WO2011145126A2 - A method for the preparation of conjugates based on transglutaminase - Google Patents

A method for the preparation of conjugates based on transglutaminase Download PDF

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WO2011145126A2
WO2011145126A2 PCT/IT2011/000158 IT2011000158W WO2011145126A2 WO 2011145126 A2 WO2011145126 A2 WO 2011145126A2 IT 2011000158 W IT2011000158 W IT 2011000158W WO 2011145126 A2 WO2011145126 A2 WO 2011145126A2
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mpeg
tgase
peg
amino
hgh
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WO2011145126A3 (en
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Gianfranco Pasut
Anna Mero
Francesco Veronese
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Università Degli Studi Di Padova
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to a new method of transglutaminase (TGase) mediated conjugation, performed under conditions that increase the specificity of the enzyme, for the preparation of new conjugates of proteins or peptides . with polymers or other molecules containing an amino group.
  • TGase transglutaminase
  • the transglutaminases [EC2.3.2.13; protein-glutamine : gamma-glutamyltransferase] belong to a family of proteins that catalyze the addition of an acyl residue to a primary amine, where the gamma-carboxamide group of glutamine in a peptide sequence is the acyl group donor and the primary amine group is the amino donor and acyl acceptor.
  • this enzyme present in prokaryotes and eukaryotes, is the formation of cross-linking bonds between proteins by catalyzing the link of a lysine (amino donor) and a glutamine (acyl donor) .
  • a typical example is the cross-linking of fibrin . mediated by factor Xllla, a TGase.
  • TGases are found, for example, in mammalians, in organs such as liver, skin and extracellular fluids [Greenberg C, S, et al. FASEB J. (1991) 5:3071-3077] and in prokaryotes [Washizu K, et al. Biosci. Biotech. Biochem. (1994) 58:82-87].
  • TGase is able to recognize, as amino donor substrates, several other molecules containing primary amino groups, in addition to the lysine residue.
  • the enzyme was therefore used' to modify site-specific manner glutamine residues of some proteins with a polymer '[US6010871, US6331422, US6322996] .
  • Sato has obtained conjugates of polyethylene glycol (PEG) with interleukin-2, using PEG terminated with a primary amine [Sato H. Adv. Drug Delivery Rev. (2002) 54:487-504].
  • PEG polyethylene glycol
  • interleukin-2 using PEG terminated with a primary amine
  • the study shows a correlation between transglutamination sites and sites of limited proteolysis by enzymes such as trypsin and chymotrypsin, concluding that only sites in flexible regions can be attacked by proteases. This study shows therefore that the flexibility of the amino acid sequence containing glutamine is a precondition for the attack by TGase.
  • This method leads to the formation of various isomers of hGH-small linker and the desired conjugate is isolated from other isomers by purification with ion-exchange chromatography. Then the conjugate is reacted with a PEG having a specific reactivity towards the molecule previously bound [ O2006/134148 ] . In this approach there is the need to of repeated process of purification to get the right intermediate and the final product in order to reach the desired monoconjugate .
  • This patent discloses as the addition of appropriate concentrations of one or more organic solvents, miscible with water, in the conjugation buffer is able to reduce the number of glutamines substrate of TGase down to one per polypeptide chain, leading the formation of products with a low degree of conjugation, preferably monocon ugates formed by these proteins and polymers or other functional molecules.
  • a. method of conjugation comprising a transglutamination reaction catalyzed by a transglutaminase (TGase) between a protein or a peptide containing two or more glutamine residues as acyl donor substrate, a molecule containing at least one amino group as amino donor substrate.
  • the method is characterized by the use of at least one organic solvent miscible with water as cosolvent in the aqueous solution of catalysis buffer to increase the selectivity of reaction and reduce the number of products of conjugation.
  • the patent claims a method for increasing the selectivity of a conjugation reaction, including a transglutamination reaction catalyzed by a transglutaminase (TGase) and involving a protein or a peptide containing two or more glutamine residues as acyl donor substrate, a molecule containing at least one amino group as the amino donor substrate, and the method characterized by the use of at least one organic solvent miscible with water as cosolvent in the catalysis buffer to increase the selectivity of reaction and reduce the number of products of conjugation.
  • TGase transglutaminase
  • the patent also discloses a method for the preparation, by a single step of TGase conjugation, specific monoconjugates : PEG- (Q20) calcitonin and PEG- (Q141) hGH.
  • the present invention provides a new method to get conjugates with TGase, and in particular for the obtainment of monoconjugates, such as the mono-PEG- (Q20 ) calcitonin and PEG- (Q141)hGH conjugates.
  • the method disclosed in this invention reduces the number of isomers that are obtained under the usual aqueous conditions of TGase catalysis (e.g. buffer solution at pH close to neutral) between molecules containing an amino group, such as amino donor substrate, and proteins or peptides having in the amino acid sequence more than a one glutamine substrate as acyl donor.
  • the new method can increase the selectivity of the conjugation by TGase and reduce or eliminate the possibility of generating isomers in order to get conjugates preferably to a single site (monoconjugates) .
  • a conjugation method comprising a transglutamination reaction, catalyzed by a transglutaminase (TGase) , between a protein or a peptide containing two or more glutamine residues as acyl donor substrate, and a molecule which contains at least one amino group as substrate amino donor.
  • This method is characterized by the use of at least one organic solvent miscible with water as cosolvent in the catalysis buffer that increases the selectivity of reaction and reduces the number of products of conjugation.
  • the patent also discloses a method for preparing, in a single step of TGase conjugation, specific products monoconjugates: PEG- (Q20) calcitonin and PEG- (Q141) hGH.
  • the TGase reaction conducted as described by this method is particularly advantageous because it allows also to increase the yield of products with a low degree of modification, especially monoconjugates.
  • the TGase reaction according to the method of the present invention is conducted in an aqueous solution, preferably buffered in the presence of at least one organic solvent, miscible with water and in appropriate concentrations, so that the catalytic activity of the enzyme is still present and that the specificity of the enzyme is modified with respect to the reaction conducted in an aqueous solution of buffer catalysis in which usually the ' TGase is employed.
  • the Change of TGase specificity after the addition of organic solvent in the catalysis buffer may be due to a direct action of the co-solvent on the enzyme, the substrates or a combination of both factors that causes a change in the enzyme's process of recognition of the sites of conjugation in the substrate protein.
  • the acyl donor substrate and the amino donor substrate are dissolved in an aqueous solution preferably buffered (e.g. with a 0.1 M phosphate buffer pH 7) containing between 5% to 80% (v / v) of one or more organic solvents miscible with water.
  • an aliquot of solubilized TGase previously dissolved in an aqueous solution, preferably buffered (e.g. with a 0.1 phosphate buffer pH 7). The reaction is allowed to react for a sufficient time to obtain the conjugation product.
  • the solvents are preferably but not limited to, ethanol, methanol and DMSO.
  • the percentage of organic solvent can vary depending on the acyl donor substrate but always the co-solvent added has to maintain the enzyme activity.
  • At least one organic cosolvent is present in an amount between 5% and 80% (v / v) , more preferably between 20% and 70% (v ./ v) , even more preferably between 30% and 65% (v / v) , and still more preferably between 40% and 60% (v / v) .
  • At least one organic co- solvent is ethanol, methanol or DMSO
  • this co-solvent is preferably present in the mixture catalyst in an amount between 5% and 80% (v / v) , more preferably between 30 % And 70% (v / v) more preferably between 55% and 65% (v / v) and even more preferably is 60% for ethanol and methanol, and preferably in an amount between 10% and 40% (v / v) and -more preferably between 25% and 35% (v / v) and even more preferably is 30% in the case of DMSO.
  • the acyl donor substrate is a protein or a peptide containing two or more glutamines as potential substrates of transglutaminase.
  • the preferred substrate acyl donor is calcitonin, human growth hormone (hGH) , or an antibody.
  • the growth hormone and calcitonin have the sequence shown in - Figure 1 (SEQ ID 01) and Figure 2 (SEQ ID N02) , respectively, as well as that of their mutants containing at least 80%, preferably 90%, more preferably 95% and still more preferably 98% of the sequences shown in Figures 1 and 2.
  • the molecule containing an amino group which acts as an amino donor substrate, has at least one amino group as TGase substrate, such as a primary amine, or it contains a group that can be modified to include an amino group in the chemical structure to act as TGase substrate, and it is a molecule selected from: a natural or synthetic polymer, preferably hydrophilic and soluble in water, a diagnostic agent such as a chelate a radioisotope or a fluorescent molecule, an oligonucleotide as an antisense oligonucleotide, an aptamer, a siRNA, a protein, a peptide, a drug, a toxin or a heterobifunctional molecule.
  • a natural or synthetic polymer preferably hydrophilic and soluble in water
  • a diagnostic agent such as a chelate a radioisotope or a fluorescent molecule
  • an oligonucleotide as an antisense oligonucleotide,
  • the molecule containing an amino group, which acts as an amino donor substrate is preferably a polymer having a molecular weight (MW) averaged between 200 Da and 300,000 Da, as determined by methods known in the literature (Leslie Howard Sperling. Introduction to physical polymer science. John Wiley and Sons, 2006), more preferably between 3000 to 100, 000 Da., even more preferably between 5000-80000 Da, and even more preferably between 10000-40000 Da
  • MW molecular weight
  • the molecule containing an amino group which acts as amino donor substrate is a polymer selected from the following group: poly (ethylene glycol) (PEG) , with linear or branched structure, preferably monofunctional, having preferably a molecular weight (MW) averaged between 2000 and 40000 Da, more preferably between 2000 Da and 30000 Da, and even more preferably between 5000 Da and 20000 Da.
  • PEG poly (ethylene glycol)
  • the most preferred poly (ethylene glycol) s are mPEG-NH 2 10000 Da, mPEG-NH 2 20000 Da, mPEG-NH 2 40000 Da, branched mPEG-NH 2 10000 Da, branched mPEG-NH 2 20000 Da and branched mPEG-NH 2 40000 Da.
  • Other branched polymers are polyvinylpyrrolidone; polyacrilmorpholine, N-hydroxy-ethyl methacrylamide copolymer, P°ly (2-ethyl- oxazoline)s, polyglutamic acid, hyaluronic acid; polysialic acid, and copolymers of PEG-poly (amino acid).
  • TGase examples include, preferably but not exclusively, microbial TGase from Streptomyces mobaraense from Streptomyces cinnamoneum Streptomyces griseocarneum (U.S. 5,156,956, included here as a citation), from Streptomyces lavendulae (U.S. 5,252,469, included here as a citation), and Streptomyces ladakanum ( JP2003199569, included here as a reference) .
  • members of the old genus Streptoverticillium are now included in the genus Streptomyces (Kaempfer, J. January Microbiol. 137, 1831-1892 (1991)).
  • TGase from Bacillus subtilis (U.S. 5,731,183, included here as a citation) and various Myxomycetes.
  • suitable TGases are those described in O96/06931 and W096/22366, both included here as references.
  • suitable non-microbial TGase include, preferably but not exclusively, TGase from the liver of guinea pig, various TGase of marine origin (EP-0555649, included here as a citation), and TGase from Japanese oyster Crassostrea gigas (U.S. 5,736,356, included here as reference.)
  • any TGase mutants may be suitable with an altered specificity than the native enzyme, such as those included in WO2008020075 , included here as references.
  • the enzyme is added to the reaction mixture until it reaches a ratio enzyme / acyl donor substrate of between 1:1 and 1:100 (w / w) , or at least at a concentration to have a detectable enzymatic activity.
  • the preferred acyl donor substrate is the calcitonin polypeptide.
  • the amino donor substrate is mPEG-NH 2 (10 kDa, 20 kDa or 40 kDa)
  • the TGase is the microbial TGase from Streptomyces mobaraense
  • the catalysis buffer is phosphate buffer 0.1 pH 7, added of ethanol . (60% v / v) .
  • the derivative mPEG5kDa- (Q20) calcitonin, mPEG20kDa- (Q20) calcitonin or mPEG40kDa- (Q20) calcitonin is obtained, depending on the molecular weight of PEG used.
  • the preferred acyl donor substrate is the protein hGH
  • the amino donor substrate is mPEG-NH 2 (5 kDa, 20 kDa or 40 kDa)
  • TGase - is the microbial TGase from Streptomyces mobaraense
  • the buffer catalysis is composed of phosphate buffer 0.1 M pH 7 added of ethanol (60% v / v) .
  • the derivative mPEG5kDa- (Q141) hGH, mPEG20kDa- (Q141) hGH or mPEG40kDa- (Q141) hGH is obtained from the reaction mixture, according to the molecular weight of PEG.
  • the preferred acyl donor substrate is the calcitonin polypeptide
  • the amino donor substrate is mPEG-NH 2 (5 kDa, 20 kDa or 40 kDa)
  • the TGase is the microbial TGase from Streptomyces mobaraense
  • the catalysis buffer is phosphate buffer 0.1 M pH 7, added of DMSO (30% v / v) .
  • DMSO (30% v / v)
  • the subject of this patent is also a monoconjugate, preferably a monoconjugate of a polymer (preferably PEG) and a protein or a peptide containing two or more glutamines, which is preferably calcitonin or hGH.
  • a monoconjugate preferably a monoconjugate of a polymer (preferably PEG) and a protein or a peptide containing two or more glutamines, which is preferably calcitonin or hGH.
  • the conjugates object of the present invention are those derivatives with a low degree of modification, preferably monoconjugates at a specific glutamine, obtainable by the method claimed and starting with a protein or a peptide containing two or more glutamines as potential acyl donor substrates of TGase.
  • the conjugates, preferably monoconjugates, of the invention are those obtained using peptides or proteins that if treated by TGase-mediated conjugation in a catalysis buffer without an organic solvent co-solvent they would lead to the formation of conjugates with a higher degree of modification and / or heterogeneity.
  • proteins or peptides containing two or more glutamines as potential acyl donor substrates for TGase are, calcitonin, human growth hormone, glucagon, alpha- synuclein, beta-casein, fibronectin, alpha chain of fibrinogen, growth hormone releasing factor (GRF) , antibodies and antibody's fragments.
  • Preferred products obtainable with the present invention are conjugates, preferably monoconjugates, composed of the above reported protein or peptide and of a polymer such as PEG-NH2, even more preferred products of the present invention are the following monoconjugates: mPEG- (Q20) calcitonin, where the molecular weight (MW) of PEG is 10000 Da, 20000 Da or 40000 Da, and mPEG- (Q141) hGH, where the molecular weight (MW) of PEG is 5000 Da, 20000 Da or 40000 Da.
  • the symbols Q20 and Q141 represent the glutamine residues in their peptide sequences SEQ ID NOl and SEQ ID NO 2 (shown in Figures 1 and 2), respectively. At this site the polymer is linked.
  • Object of the present invention is a pharmaceutical or diagnostic preparation comprising at least a monoconjugates obtainable by the method of here described, the preparation possibly including a further therapeutically active or diagnostic agent, and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical preparation may be for oral, parenteral, rectal, topical, vaginal, ocular or inhalation administration.
  • the invention is also a method of treatment or a method of diagnosis comprising the administration of a pharmaceutical formulation as defined above.
  • Fig. 1 shows the amino acid sequence of salmon calcitonin (SCT) with glutamines in bold.
  • Fig. 2 shows the amino acid sequence of growth hormone (hGH) with glutamines in bold.
  • Fig. 3 shows the chromatographic profiles of SCT, the reaction mixture PEG 5kDa -sCT in the presence and absence of co-solvent.
  • a monoconj ugates derivative is formed, while a mixture of mono and biconjugates is obtained in absence of organic co-solvents.
  • the separation was performed using a reversed phase Agilent C18 column (4.6 x 250 mm) at flow rate of 1 mL / rain with a linear gradient in acetonitrile (10- 70%) for a period of 25 min.
  • the effluent from the column was monitored by measuring the absorbance at 226 nm.
  • Fig. 4 is the ESI-TOF analysis of PEG-sCT obtained in the presence of co-solvents. Analyses were conducted using an API- TOF instrument MarinerTM (Applied Biosystems) .
  • Fig. 5 is an overlap of chromatographic profiles of hGH and the reaction mixture PEG 5kDa -hGH in the absence of co-solvent.
  • a mixture of mono and biconjugates is obtained after 18 hours at room temperature.
  • the separation was performed using a gel filtration column Agilent GF-250 (4.6 x 250 mm), flow rate of 0.3 mL / min of 0.1 M phosphate buffer, 0.2 M NaCl containing 20% acetonitrile .
  • the effluent from the column was monitored by measuring the absorbance at 280 nm.
  • Fig. 6 is a comparison of the chromatographic profiles of hGH, the reaction mixture PEG 5k Da-E > EG54o- GH and hGH in the presence of co-solvents.
  • the separation was performed using a reversed phase Agilent C18 column (4.6 x 250 mm) ' at flow rate of 1 mL / min with a linear gradient in acetonitrile (40-70%) for period of 25 min.
  • the effluent from the column was monitored by measuring the absorbance at 280 nm.
  • Fig. 8 shows the chromatographic profiles of SCT and the reaction mixture ' POZ 5kDa -sCT in the presence of DMSO. In these conditions a monoconjugates is formed. The separation was carried out under the same conditions as described in Figure 3.
  • Fig. 9 shows the elution of the fragments obtained after tryptic digestion of sCT and PEGs 4 0Da-sCT . The separation was carried out under the same conditions as described in Figure 3.
  • Fig. 10 shows the mass spectrum of digested PEG-hGH. In particular, it is reported the region in the range from 560-700 Da to show the derivatization with PEG fragment Glnl41-Lysl45.
  • the signal at 583.82 m / z corresponds to the fragment Glnl41- Lysl45 conjugated with PEG and double charge.
  • Fig. 11 shows the far-UV CD spectrum and fluorescence spectrum of sCT with and without co-solvents.
  • the spectra were recorded at protein concentration of 0.1 mg / mL in 10 m phosphate buffer pH 7 - '
  • Fig. 12 shows the far-UV CD spectrum and fluorescence spectrum of hGH with and without co-solvents.
  • the spectra were recorded at protein concentration of 0.1 mg / mL in 10 rtiM phosphate buffer pH 7.
  • Fig. 13 shows the graph of the hypocalcaemic effect of sCT and PEG-sCT derivatives in Sprangue-Dawley rats.
  • Example 1 mTGase-mediated PEGylation of salmon calcitonin in aqueous buffer.
  • Example 2 mTGase-mediated PEGylation of salmon calcitonin in aqueous buffer in the presence of co-solvents.
  • mPEG-NH 2 (5 kDa) at a 10-fold molar excess is incubated with sCT in 0.1 M phosphate buffer pH 7 containing 30% (v/v) of DMSO.
  • a solution of TGase from Streptomyces mobaraense is added to E/S ratio of 1:15 (w/w)- and the reaction is allowed to proceed for 3 hours at room temperature.
  • the analysis of the reaction mixture by RP-HPLC confirmed the formation of a new conjugate ( Figure 1) .
  • the peak, collected and characterized by MALDI mass spectrometry, is a mono-conjugate derivative.
  • Example 3 mTGase-mediated PEGylation of salmon calcitonin in aqueous bu fer in the presence of co-solvents .
  • Example 5 mTGase-mediated PEGylation of hGH in aqueous buffer.
  • mPEG-NH 2 5 kDa at a 10-fold molar excess is added to a solution of hGH (1 mg/mL) in 0.1 M phosphate buffer pH 7.
  • the TGase enzyme from Streptomyces mobaraense is added in catalytic amount (ratio E/S, 1:50).
  • the reaction is left stirring for 4 hours at room temperature and monitored by SEC-HPLC chromatography. Chromatographic profile, shown in Figure 3, confirms TGase mediated conjugation with the appearance of mono- and bi-PEGylated products.
  • Example 6 mTGase-mediated PEGylation of hGH in aqueous buffer in the presence of co-solvents .
  • Example 7 mTGase-mediated PEGylation of hGH in aqueous buffer in the presence of co-solvents .
  • the conjugation of PEG to hGH was carried out using the conditions described in Example 6 except that ' the amino donor substrate is a monodisperse boc-PEG-NH2 (556 Da) .
  • Example 8 Preparation of POZ-sCT by TGase in aqueous buffer in the presence of co-solvents.
  • Example 9 Proteolytic digestion of PEG-sCT and determination of the conjugation site .
  • the three peaks correspond to the fragment Leul2-Lysl8 (854 Da), Leul9-Arg24 (776 Da) and Cysl-Lysll (1124 Da).
  • the Thr25-Pro32 fragment (733.7 Da) is not eluted in these conditions because it is very hydrophilic. However, the lack of such residual does not influence the determination of the binding site because it does not contain glutamine residues.
  • the superimposition of the chromatographic elution profile of digested boc-PEG540Da-sCT and sCT shows that the a and c fragments are identified, while the fragment b is disappeared.
  • a new product in boc-PEG540Da-sCT profile is identified (peak d) that analysed by mass spectrometry shows a molecular weight of 1316 Da, which corresponds to residue Leul9-Arg24 linked to a chain of monodisperse PEG.
  • peak d A new product in boc-PEG540Da-sCT profile is identified (peak d) that analysed by mass spectrometry shows a molecular weight of 1316 Da, which corresponds to residue Leul9-Arg24 linked to a chain of monodisperse PEG.
  • -Gln20 is a specific substrate of TGase in the presence of co-solvents being the only Gin present in this peptide.
  • the tryptic digestion is carried out also on the derivative mPEG5kDa-sCT .
  • HPLG analysis shows the presence of the fragment a and c, a new fragment with greater retention times and the disappearance of the fragment b (data not shown) .
  • Example 10 Proteolytic digestion of PEG-hGH and determination of the conjugation site.
  • the peptide fragments of hGH and boc-PEG 54 0Da-hGH are determined by ESI-TOF mass spectrometry. By the mass analysis of digested hGH the most tryptic fragments are identified with a percentage of sequence coverage of 85%.
  • the digest of boc-PEG 5 4 0 Da _ hGH shows all the fragments identified in the native hGH except that the fragments corresponding to the sequence Glnl41-Lysl45, Phel46-Lysl68- Phel91 and Serl84. A new fragment with a double charge .
  • Example 11 Effect of organic co-solvents on the conformation of calcitonin and hGH.
  • Example 12 Pharmacodynamic profiles of PEG-calcitonin in rats .
  • the biological effects of the derivatives obtained in the present invention were assessed as the ability to develop a hypocalcaemic response in vivo.
  • Sprangue- Dawley rats weighing between 180-250 g, were divided into groups and each group received by injection into a tail vein one of the following samples: sCT, mPEG 51cDa -sCT or mPEGio kDa -sCT at a dose of 40yg/kg (sCT equiv.).
  • sCT equiv. a dose of 40yg/kg
  • plasma samples were taken and analysed by colorimetric assay (Vinci Biochem, Vinci IT) to assess the levels of calcium in the blood.

Abstract

The present invention reports a new method to selectively obtain conjugates, preferably monoconjugates, between a protein or a peptide and a polymer or other molecules containing an amino group by the use of transglutaminase in catalysis buffers containing an organic cosolvent miscible with water.

Description

A METHOD FOR THE PREPARATION OF CONJUGATES BASED ON TRANSGLUTAMINASE
DESCRIPTION OF THE INDUSTRIAL INVENTION FIELD OF THE INVENTION
The present invention relates to a new method of transglutaminase (TGase) mediated conjugation, performed under conditions that increase the specificity of the enzyme, for the preparation of new conjugates of proteins or peptides . with polymers or other molecules containing an amino group.
FOUNDAMENTALS OF THE INVENTION
The use of enzymes to form covalent bond between two molecules has the advantage of a high site-specific conjugation that usually leads to the formation of conjugates chemically more defined than those obtained with other chemical methods.
The transglutaminases (TGases) [EC2.3.2.13; protein-glutamine : gamma-glutamyltransferase] belong to a family of proteins that catalyze the addition of an acyl residue to a primary amine, where the gamma-carboxamide group of glutamine in a peptide sequence is the acyl group donor and the primary amine group is the amino donor and acyl acceptor. In nature the role of this enzyme, present in prokaryotes and eukaryotes, is the formation of cross-linking bonds between proteins by catalyzing the link of a lysine (amino donor) and a glutamine (acyl donor) . A typical example is the cross-linking of fibrin . mediated by factor Xllla, a TGase. TGases are found, for example, in mammalians, in organs such as liver, skin and extracellular fluids [Greenberg C, S, et al. FASEB J. (1991) 5:3071-3077] and in prokaryotes [Washizu K, et al. Biosci. Biotech. Biochem. (1994) 58:82-87].
It has been demonstrated by several authors that TGase is able to recognize, as amino donor substrates, several other molecules containing primary amino groups, in addition to the lysine residue. The enzyme was therefore used' to modify site-specific manner glutamine residues of some proteins with a polymer '[US6010871, US6331422, US6322996] . For example, Sato has obtained conjugates of polyethylene glycol (PEG) with interleukin-2, using PEG terminated with a primary amine [Sato H. Adv. Drug Delivery Rev. (2002) 54:487-504]. The article brings out clearly that the TGase has very strict requirements for the acyl donor substrate, i.e. it recognizes only few glutamines inserted is specific amino acid sequences. On the other hand, the enzyme is less stringent with respect to the amino donor substrate. Subsequently, a study, thoroughly examining various proteins, showed that the selectivity of TGase for some glutamines, among many in a protein, is directly linked to the flexibility of the peptide sequence that contains the glutamines [Fontaha A, et al. Adv. Drug Delivery Rev. (2008) 60:13-28]. In this study it is concluded that a glutamine should be included in sequences /with high conformational flexibility, as evidenced by high levels of B-factor of these sequences, to be a substrate for TGase. The study also shows a correlation between transglutamination sites and sites of limited proteolysis by enzymes such as trypsin and chymotrypsin, concluding that only sites in flexible regions can be attacked by proteases. This study shows therefore that the flexibility of the amino acid sequence containing glutamine is a precondition for the attack by TGase.
It should be emphasized that the paper does not describe the possibility to reversibly change the flexibility of the peptide sequence of the acyl donor substrate in order . to vary the affinity of TGase towards a particular substrate.
It would be very .desirable to have a method to selectively or preferably select a site of conjugation in a protein or peptide containing several glutamines as acyl donor substrates. As reported in the literature, many proteins of therapeutic interest possess more than a glutamine with the requirements to be substrate of the enzyme (e.g. growth hormone, calcitonin, glucagon, insulin chain A) [Fontana A, et al. Adv. Drug Delivery Rev. (2008) 60:13-28; Gorman JJ, et al. J Biol. Chem. (1980) 255:1175-1180; FOLK JE, et al. J Biol. Chem. 1965 240:2951- 2960]. This fact prevents the possibility for these proteins to get only a monoconjugate product through the use of TGase for the conjugation with polymers such as PEG-NH2.
The use of this enzyme to obtain conjugates of PEG with proteins or peptides has been described in patents and articles but, in any case, the enzymatic conjugation reaction has been, carried out in aqueous medium in order to preserve the biological activity and specificity of the enzyme [US6010871; US6331422; US6322996; ITMI20061624 ; Sergi M, et al . In: PEGylated protein drugs: basic science and clinical applications. Veronese FM (ed) Birkhauser: Berlin (G) . (2009) : 75-88 ; Fontana A, et al. Adv. Drug Delivery Rev. (2008) 60:13-28]. It is clear that this approach can lead to PEG-protein monoconjugates , which are particularly desired products because a lot easier to purify and characterize, only in cases in which the target. protein has only one glutamine that meets the substrate requirements for TGase. In the case of proteins or peptides containing multiple acyl donor substrates, mixtures of mono and biconjugates are obtained. From these mixtures the desired product is purified only through difficult purifications. For example, in order to obtain monoconjugates in a patent is disclosed a method based on TGase. In this case TGase binds to human growth hormone a small bifunctional molecule carrying a specific reactivity towards a special polymer employed in a subsequent modification. This method leads to the formation of various isomers of hGH-small linker and the desired conjugate is isolated from other isomers by purification with ion-exchange chromatography. Then the conjugate is reacted with a PEG having a specific reactivity towards the molecule previously bound [ O2006/134148 ] . In this approach there is the need to of repeated process of purification to get the right intermediate and the final product in order to reach the desired monoconjugate .
To overcome these limitations and extend the TGase mediated monoconj ugation to other proteins and peptides, it would be very- useful to have a method to reduce the number of glutamines, present in the target protein/peptide, which possess the requirements of the TGase substrate. To achieve this result, some authors have prepared, by genetic engineering, mutants of the target protein in which there are less glutamines [US6322996, WO2004/108667] . Alternatively, mutants of TGase with different substrate specificities have been studied [Zhao X, et al. J Biomol.,. Screening. (2010) 15:206-212] . In both cases, it is required a case-by-case development of the desired mutant, thus impacting negatively on the economics of the process of enzymatic conjugation. Furthermore, in the first case it could be created also mutants of the therapeutic protein that could trigger immune reactions. In the second case, in which the TGase mutants would be designed only for the desired specificity for a particular protein, it is lost the easy applicability of the method to different proteins.
The studies presented above show that in the field of polymer bioconjugates of therapeutic peptide and protein, scientific research aimed at obtaining more selective monoconj ugates in which the polymer is linked uniquely to a single, repeatable and specific amino acid residues in peptide sequence [Harris MJ, et al. Nat. Rev. Drug Discov. (2003) 2:214-221; Pasut G., et al. Drug Discov Today (2005) 10:1451-1458], in order to obtain derivatives more easily purified, characterized, with defined biological properties and most suitable for clinical use.
From the above considerations, it is clear that a method based on TGase mediated enzymatic conjugation able to yield a single monoconjugate even in those cases where there is more than one glutamine as TGase substrate would be seen as an innovation that fulfils the potential of the TGase approach. It is the subject of the present invention a method for increasing the selectivity of the TGase, which allows to obtain in a single step, products with low degree of conjugation, preferably monoconjugates also for those proteins or peptides that in aqueous conditions of TGase mediated conjugation forms positional isomers and / or biconjugates .
ABSTRACT. OF THE INVENTION
In order to reduce the number of glutamines substrate of TGase in proteins or peptides, we found that the presence of suitable organic solvents, miscible with water, in the catalysis buffer allows the obtainment of a single conjugation product (monoconjugate) at one specific glutamine, even in the case of those proteins that contain more than one glutamine residue as TGase substrate under the standard conditions of catalysis. The latter situation' leads to the formation of a mixture of conjugates, namely, monoconjugates, biconjugates and polyconcj ugates .
This patent discloses as the addition of appropriate concentrations of one or more organic solvents, miscible with water, in the conjugation buffer is able to reduce the number of glutamines substrate of TGase down to one per polypeptide chain, leading the formation of products with a low degree of conjugation, preferably monocon ugates formed by these proteins and polymers or other functional molecules.
Based on the results obtained the present patent claims a. method of conjugation comprising a transglutamination reaction catalyzed by a transglutaminase (TGase) between a protein or a peptide containing two or more glutamine residues as acyl donor substrate, a molecule containing at least one amino group as amino donor substrate. The method is characterized by the use of at least one organic solvent miscible with water as cosolvent in the aqueous solution of catalysis buffer to increase the selectivity of reaction and reduce the number of products of conjugation.
In other words, the patent claims a method for increasing the selectivity of a conjugation reaction, including a transglutamination reaction catalyzed by a transglutaminase (TGase) and involving a protein or a peptide containing two or more glutamine residues as acyl donor substrate, a molecule containing at least one amino group as the amino donor substrate, and the method characterized by the use of at least one organic solvent miscible with water as cosolvent in the catalysis buffer to increase the selectivity of reaction and reduce the number of products of conjugation.
The patent also discloses a method for the preparation, by a single step of TGase conjugation, specific monoconjugates : PEG- (Q20) calcitonin and PEG- (Q141) hGH. These examples show that, with the method here claimed, only one isomer is obtained as a product diversely to the same TGase reaction conducted in a catalysis buffer without organic cosolvent.
Products obtained by the' method disclosed in this invention, their pharmaceutical compositions and their Use in therapy are also described in this patent.
DESCRIPTION
The present invention provides a new method to get conjugates with TGase, and in particular for the obtainment of monoconjugates, such as the mono-PEG- (Q20 ) calcitonin and PEG- (Q141)hGH conjugates.
The method disclosed in this invention reduces the number of isomers that are obtained under the usual aqueous conditions of TGase catalysis (e.g. buffer solution at pH close to neutral) between molecules containing an amino group, such as amino donor substrate, and proteins or peptides having in the amino acid sequence more than a one glutamine substrate as acyl donor. In particular, the new method can increase the selectivity of the conjugation by TGase and reduce or eliminate the possibility of generating isomers in order to get conjugates preferably to a single site (monoconjugates) .
On this basis the patent claims a conjugation method comprising a transglutamination reaction, catalyzed by a transglutaminase (TGase) , between a protein or a peptide containing two or more glutamine residues as acyl donor substrate, and a molecule which contains at least one amino group as substrate amino donor. This method is characterized by the use of at least one organic solvent miscible with water as cosolvent in the catalysis buffer that increases the selectivity of reaction and reduces the number of products of conjugation.
The patent also discloses a method for preparing, in a single step of TGase conjugation, specific products monoconjugates: PEG- (Q20) calcitonin and PEG- (Q141) hGH. These examples show that with the method claimed, only one isomer is obtained as a product of monoconjugation, diversely the same reaction conducted in catalysis buffers without organic cosolvets forms mixtures of isomers.
Products obtained by the method of the invention, pharmaceutical compositions that include them and their use in therapy are also described.
The TGase reaction conducted as described by this method is particularly advantageous because it allows also to increase the yield of products with a low degree of modification, especially monoconjugates.
The TGase reaction according to the method of the present invention is conducted in an aqueous solution, preferably buffered in the presence of at least one organic solvent, miscible with water and in appropriate concentrations, so that the catalytic activity of the enzyme is still present and that the specificity of the enzyme is modified with respect to the reaction conducted in an aqueous solution of buffer catalysis in which usually the ' TGase is employed. The Change of TGase specificity after the addition of organic solvent in the catalysis buffer may be due to a direct action of the co-solvent on the enzyme, the substrates or a combination of both factors that causes a change in the enzyme's process of recognition of the sites of conjugation in the substrate protein.
In the method disclosed in this patent the acyl donor substrate and the amino donor substrate are dissolved in an aqueous solution preferably buffered (e.g. with a 0.1 M phosphate buffer pH 7) containing between 5% to 80% (v / v) of one or more organic solvents miscible with water. To this solution is added an aliquot of solubilized TGase previously dissolved in an aqueous solution, preferably buffered (e.g. with a 0.1 phosphate buffer pH 7). The reaction is allowed to react for a sufficient time to obtain the conjugation product.
The solvents are preferably but not limited to, ethanol, methanol and DMSO.
The percentage of organic solvent can vary depending on the acyl donor substrate but always the co-solvent added has to maintain the enzyme activity.
For example, in the method of the invention at least one organic cosolvent is present in an amount between 5% and 80% (v / v) , more preferably between 20% and 70% (v ./ v) , even more preferably between 30% and 65% (v / v) , and still more preferably between 40% and 60% (v / v) .
In a preferred form of the invention, at least one organic co- solvent is ethanol, methanol or DMSO, and this co-solvent is preferably present in the mixture catalyst in an amount between 5% and 80% (v / v) , more preferably between 30 % And 70% (v / v) more preferably between 55% and 65% (v / v) and even more preferably is 60% for ethanol and methanol, and preferably in an amount between 10% and 40% (v / v) and -more preferably between 25% and 35% (v / v) and even more preferably is 30% in the case of DMSO.
The acyl donor substrate is a protein or a peptide containing two or more glutamines as potential substrates of transglutaminase. In some cases the preferred substrate acyl donor is calcitonin, human growth hormone (hGH) , or an antibody.
Preferably the growth hormone and calcitonin have the sequence shown in - Figure 1 (SEQ ID 01) and Figure 2 (SEQ ID N02) , respectively, as well as that of their mutants containing at least 80%, preferably 90%, more preferably 95% and still more preferably 98% of the sequences shown in Figures 1 and 2.
The molecule containing an amino group, which acts as an amino donor substrate, has at least one amino group as TGase substrate, such as a primary amine, or it contains a group that can be modified to include an amino group in the chemical structure to act as TGase substrate, and it is a molecule selected from: a natural or synthetic polymer, preferably hydrophilic and soluble in water, a diagnostic agent such as a chelate a radioisotope or a fluorescent molecule, an oligonucleotide as an antisense oligonucleotide, an aptamer, a siRNA, a protein, a peptide, a drug, a toxin or a heterobifunctional molecule.
Preferably, the molecule containing an amino group, which acts as an amino donor substrate, is preferably a polymer having a molecular weight (MW) averaged between 200 Da and 300,000 Da, as determined by methods known in the literature (Leslie Howard Sperling. Introduction to physical polymer science. John Wiley and Sons, 2006), more preferably between 3000 to 100, 000 Da., even more preferably between 5000-80000 Da, and even more preferably between 10000-40000 Da
In preferred forms of the invention the molecule containing an amino group which acts as amino donor substrate is a polymer selected from the following group: poly (ethylene glycol) (PEG) , with linear or branched structure, preferably monofunctional, having preferably a molecular weight (MW) averaged between 2000 and 40000 Da, more preferably between 2000 Da and 30000 Da, and even more preferably between 5000 Da and 20000 Da.
The most preferred poly (ethylene glycol) s are mPEG-NH2 10000 Da, mPEG-NH2 20000 Da, mPEG-NH2 40000 Da, branched mPEG-NH2 10000 Da, branched mPEG-NH2 20000 Da and branched mPEG-NH2 40000 Da. Other branched polymers are polyvinylpyrrolidone; polyacrilmorpholine, N-hydroxy-ethyl methacrylamide copolymer, P°ly (2-ethyl- oxazoline)s, polyglutamic acid, hyaluronic acid; polysialic acid, and copolymers of PEG-poly (amino acid).
Examples of possible TGase include, preferably but not exclusively, microbial TGase from Streptomyces mobaraense from Streptomyces cinnamoneum Streptomyces griseocarneum (U.S. 5,156,956, included here as a citation), from Streptomyces lavendulae (U.S. 5,252,469, included here as a citation), and Streptomyces ladakanum ( JP2003199569, included here as a reference) . Please note that members of the old genus Streptoverticillium are now included in the genus Streptomyces (Kaempfer, J. January Microbiol. 137, 1831-1892 (1991)). More appropriate are TGase from Bacillus subtilis (U.S. 5,731,183, included here as a citation) and various Myxomycetes. Other examples of suitable TGases are those described in O96/06931 and W096/22366, both included here as references. Examples of suitable non-microbial TGase include, preferably but not exclusively, TGase from the liver of guinea pig, various TGase of marine origin (EP-0555649, included here as a citation), and TGase from Japanese oyster Crassostrea gigas (U.S. 5,736,356, included here as reference.) In addition, any TGase mutants may be suitable with an altered specificity than the native enzyme, such as those included in WO2008020075 , included here as references.
Preferably, the enzyme is added to the reaction mixture until it reaches a ratio enzyme / acyl donor substrate of between 1:1 and 1:100 (w / w) , or at least at a concentration to have a detectable enzymatic activity.
In one case, the preferred acyl donor substrate is the calcitonin polypeptide. The amino donor substrate is mPEG-NH2 (10 kDa, 20 kDa or 40 kDa) , The TGase is the microbial TGase from Streptomyces mobaraense, and the catalysis buffer is phosphate buffer 0.1 pH 7, added of ethanol . (60% v / v) . In this case, only the derivative mPEG5kDa- (Q20) calcitonin, mPEG20kDa- (Q20) calcitonin or mPEG40kDa- (Q20) calcitonin is obtained, depending on the molecular weight of PEG used.
In another case, the preferred acyl donor substrate is the protein hGH, the amino donor substrate is mPEG-NH2 (5 kDa, 20 kDa or 40 kDa) , TGase - is the microbial TGase from Streptomyces mobaraense, and the buffer catalysis is composed of phosphate buffer 0.1 M pH 7 added of ethanol (60% v / v) . In this case, only the derivative mPEG5kDa- (Q141) hGH, mPEG20kDa- (Q141) hGH or mPEG40kDa- (Q141) hGH is obtained from the reaction mixture, according to the molecular weight of PEG.
In another case, the preferred acyl donor substrate is the calcitonin polypeptide, the amino donor substrate is mPEG-NH2 (5 kDa, 20 kDa or 40 kDa) . The TGase is the microbial TGase from Streptomyces mobaraense, and the catalysis buffer is phosphate buffer 0.1 M pH 7, added of DMSO (30% v / v) . In this case, only the derivative mPEG5kDa- (Q20 ) calcitonin, mPEG20kDa-
(Q20) calcitonin or mPEG40kDa- (Q20 ) calcitonin is obtained, depending on the molecular weight of PEG used.
The subject of this patent is also a monoconjugate, preferably a monoconjugate of a polymer (preferably PEG) and a protein or a peptide containing two or more glutamines, which is preferably calcitonin or hGH. These conjugates can be obtained using the method of the present invention. The conjugates object of the present invention are those derivatives with a low degree of modification, preferably monoconjugates at a specific glutamine, obtainable by the method claimed and starting with a protein or a peptide containing two or more glutamines as potential acyl donor substrates of TGase. In other words, the conjugates, preferably monoconjugates, of the invention are those obtained using peptides or proteins that if treated by TGase-mediated conjugation in a catalysis buffer without an organic solvent co-solvent they would lead to the formation of conjugates with a higher degree of modification and / or heterogeneity. Examples of proteins or peptides containing two or more glutamines as potential acyl donor substrates for TGase are, calcitonin, human growth hormone, glucagon, alpha- synuclein, beta-casein, fibronectin, alpha chain of fibrinogen, growth hormone releasing factor (GRF) , antibodies and antibody's fragments.
Preferred products obtainable with the present invention are conjugates, preferably monoconjugates, composed of the above reported protein or peptide and of a polymer such as PEG-NH2, even more preferred products of the present invention are the following monoconjugates: mPEG- (Q20) calcitonin, where the molecular weight (MW) of PEG is 10000 Da, 20000 Da or 40000 Da, and mPEG- (Q141) hGH, where the molecular weight (MW) of PEG is 5000 Da, 20000 Da or 40000 Da. In these abbreviations the symbols Q20 and Q141 represent the glutamine residues in their peptide sequences SEQ ID NOl and SEQ ID NO 2 (shown in Figures 1 and 2), respectively. At this site the polymer is linked.
Object of the present invention is a pharmaceutical or diagnostic preparation comprising at least a monoconjugates obtainable by the method of here described, the preparation possibly including a further therapeutically active or diagnostic agent, and optionally a pharmaceutically acceptable excipient. The pharmaceutical preparation may be for oral, parenteral, rectal, topical, vaginal, ocular or inhalation administration.
Finally, the invention is also a method of treatment or a method of diagnosis comprising the administration of a pharmaceutical formulation as defined above.
Short description of the figures
Fig. 1 shows the amino acid sequence of salmon calcitonin (SCT) with glutamines in bold.
Fig. 2 shows the amino acid sequence of growth hormone (hGH) with glutamines in bold.
Fig. 3 shows the chromatographic profiles of SCT, the reaction mixture PEG5kDa-sCT in the presence and absence of co-solvent. In the presence of co-solvents a monoconj ugates derivative is formed, while a mixture of mono and biconjugates is obtained in absence of organic co-solvents. The separation was performed using a reversed phase Agilent C18 column (4.6 x 250 mm) at flow rate of 1 mL / rain with a linear gradient in acetonitrile (10- 70%) for a period of 25 min. The effluent from the column was monitored by measuring the absorbance at 226 nm.
Fig. 4 is the ESI-TOF analysis of PEG-sCT obtained in the presence of co-solvents. Analyses were conducted using an API- TOF instrument MarinerTM (Applied Biosystems) .
Fig. 5 is an overlap of chromatographic profiles of hGH and the reaction mixture PEG5kDa-hGH in the absence of co-solvent. A mixture of mono and biconjugates is obtained after 18 hours at room temperature. The separation was performed using a gel filtration column Agilent GF-250 (4.6 x 250 mm), flow rate of 0.3 mL / min of 0.1 M phosphate buffer, 0.2 M NaCl containing 20% acetonitrile . The effluent from the column was monitored by measuring the absorbance at 280 nm.
Fig. 6 is a comparison of the chromatographic profiles of hGH, the reaction mixture PEG5kDa-E>EG54o- GH and hGH in the presence of co-solvents. The separation was performed using a reversed phase Agilent C18 column (4.6 x 250 mm)' at flow rate of 1 mL / min with a linear gradient in acetonitrile (40-70%) for period of 25 min. The effluent from the column was monitored by measuring the absorbance at 280 nm.
Fig. 7 ESI-TOF analysis of PEG-hGH obtained in the presence of co-solvents.
Fig. 8 shows the chromatographic profiles of SCT and the reaction mixture ' POZ5kDa-sCT in the presence of DMSO. In these conditions a monoconjugates is formed. The separation was carried out under the same conditions as described in Figure 3.
Fig. 9 shows the elution of the fragments obtained after tryptic digestion of sCT and PEGs40Da-sCT . The separation was carried out under the same conditions as described in Figure 3.
Fig. 10 shows the mass spectrum of digested PEG-hGH. In particular, it is reported the region in the range from 560-700 Da to show the derivatization with PEG fragment Glnl41-Lysl45. The signal at 583.82 m / z corresponds to the fragment Glnl41- Lysl45 conjugated with PEG and double charge.
Fig. 11 shows the far-UV CD spectrum and fluorescence spectrum of sCT with and without co-solvents. The spectra were recorded at protein concentration of 0.1 mg / mL in 10 m phosphate buffer pH 7 - ' Fig. 12 shows the far-UV CD spectrum and fluorescence spectrum of hGH with and without co-solvents. The spectra were recorded at protein concentration of 0.1 mg / mL in 10 rtiM phosphate buffer pH 7.
Fig. 13 shows the graph of the hypocalcaemic effect of sCT and PEG-sCT derivatives in Sprangue-Dawley rats.
EXAMPLES
Example 1 : mTGase-mediated PEGylation of salmon calcitonin in aqueous buffer.
To a solution of salmon calcitonin (sCT) (1 mg / mL) in 0.1 M phosphate buffer pH 7 mPEG-NH2 (5 kDa) in 10 times molar excess is added. After complete dissolution of the polymer TGase from Streptomyces mobaraense is added in catalytic amount (ratio E/S, 1:50, w/w) . The reaction is left to stir for 3 hours at room temperature and monitored by RP-HPLC. Profile chromatogram shown, in Figure 1, confirms the modification of sCT mediated by TGase and the appearance of mono- and bi-conj ugates that indicate the conjugation of PEG to the two glutamine residues: Glnl and Gln20.
Example 2: mTGase-mediated PEGylation of salmon calcitonin in aqueous buffer in the presence of co-solvents.
mPEG-NH2 (5 kDa) at a 10-fold molar excess is incubated with sCT in 0.1 M phosphate buffer pH 7 containing 30% (v/v) of DMSO. A solution of TGase from Streptomyces mobaraense is added to E/S ratio of 1:15 (w/w)- and the reaction is allowed to proceed for 3 hours at room temperature. The analysis of the reaction mixture by RP-HPLC confirmed the formation of a new conjugate (Figure 1) . The peak, collected and characterized by MALDI mass spectrometry, is a mono-conjugate derivative. Example 3 : mTGase-mediated PEGylation of salmon calcitonin in aqueous bu fer in the presence of co-solvents .
The conjugation of PEG to sCT was conducted using the conditions described in Example 2 except that in this case is used boc-PEG- NH2 (556 Da) as a substrate amino donor. This PEG was chosen because it is easily detectable by ESI-TOF mass spectrometry. Under these conditions, the RP-HPLC analysis of the reaction mixture shows the formation of a new product. The ESI-TOF mass analysis of this product confirms the presence of a derivative of 3972.72 Da, which corresponds to the sCT (34.30.7 Da) attached to a polymer chain (Figure 2) .
Example : mTGase-mediated PEGylation of salmon calcitonin in aqueous buf er in the presence of co-solvents .
The conjugation of PEG. to sCT was conducted using the conditions described in Example 3, but using ethanol as co-solvent (60% v v) . Under these conditions, the results by MALDI-MS analysis show the presence of a mono-conjugate.
Example 5: mTGase-mediated PEGylation of hGH in aqueous buffer. mPEG-NH2 5 kDa at a 10-fold molar excess is added to a solution of hGH (1 mg/mL) in 0.1 M phosphate buffer pH 7. After complete dissolution of the polymer, the TGase enzyme from Streptomyces mobaraense is added in catalytic amount (ratio E/S, 1:50). The reaction is left stirring for 4 hours at room temperature and monitored by SEC-HPLC chromatography. Chromatographic profile, shown in Figure 3, confirms TGase mediated conjugation with the appearance of mono- and bi-PEGylated products.
Example 6: mTGase-mediated PEGylation of hGH in aqueous buffer in the presence of co-solvents .
To a solution of hGH (1 mg / mL) in 0.1 M phosphate buffer pH 7, containing ethanol or methanol (60% v / v) , mPEG-NH2 (5 kDa) in molar excess of 100 times is added. A solution of TGase from Streptomyces mobaraense is added to E/S ratio of 1:5 (w./w) and the reaction is allowed to proceed for 18 hours at room temperature. The RP-HPLC chromatographic profile shows the formation of a single derivative (Figure 4). This product, analysed by gel filtration, eluted at the same retention time of the mono-conjugate hGH, confirming the presence of a single PEG chain linked to the protein.
Example 7 : mTGase-mediated PEGylation of hGH in aqueous buffer in the presence of co-solvents .
The conjugation of PEG to hGH was carried out using the conditions described in Example 6 except that' the amino donor substrate is a monodisperse boc-PEG-NH2 (556 Da) .
Under these conditions the chromatographic analysis of reaction mixture in the RP-HPLC shows the formation of a new product (Figure 4) that, analysed by ESI-TOF mass, gives a molecular weight of 22665.6 Da corresponding to hGH (22,126 Da) attached to one chain of PEG (Figure 5) .
Example 8 : Preparation of POZ-sCT by TGase in aqueous buffer in the presence of co-solvents.
Poly (2-ethyl-oxazolines) · (POZ-NH2 5 kDa) at a 10-fold molar excess is incubated with sCT (1 mg/mL) in 0.1 M phosphate buffer pH 7 containing 30% DMSO (v/v) . A solution of TGase from Streptomyces mobaraense is added to E/S 1:15 (w/w) and the reaction is allowed to proceed for 3 hours at room temperature. The reaction mixture, analysed by RP-HPLC, confirmed the modification of about 50% of calcitonin and formation of a mono- derivative (Figure 6) .
Example 9 : Proteolytic digestion of PEG-sCT and determination of the conjugation site .
At 20 g of boc-PEG540Da-sCT, dissolved in 20 L of 50 mM Tris pH 7.8, are added 1.6 uL of trypsin (Sigma-Aldrich, Milan, IT), E/S ratio, 1:50 (w / w) . The solution is incubated for 3 h at 37 ° C. At the same time the tryptic digestion of native sCT is also performed. Each tryptic digest is analysed in RP-HPLC and products are collected and analysed by ESI-TOF mass. In Figure 7 the chromatographic profile of native SCT is showed. The three peaks (namely a, b, c) correspond to the fragment Leul2-Lysl8 (854 Da), Leul9-Arg24 (776 Da) and Cysl-Lysll (1124 Da). The Thr25-Pro32 fragment (733.7 Da) is not eluted in these conditions because it is very hydrophilic. However, the lack of such residual does not influence the determination of the binding site because it does not contain glutamine residues. The superimposition of the chromatographic elution profile of digested boc-PEG540Da-sCT and sCT shows that the a and c fragments are identified, while the fragment b is disappeared. A new product in boc-PEG540Da-sCT profile is identified (peak d) that analysed by mass spectrometry shows a molecular weight of 1316 Da, which corresponds to residue Leul9-Arg24 linked to a chain of monodisperse PEG. This indicates that -Gln20 is a specific substrate of TGase in the presence of co-solvents being the only Gin present in this peptide. Finally, the tryptic digestion is carried out also on the derivative mPEG5kDa-sCT . Again the HPLG analysis shows the presence of the fragment a and c, a new fragment with greater retention times and the disappearance of the fragment b (data not shown) . The complete absence of fragment b is due solely to conjugation of PEG to a chain of Glr.20.
Example 10: Proteolytic digestion of PEG-hGH and determination of the conjugation site.
A 400 μg of hGH and boe-PEG540Da-hGH, dissolved separately in 400i of buffer 50 mM TRIS, -6 M Gdn, pH 9, TCEP (final concentration 5 mM) is added and the reaction, mixture is allowed to stir for one hour at 37 ° C. Then iodoacetammide (final concentration 25 mM) is added and the reaction is conducted in the dark for 30 min at 37 ° C. After purification by HPLC, the reduced and carboxymethylated products are subjected to tryptic digestion overnight at 37 ° C. After pepclean™ desalting column ' (Pierce C18 spin column) purification, the peptide fragments of hGH and boc-PEG540Da-hGH are determined by ESI-TOF mass spectrometry. By the mass analysis of digested hGH the most tryptic fragments are identified with a percentage of sequence coverage of 85%. The digest of boc-PEG540Da_hGH shows all the fragments identified in the native hGH except that the fragments corresponding to the sequence Glnl41-Lysl45, Phel46-Lysl68- Phel91 and Serl84. A new fragment with a double charge . molecular weight [583Da +2 H]+2 present only in the boc-PEG540Da-hGH digest is the fragment Glnl41-Lysl45 (626.31 Da) attached to a chain of PEG (540Da) (Figure 8). The fragment Glu39-Cys41, containing the residue Gln40, is reported to be acyl donor substrate of TGase in aqueous conditions of catalysis is not identified in the digested tryptic samples because it has a low molecular weight and is not detected by the instrument. The total absence of the fragment Glnl41-Lysl45 and the lack of Glu39-Cys41 fragment conjugated to PEG (with mass of 944.2 Da and the corresponding double-charged [M +2 H]2 +) indicate that all the Glnl41 residue is completely linked to PEG. The fragments Lysl68-Phel46 and Serl84-Phel91 are not identified. However, they do not contain glutamine and therefore are not relevant to analysis.
Example 11: Effect of organic co-solvents on the conformation of calcitonin and hGH.
A) Effect of organic solvents on the fluorescence and circular dichroism spectra of sCT.
Fluorescence and circular dichroism measurements of native sCT are carried out at the concentration solutions of 0.1 mg/mL as spectrophotometrically determined at 280 nm. An increase in tyrosine fluorescence is observed with the increase in the percentage of organic solvent added to the buffer phosphate. Although the polarity of the solvent increases the intensity of fluorescence, this is not accompanied by shift of the maximum emission, except a slight red shift of tyrosine in presence of DMSO (Figure 9b) . Circular dichroism spectra show that in the presence of organic co-solvents secondary structure of SCT is changed. While in physiological conditions sCT is largely disordered, . it takes a strong a-helix structure in an environment with low dielectric constant (Figure 9a) . The data may suggest that some portion of the polypeptide adopting a more ordered structure is less accessible to the enzyme with a consequent reduction of site conjugation.
B) Effect of organic solvents on the fluorescence and circular dichroism spectra of hGH.
Fluorescence and circular dichroism measurement of native hGH are carried out at the concentration solutions of 0.1 mg/mL as spectrophotometrically determined at 280 nm. hGH has only one tryptophan located in a buried region, almost completely solvent-shielded, which represents a convenient probe for structural analysis. While the presence of DMSO does not change the environment around the Trp, the presence of ethanol yields a red shift of the maximum emission due to an increased exposition of Trp, thus suggesting a modification of hGH conformation (Figure 12b)'1. The CD spectra show that the protein maintains its secondary structure and compactness even at high concentrations of ethanol, with an increase of a percentage of a-helix from 46% to 56% (Figure 12a) . -
Example 12 : Pharmacodynamic profiles of PEG-calcitonin in rats .
The biological effects of the derivatives obtained in the present invention were assessed as the ability to develop a hypocalcaemic response in vivo. After anaesthesia, Sprangue- Dawley rats, weighing between 180-250 g, were divided into groups and each group received by injection into a tail vein one of the following samples: sCT, mPEG51cDa-sCT or mPEGiokDa-sCT at a dose of 40yg/kg (sCT equiv.). At predetermined times (0, 0.5, 1, 2, 3, 4, 6 and 24 hours), plasma samples were taken and analysed by colorimetric assay (Vinci Biochem, Vinci IT) to assess the levels of calcium in the blood. The data show that both •conjugates are active reducing plasma levels of calcium after administration. In particular after 6 hours post administration, the percentage of calcium in plasma was reduced to 60% of the initial value. Diversely, levels of plasma calcium at the same time are returned to baseline levels after native administration of sCT (Figure 11). Note that the molecular weight of PEG is critical for prolonged activity; in fact mPEG5kDa-sCT derivative shows a similar pharmacodynamic profile , in comparison with unconjugated sCT.

Claims

1) A conjugation method comprising a reaction catalyzed by a transglutaminase (TGase) between a protein or a peptide, containing two or more glutamine residues as acyl donor substrate, and a molecule containing at least one amino group as an amino donor substrate, this method is characterized by the use of at least an organic solvent miscible with water as cosolvent in the aqueous solution, to increase the reaction selectivity and reduce the number of products of conjugation.
2) A method as described in claim 1. for preparing a monoconjugate product, that is a product in which the molecule containing at least one amino group is linked only to a specific site of the protein or peptide, or for the preparation of a biconjugate product or a product in which two molecules-containing at least one amino group are linked to two specific sites in the protein or peptide.
3) A method as described in claims 1-2 in which at least one organic cosolvent is present in a quantity ranging from 5% to 80% (v/v) , more preferably between 20% and 70% (v/v) , even more preferably between 30% and 65% (v/v), and even more' preferably between 30% and 60% (v/v) , and in which the aqueous solution optionally also contains the reaction buffer.
4) A method as described in claims 1-3 in which at least one organic co-solvent is ethanol, methanol or DMSO, and as such co-solvent is preferably present in the enzymatic mixture in a quantity ranging from 5% to 80% (v/v) , more preferably between 30% and 70% (v/v) , even more preferably between 50% and 65% (v/v) and even more preferably is 50% (v/v) in case of ethanol and 60% (v/v) in case methanol, and it is preferably a quantity ranging from 10% to 40% (v/v) and more preferably between 25% and 35% (v/v) and even more preferably of 30 % (v/v) in the case of DMSO.
5) A method as described in claims 1-4 in which the transglutaminase (TGase) is obtained from the following sources: Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces grisepcarneum , Streptomyces lavendulae , Streptomyces ladakanum, Bacillus subtilis, Myxomycetes, guinea pig liver, from a marine organism such as the japanese oyster Crassostrea gigas, or it is a mutant of a transglutaminase produced from these sources including at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% of the amino acid sequence of the native enzyme.
6) A method as described in any of the preceding claims in which the protein or peptide containing two or more glutamine residues is calcitonin with the sequence SEQ ID Nl, the human growth hormone (hGH) with the sequence SEQ ID N2 or a mutant including at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% of the amino acid sequence of the sequence SEQ ID Nl or SEQ ID N2.
7) A method as described in any of the preceding claims in which the molecule containing an amino group and acting as the amino donor substrate is selected from: a natural or synthetic polymer, preferably hydrophilic and soluble in water, a diagnostic agent such as a radioisotope or a chelate, a fluorescent molecule, an oligonucleotide, such as an antisense oligonucleotide an aptamer or a siRNA, a protein or a peptide, a drug, a toxin or a molecule heterobifunctional .
8) A method as described in any of the preceding claims in which the molecule contains an amino group acting as a substrate amino donor is preferably a polymer having an average molecular weight (MW) between 200 Da and 300000 Da, more preferably between 3000-100000 Da, even more preferably between 5000 Da and 80000 Da, and even more preferably between 10000 Da and 40000 Da.
9) A method as described in any of the preceding claims in which the molecule containing an amino group and acting as the amino donor substrate is a polymer selected from the following group: poly (ethylene glycol) (PEG) with a linear or branched structure, preferably mono-functional, preferably having an average molecular weight (MW) between 2000 Da and 40000 Da, more preferably between 2000 Da and 30000 Da and even more preferably between 5000 Da and 20000 Da, and where the more favorites poly (ethylene glycol) s are mPEG-NH2 10000 Da, mPEG- NH2 20000 Da, mPEG-NH2 40000 Da, branched mPEG-NH2 10000 Da, branched mPEG-NH2 20000Da and branched mPEG-NH2; polyvinylpyrrolidone; polyacriloylmorpholin, N-hydroxy-ethyl methacrylamide copolymer; poly (2-ethyl-oxazolin) , polyglutamic Acid, hyaluronic acid, polysialyc acid, and copolymers of PEG- poly (amino acid).
10) A monoconj ugate, preferably a monoconjugate of a polymer, which is preferably PEG, and a protein or a peptide containing two or more glutamine residues, which preferably is calcitonin with the sequence SEQ ID Nl, the human growth hormone (hGH) with the sequence SEQ ID N2 or a mutant including at least 80%, preferably at least' 85%, more preferably at least 90%, and even more preferably at least 95% of the amino acid sequence of the sequence SEQ ID Nl or SEQ ID N2, and such conjugate is obtained using the method as described in any of the previous claims.
11) A conjugate obtainable by a method as described in any of claims 1-9, where the TGase is from Streptomyces mobaraense, the aqueous solution containing the TGase is 0.1 M phosphate buffer pH 7, the organic cosolvent in the reaction mixture is ethanol in a quantity of 50% (v/v) , or is DMSO in a quantity of 30% (v/v) , the acyl donor substrate is calcitonin and the amino donor substrate is selected from mPEG-NH2 10000 Da, mPEG-NH2 20000 Da and mPEG-NH2 40000 Da, whose conjugate having the formula: mPEG- (Q20) -calcitonin, and the molecular weight (MW) of PEG is 10000 Da, 20000 Da or 40000 Da, respectively.
12) A conjugate obtainable by a method as described in any of claims 1-9, where the TGase is from Streptomyces mobaraense, the aqueous solution containing the TGase is 0.1 M phosphate buffer pH 7, the organic co-solvent present in the reaction mixture is methanol in an amount of 60% (v/v) or ethanol in an amount of 50% (v/v) , the acyl donor substrate is hGH and the amino donor' substrate is selected from mPEG-NH2 10000 Da, mPEG-NH2 20000 Da and mPEG-NH2 40000 Da, whose conjugate having the formula: mPEG- (Q141) -hGH, where the molecular weight (MW) of the PEG is 5000 Da, 20000 Da or 40000 Da, respectively.
13) A pharmaceutical or diagnostic preparation comprising at least one conjugate obtained as described in any of claims 10- 12, and possibly including a further therapeutically active or diagnostic agent, and optionally a pharmaceutically acceptable excipient .
14) A pharmaceutical preparation as defined in claim 13 for oral, parenteral, rectal, topical, vaginal, ocular or inhalation administration .
15) A treatment method or a method of diagnosis comprising the administration of a formulation according to any of claims 13- 14.
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CN109943546A (en) * 2019-04-12 2019-06-28 天津科技大学 A kind of glutamine transaminage mutant and its preparation method and application
CN110358799A (en) * 2019-07-26 2019-10-22 山东大学 A method of based on enzymatic hyaluronic acid pointed decoration polypeptide
CN110358799B (en) * 2019-07-26 2021-02-26 山东大学 Method for site-directed modification of polypeptide based on enzyme-catalyzed hyaluronic acid

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