half -life of the respective complex in vivo, its biodistribution, the mode of administration, the severity of the disease/disorder being treated (i.e. the amount of digoxin or digitoxin to be neutralized) as well as the medical condition of the patient. For example, treatment of acute short-term conditions or disorders such as a digoxin or digitoxin overdoses during the treatment of cardiac diseases might be best accomplished when using a dose as high as maintainable.
  • the smaller size of the BBP mutein compared to antibody (Fab) fragments may be of advantage, since the smaller size should lead to a faster clearance from the blood stream and the body.
  • the BBP mutein can be applied in higher dosages and at the same time be more effective than antibody fragments.
  • the mutein may also be given in a sustained release formulation.
  • the half-life of the BBP mutein can be extended for this purpose for example, by fusion to the Fc region of an preferably human immunoglobulin or by conjugation to a polymer such as polyalkylene glycol (substituted or unsubstituted), for example, polyethylene glycol (PEG) as described in WO 99/64016, US Patent 6,177,074, US Patent 6,403,564 in relation to interferon, or as known for other proteins such as PEG- modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase, for example (see for example, Fuertges et al. (1990) J Control. Release 11, 139- 148).
  • PEG polyethylene glycol
  • PEG-ADA PEG-adenosine deaminase
  • PEG-superoxide dismutase for example (see for example, Fuertges et al. (1990) J Control.
  • the molecular weight of such a polymer which can be preferably polyethylene glycol, may range from about 300 to 70.000 Dalton.
  • carbohydrate oligo- and polymers such as starch or hydroxyethyl starch (HES) can also conjugated to a mutein of the invention for this purpose.
  • HES hydroxyethyl starch
  • Further suitable fusion partners for extending the half-life of a BBP mutein of the invention include albumin (Osborn, B.L. et al. (2002) J Pharmacol. Exp. Ther. 303, 540-548), or a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig, T.
  • a BBP mutein of the invention may also be used as storage and/or transporter molecule for digitalis compounds such as digoxin, digoxigenin, digitoxin or digitoxigenin or derivatives and structural or functional analogues thereof as described above.
  • a dose of about 0.05 mg to 50 mg lipocalin mutein per kilogram body weight may be appropriate.
  • Preferred dosage levels range from 0.5 mg to 5 mg per kg body weight for a long-term regimen and from 5 mg to 25 mg per kg body weight for short-term treatments.
  • the inventive mutein can be applied as a single dose or may be divided into several, e.g. two to four, part administrations.
  • a BBP mutein described here can also be continuously infused over a certain period of time.
  • the muteins of the present invention can be formulated into compositions using pharmaceutically acceptable ingredients as well as established methods of preparation (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA).
  • pharmaceutically inert inorganic and/or organic excipients can be used.
  • pills, powders, gelatin capsules or suppositories for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils can be used.
  • Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols, and suitable mixtures thereof as well as vegetable oils.
  • the pharmaceutical composition may also contain additives, such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • the BBP muteins may be incorporated into slow or sustained release or targeted delivery systems, such as liposomes and microcapsules.
  • the formulations can be sterilized by numerous means, including filtration through a bacteria- retaining filter, or by incorporating antiseptic agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile medium just prior to use.
  • a BBP mutein of the present invention or a fusion protein or conjugate thereof can also be employed in many diagnostic applications.
  • such a mutein can be used in all applications in which anti-digoxin or anti-digoxigenin antibodies are used, except those with specifically rely on the glycosylated Fc region.
  • the inventive muteins can be used for the binding and/or detection of digoxin, digoxigenin, digitoxin, digitoxigenin or a derivative or analogue thereof as defined above, in a method comprising contacting the mutein with a test sample supposed to contain said target molecules and detecting the mutein/target molecule complex qualitatively and/or quantitatively by a suitable signal.
  • a mutein can also be used for the separation of target molecules, in a method comprising contacting the mutein with a sample supposed to contain said target molecules in order to allow complex formation and separating the mutein/target molecule complex from the sample.
  • the complex comprising the mutein and the target molecule may be immobilized on any suitable solid phase or extracted in a poorly miscible liquid phase.
  • kits comprising a BBP mutein as described here.
  • a kit can be a diagnostic kit that is used for determination of digoxin concentration in a blood sample, for example. It can also be a kit that is used for analytical purposes in research, for example, a digoxigenin-based system for the non-radioactive labeling and detection of nucleic acids and proteins.
  • the detectable signal that is used for the detection of the desired compound can be provoked by a label, as explained above, or by a change of physical properties due to the binding, i.e. the complex formation itself.
  • a label as explained above
  • a change of physical properties due to the binding i.e. the complex formation itself.
  • plasmon surface resonance the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold-coated glass chip.
  • the BBP mutein of the invention may be labeled directly, for example by covalent coupling. It is, however, also possible to use indirect labeling, for example by means of labeled antibodies against the BBP or muteins thereof or against domains of fusion proteins of these muteins.
  • inventive fusion proteins containing an enzyme for example alkaline phosphatase, ⁇ -lactamase, ⁇ -galactosidase or horseradish peroxidase, instead of a labeled BBP mutein is particularly advantageous.
  • the detection method with a particularly small number of processing steps, by utilizing directly, for example, the ability of the enzyme as part of the fusion protein to catalyze a chromogenic, fluorogenic or chemiluminescent reaction.
  • the muteins of the invention or fusion proteins thereof may also be used for immobilizing a given molecule conjugated with digoxin, digoxigenin, digitoxin or digitoxigenin or derivatives and structural or functional analogues thereof as described above.
  • This immobilization is preferably carried out on a solid phase coated with the inventive BBP mutein or fusion protein thereof.
  • suitable solid phases include plastic microtiter plates, "immunosticks", (micro)beads made of organic, inorganic or paramagnetic materials, or sensor surfaces.
  • column materials are also considered for immobilizing the muteins or their fusion proteins.
  • said coating is carried out on suitable column materials via coupling by means of chemically reactive groups. Column materials prepared in this way may be used for removing from a solution digoxin itself, substances conjugated with digoxigenin groups and also, where appropriate, complexes of such substances with other molecules.
  • Figure 1 schematically depicts the expression vector pBB21-DigA16 (Schlehuber, S. and Skerra, A. (2002) Biophys. Chem. 96, 213-228), which encodes a fusion protein comprising the OmpA signal sequence, the BBP variant DigA16, and the Strep-tag® ⁇ , and furthermore encodes the disulphide isomerase DsbC of E. coli (Zapun et al. (1995) Biochemistry 34, 5075-5089). Expression of the gene fusion is under the control of the tetracycline promoter/operator (tet p/o ). Transcription is terminated at the lipoprotein transcription terminator (t lpp ).
  • the vector further comprises an origin of replication (ori), the intergenic region of bacteriophage fl (fl-IG), a ⁇ -lactamase gene (bla) conferring ampicillin resistance, and the tetracycline repressor gene (tetR).
  • ori origin of replication
  • fl-IG intergenic region of bacteriophage fl
  • bla ⁇ -lactamase gene conferring ampicillin resistance
  • tetR tetracycline repressor gene
  • Figure 2 illustrates the chemical structures of the steroid compounds used in the binding experiments.
  • the purified BBP muteins were used in a concentration of 1 ⁇ M (A) or 0.1 ⁇ M (B) and titrated with the respective ligand (100 ⁇ M (A) and 10 ⁇ M (B) stock solutions in DMF).
  • the data were normalized to an initial fluorescence intensity of 100 % and fitted by non-linear least-squares regression (cf. Schlehuber, S. and Skerra, A. (2002), Biophys. Chem. 96, 213-228).
  • Figure 4 shows a representative analysis of the affinity of the exemplary muteins of the inventions, DigA16 N56L (filled squares) and DigA16 N56M (filled triangles), in comparision with the mutein DigA16 (filled and closed circles, respectively) for digoxigenin (A) and digitoxigenin (B), respectively, as determined by fluorescence titration.
  • the experimental conditions were as in Fig. 3.
  • Figure 5 shows a representative analysis of the binding affinity of the exemplary mutein DigA16 N56L/H86Q (filled triangles) in co parison with the variants DigA16 N56L (filled squares) and DigA16 H86Q (filled circles) for digoxigenin (A) and digitoxigenin (B), respectively, as determined by fluorescence titration.
  • the experimental conditions were as in Fig. 3.
  • Figure 6 shows the results of proliferation assays with the cardiac steroids digitoxin and digoxin.
  • A 20,000 Jurkat cells were adjusted to 0.1 ⁇ M digitoxin, and then 0.05 ⁇ M (black bars), 0.1 ⁇ M (grey bars) or 0.5 ⁇ M (white bars) of each DigA16 H86 ⁇ , DigA16 H86Q, Digitalis Antidot (Roche) or recombinant bilin-binding protein (as control) was added. After over-night incubation the proliferation status of the culture was determined spectroscopically at 490 nm (corrected by a reference absorption at 690 nm) employing a commercially available proliferation assay.
  • Single-stranded template D ⁇ A was prepared as previously described (Geisselsoder, J. et al. (1987) Biotechniques 5, 786-791).
  • E. coli CJ236i (Fiedler, M. & Skerra, A. (1999) Protein Expr. Purif 17, 421-427) was transformed with the expression vector pBBP21- DigA16.
  • 4 ml LB medium containing 100 ⁇ g/ml ampicillin (LB/ Amp) was inoculated with a single transformant and incubated overnight at 30°C, 200 rpm.
  • the phagemid particles were precipitated for 30 min on ice by adding 5 ml 3.5 M ammonium acetate containing 20 % (w/v) PEG 8000.
  • the phagemids were sedimented by centrifugation (18000 g, 4°C, 15 min), resuspended in 200 ⁇ l 300 mM ⁇ aCl, 100 mM Tris HCl pH 8.0, 1 mM EDTA and incubated on ice for 30 min.
  • the phagemid solution was sequentially extracted with phenol (2 x 200 ⁇ l), phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v; 200 ⁇ l) and chloroform isoamyl alcohol (24:1 v/v; 200 ⁇ l).
  • the aqueous phases were combined (approx. 300 ⁇ l) and the single-stranded D ⁇ A was precipitated for 1 h at -20°C by adding 30 ⁇ l 7.8 M ammonium acetate and 750 ⁇ l ethanol.
  • the yields were in the range of 10-100 ⁇ g single-stranded DNA.
  • His86 ⁇ Ser 5'-CCAATAGTGTAGCTGCTGTAGATCTTTCCAATC-3' (SEQ ID NO: 5) His86 ⁇ Asn: 5'-TAGTGTAGCTGTTGTAGATCTTTCC-3' (SEQ ID NO: 6) His86 ⁇ Gln: 5'-CCAATAGTGTAGCTCTGGTAGATCTTTCC-3' (SEQ ID NO: 7) Nal56 ⁇ Leu: 5'-GAGTAGCGCGAAAGTTTGACACTCTTGC-3' (SEQ ID NO: 11)
  • Nal56 ⁇ Met 5'-TACAGAGTAGCGCGACATTTTGACACTCTTGC-3' (SEQ ID NO: 12)
  • the oligodeoxynucleotides were phosphorylated at their 5'-termini using T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's recommendations.
  • Hybridization of the oligodeoxynucleotides to the single-stranded template DI A was performed in a total volume of 10 ⁇ l using 200 ng template DNA and 3.3 pmoles of the respective phosphorylated oligodeox-ynucleotide in 200 mM Tris/HCl pH 7.5, 500 mM NaCl, 20 mM MgC ⁇ .
  • the reaction mixture was heated at 80°C for 5 min and subsequently allowed to cool down to about 20°C within 4- h.
  • E. coli JM83 was transformed with the expression plasmid pBBP21 harboring the coding region for the respective DigA16 variant (cf. example
  • the cells from one flask were centrifuged (15 minutes, 5500 g, 4°C) and re-suspended in 20 ml periplasmic release buffer (100 mM Tris/HCl pH 8.0, 500 mM sucrose, 1 mM EDTA), followed by cooling on ice for 30 min. Subsequently, the spheroplasts were removed in two successive centrifugation steps (15 min, 5300 g, 4°C and 15 min 270OO g, 4°C, respectively).
  • the supernatant containing the periplasmic protein extract was dialyzed against SA-buffer (100 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), sterile-fiLtered, and subjected to chromatographic purification.
  • SA-buffer 100 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA
  • the purification was performed by using the Strep-Tag® II-affinity tag (Skerra, A. and Schmidt, T.G.M. (2000) Methods Enzymol. 326, 271-304) located at the C-terminus of the BBP mutein.
  • a chromatographic column with a bed volume of 2 ml was filled with Streptactin Superflow affinity matrix (IB A) and equilibrated with 20 rol SA-buffer (pH 8.0) at 4°C at a flow rate of 20 ml/h. Chromatography was monitored by measuring the absorption at 280 nm of the eluate in a flow-through photometer.
  • the column was washed with SA-buffer (pH 8.0) until reaching the base line and the bound BBP mutein was subsequently eluted with ca. 15 ml of a solution of 2.5 mM D-desthiobiotin (B3A) in SA-buffer (pH 8.0) collecting fractions of the eluate.
  • the fractions containing purified protein were analyzed via SDS-polyacrylamide gel electrophoresis (Fling, S.P. and Gregerson, D.S. (1986) Anal. Biochem. 155, 83-88), combin-ed and dialyzed against an appropriate buffer, if needed.
  • the protein yield was in the range between 0.2-0.6 mg/1 culture.
  • a chromatographic column with a bed volume of 20 ml was filled with Q-Sepharose (Amersham) and equilibrated with 40 ml of endotoxin-free SA-buffer (pH 6.0) at 4°C at a flow rate of 2 ml/min prepared with ultrafiltrated water (Milli- Q Biocel, Millipore).
  • the protein solution was applied to the column and the column was washed with SA-buffer (pH 6.0) until the base line was reached again.
  • the flow-through containing the endotoxin-depleted recombinant protein was collected.
  • Protein-containing fractions were pooled and the pH of the solution was adjusted to pH 8.0 by titration with 1 M endotoxin-free Tris (prepared with ultrafiltrated water).
  • the purified protein was dialyzed against endotoxin-free PBS (prepared with ultrafiltrated water).
  • the endotoxin content of the recombinant BBP mutein was determined by means of the COAMATIC Chromo-LAL Kit (Chromogenix) and was typically in the range of 200-400 EU/mg of purified protein.
  • the ligand binding properties of DigA16 as well as of the five variants described above were determined by means of fluorescence titration. In this case, the decrease in intrinsic tryptophan fluorescence of the protein forming a complex with the ligand was measured.
  • the measurements were carried out in an LS 50 B fluorimeter (Perkin Elmex) using a lxl cm 2 quartz cuvette thermostatted at 25°C using an excitation wavelength of 295 nm (slit width 4 nm) and an emission wavelength of 345 nm (slit width 6 nm for 1 ⁇ M protein solutions and 8 nm for 0.1 ⁇ M protein solutions, respectively).
  • the ligands used (Fig. 2) were digoxigenin, digoxin, digitoxigenin, digitoxin, ouabain, 4-aminofluorescein (all obtained from Fluka), progesterone, and testosterone (both from Sigma). The ligands showed no significant intrinsic fluorescence or absorption at the selected wavelengths.
  • the buffer system used was PBS (4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 , 115 mM NaCl, pH 7.4) containing 1 mM EDTA (PBS/E).
  • PBS 4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 , 115 mM NaCl, pH 7.4
  • the solution of the relevant purified mutein was dialyzed four times against this buffer and adjusted to a concentration of 1 ⁇ M or O.l ⁇ M by dilution. All solutions used were sterile-filtered (Filtropur S 0.45 ⁇ m, Sarstedt). The concentration was determined by means of absorption at 280 nm using a calculatory extinction coefficient of 53580 M ⁇ cnr 1 (Wisconsin Software Package, Genetics Computer Group ; Gill, S.C. and von Hippel, P.H. (1989) Anal. Biochem. 182, 319-326).
  • Fig. 3 illustrates the results of a representative titration of DigA16 and its muteins DigA16 H86S, DigA16 H86N, and DigAl ⁇ H86Q with digoxigenin (A) and digitoxigenin (B), whereas Fig. 4 shows an analogous analysis for DigA16 and its muteins DigAl ⁇ V56L and DigA16 N56M.
  • the dissociation constants of the complexes between the DigAl ⁇ muteins and the various ligands as determined by the fluorescence titration experiments are summarized in the following tables.
  • Table 2 Comparison of specificity with BBP muteins of the invention in terms of the ratio of progesterone/digoxin binding.
  • the BBP variants of the invention which carry a mutation at either of two sequence positions 56 and 86, bind digoxigenin with a dissociation constant of less than 25 nM or even less than 15 nM, i.e. with the same or even significantly higher affinity than the known BBP muteins DigAl ⁇ and DigA16/19 (Table 1 and 3). Digitoxin and digitoxigenin are usually bound by the muteins of the invention with higher affinities than digoxigenin and digoxin, respectively.
  • the affinity of the muteins of the invention to digitoxin and digitoxigenin is increased by approximately one order of magnitude (K of DigAl ⁇ H86S, DigAl ⁇ H86N, and DigAl ⁇ H86Q between 600 pM and 140 pM compared to the respective Ro of 3.2 nM to 2.0 nM of the mutein DigAl ⁇ , for example).
  • K of DigAl ⁇ H86S, DigAl ⁇ H86N, and DigAl ⁇ H86Q between 600 pM and 140 pM compared to the respective Ro of 3.2 nM to 2.0 nM of the mutein DigAl ⁇ , for example.
  • muteins of the invention carrying a mutation at sequence positions 56 and/or 86 bind digoxin with substantially the same affinity as digoxigenin.
  • the muteins of the present invention bind digitoxin and digitoxigenin with substantially the same affinity.
  • Sarcolemnal Na + /K + - ATPase actively transports 3 Na+ ions out of the cell and 2 K + inside upon hydrolysis of 1 ATP molecule, which ultimately results in the generation of an ion gradient and an electrochemical potential across the plasma membrane, which is needed for electrical stimulation of nerve cells or glucose and amino acid import.
  • Cardioactive glycosides like digoxin bind to the ⁇ -subunit of the ATPase and thereby block this activity, leading to higher intracellular Na + concentrations, which in turn lead to an increased intracellular Ca 2+ - concentration due to the Na + /Ca 2+ antiporter system.
  • the resulting raise of intracellular Ca 2+ level ultimately causes the positive inotropic effects of digoxin derivatives (Hauptman, P.J. and Kelly, R.A. (1999), supra).
  • digoxin causes cell depolarisation and also cell damage.
  • the novel assay described herein is based on the surprising finding that the proliferation of mammalian cells (Jurkat) can be reduced by approximately 50 % by treatment with cardioactive steroids. This toxic effect can be neutralized with the digoxigenin- binding BBP muteins of the invention or by a commercially available anti-digoxigenin Fab fragment, both of which complex free glycosides and hence prevent inhibition of the sarcolemnal Na+/K+- ATPase.
  • 20,000 Jurkat cells of an exponentially growing cell suspension in 100 ⁇ l RPIvfl-1640 medium (Gibco) containing 10 % FCS were plated out per well in a 96 well plate (Bio-One, Greiner). Afterwards, the cultures were adjusted to either 0.1 ⁇ M digitoxin or 0.2 ⁇ M digoxin by adding either 1 ⁇ l or 2 ⁇ l of a 10 ⁇ M solution of the respective steroid in PBS (obtained by 1:1000 dilution of a 10 mM stock solution in DMSO) to the cells.
  • DigAl ⁇ H86N, DigAl ⁇ H86Q, recombinant BBP or commercially available digoxin-specific antibody (Fab) fragment Digitalis Antidot (Roche) in PBS was added to the digitoxin-treated cells to a final concentration of 0.05 ⁇ M, 0.2 ⁇ M or 0.5 ⁇ M.
  • DigAl ⁇ H86N, DigAl ⁇ H86Q, recombinant BBP or Digitalis Antidot was added to the digoxin-treated cells to a final concentration of 0.1 ⁇ M, 0.2 ⁇ M or 1 ⁇ M.
  • the proliferation status of the cultures was determined by means of CellTiter 96 ® AQueous Non-Radioactive Cell Proliferation Assay (Promega) according to the instructions of the manufacturer. Briefly, 20 ⁇ l of the reagent were added to each well containing 100 ⁇ l of culture and incubated at 37°C and 5 % CO for 1-4 h. The amount of proliferating cells was measured as the absorbance of a formazan product, which is produced from a tetrazolium compound and an electron coupling reagent in the reagent and is directly proportional to the number of viable cells in the culture, at a wavelength of 490 nm in a 96 well plate reader (Tecan). To account for background absorbance contributed e.g. by cell debris, the absorbance at a wavelength of 690 nm was also recorded and subtracted. For analysis of the data, measured absorbance values were averaged over the three parallel cultures and standard errors were determined.
  • Fig. 6 (A) shows the results of the proliferation assay with digitoxin.
  • DigAl ⁇ H86N completely restored cell proliferation in the presence of 0.1 ⁇ M digitoxin at a concentration of 0.5 ⁇ M and partially neutralized digitoxin toxicity at equimolar concentrations but was inactive at a concentration of 0.05 ⁇ M.
  • DigAl ⁇ H86Q completely neutralized digitoxin toxicity at any of the tested concentrations.
  • Digitalis Antidot showed apparently the same digitoxin-neutralizing activity as DigAl ⁇ H86N, whereas the BBP did not show any anti-digitoxin activity.
  • Fig. 6 (B) shows the results of the proliferation assay with digoxin.
  • the proliferation of Jurkat cells was reduced by 50 % after the addition of 0.2 ⁇ M digoxin.
  • DigAl ⁇ H86N completely restored cell viability in the presence of 0.2 ⁇ M digoxin at a concentration of 1 ⁇ M and partially at equimolar concentration but showed only weak neutralizing effect at a concentration of 0.1 ⁇ M.
  • DigAl ⁇ H86Q completely neutralized digoxin toxicity at 1 ⁇ M and 0.2 ⁇ M and showed partial restoration of proliferation 0.1 ⁇ M.
  • Digitalis Antidot again showed apparently the same digoxin-neutralizing activity as DigAl ⁇ H86N whereas the BBP did not exhibit any anti-digoxin activity.
  • the BBP mutein DigAl ⁇ H86N was chosen to investigate its ability as antidote to reverse digoxin toxicity in vivo in an animal model based on anesthetized guinea pigs. This model was validated in previous experiments that made use of anti-digoxin monoclonal antibodies and antibody fragments (Lechat, P. et al. (1984) J. Pharm. Exp. Ther. 229, 210-213). The published protocol was followed in the experiments described herein, with just minor alterations.
  • Ventilation was delivered via a tracheotomy with a mixture of 40 % oxygen in room air supplied with a Harvard small animal ventilator. Rectal temperature was recorded and maintained at 38 ⁇ 1°C by a thermostatically controlled heating blanket below the animal. The ECG lead II was recorded and the signal was used to calculate heart rate using Powerlab software (AD Instruments, Australia). The right jugular vein was exposed through a midline incision in the neck. A cannula was inserted into the jugular vein for administration of drugs. At the end of each experiment animals still alive were sacrificed by cervical dislocation under anaesthesia.
  • guinea pigs Two guinea pigs (guinea pig no. 5 and 6) were used to study the digoxin-neutralizing effect of the commercially available digoxin-specific Fab fragment Digitalis Antidot (Roche) as a control. After having received the digoxin bolus dose both animals developed first signs of toxicity after 9 and 10 min, respectively. At this time the guinea pigs were injected with 28.5 mg (1.7 times molar excess compared to the applied digoxin) of Digitalis Antidot at a concentration of 9.5 mg/ml in PBS. The toxic effects of digoxin were completely reversed in both animals 19 min after the digoxin bolus injection, i.e.

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Abstract

L'invention concerne de nouvelles mutéines de la protéine de Pieris brassicae se fixant sur la biline, qui se fixent sur la digoxyne et la digitoxine ayant sensiblement la même affinité et qui se fixent sur la digoxygénine avec un KD de 25 nM ou moins. L'invention concerne également les molécules d'acide nucléique correspondantes qui codent de telles mutéines, de même qu'un procédé pour produire lesdites mutéines par génie génétique. L'invention concerne pour finir une composition pharmaceutique et un kit comprenant de telles mutéines, de même que l'utilisation de ces mutéines dans diverses applications.
PCT/EP2004/003741 2004-04-07 2004-04-07 Muteines se fixant sur la digoxygenine de la proteine de pieris brassicae qui se fixe sur la biline WO2005097994A1 (fr)

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PCT/EP2004/003741 WO2005097994A1 (fr) 2004-04-07 2004-04-07 Muteines se fixant sur la digoxygenine de la proteine de pieris brassicae qui se fixe sur la biline

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US20150376246A1 (en) * 2013-03-14 2015-12-31 University Of Washington Through Its Center For Commercialization High affinity digoxigenin binding proteins
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CN103439492A (zh) * 2013-08-22 2013-12-11 顺昊细胞生物技术（天津）有限公司 一种试剂盒
CN103439492B (zh) * 2013-08-22 2015-03-11 顺昊细胞生物技术（天津）股份有限公司 一种试剂盒

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