Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/805—Haemoglobins; Myoglobins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock

Definitions

• the present invention relates to oxygen transporters having a structure similar to hemoglobin and which can be used as a blood substitute having a reduced affinity for oxygen.
• ⁇ is therefore desirable to be able to have an artificial oxygen transporter which can be substituted for blood for transfusion use.
• Hemoglobin is the main constituent of the red blood cell, its essential function is to fix, transport and deliver the quantity of oxygen necessary for the normal functioning of tissues.
• the hemoglobin molecule has two parts, a protein part, globin, and groups that house ferrous iron atoms responsible for fixing oxygen, heme.
• the globin is a tetramer composed of four identical chains two by two which are named, for the peptide chains of 141 amino acids, alpha, and for the chains of 146 amino acids, chains of the beta type; for this reason normal human globin is denoted alpha 2 beta 2 .
• beta chain or “non-alpha” covers not only beta chains, but also chains called epsilon, gamma or delta.
• a hemin group is linked to each of the polypeptide chains.
• hemoglobin Normally in adults, more than 95% of hemoglobin is constituted by alpha 2 beta 2 tetramer, that is to say the association of two heterologous alpha-beta dimers, associated with the catalytic complex, Theme, that is to say, hemoglobin A. There is 2% to 3% of a hemoglobin consisting of tetramers alpha 2 delta 2 , and traces of fetal hemoglobin alpha 2 gamma 2 .
• patent application WO 88/09179 proposed this type of approach, namely the separate synthesis of alpha chains and beta chains, in particular in the form of non-natural mutants.
• the subject of the present invention is an oxygen transporter which requires the synthesis of only one type of chain, this type of chain being able to be associated in the form of a tetramer carrying heme and capable of reversibly fixing the 'oxygen.
• the present invention relates to an oxygen transporter of the type comprising at least four practically identical globin-type polypeptide chains, capable of associating or being associated with one another by incorporating Theme, in order to ensure a binding activity. reversible oxygen.
• polypeptide chains of globin type one refers to chains of alpha type or chains of beta type, and that these chains are practically identical, preferably are completely identical.
• This type of transporter therefore differs from the transporters of the prior art in which two distinct types of chains are always provided which are capable of joining together, such as for example alpha and beta chains, even if, moreover, these chains contain certain modifications or appear as a dimer.
• the oxygen transporter according to the present invention can have two different embodiments.
• the four polypeptide chains are identical and their structure, which borrows from alpha chains and beta-type chains, is such that they will be able to associate by reconstituting an interface similar to the alpha 1 interface, beta 2 which is involved in the association of alpha chains and beta chains of natural HbA.
• the chains are identical and are, in particular, all beta chains which will be associated, in the form of a tetramer.
• the present invention relates to artificial oxygen transporters as described above, comprising at least four polypeptide chains incorporating Theme and each having a sequenced globin type structure:
• - V, X, and Z are globin-like polypeptide sequences
• (W) and (Y) are two complementary polypeptide sequences capable of interacting to constitute an interface which, when it belongs to two distinct chains, is at least in part similar to the alpha 1 beta 2 interface, said chains being able to associate by reconstituting said interface similar to the alpha 1 beta 2 interface in order to ensure a reversible and cooperative binding activity of Toxygen.
• the four polypeptide chains associated in a tetramer are associated "head to tail" so that the comparison of the sequences W 1 of one of the chains with the sequence Y 2 of l the other chain leads, in parallel, to the connection of the sequence Y 1 of the first with the sequence W 2 of the second to reconstitute at least in part the alpha 1 , beta 2 interface of the natural hemoglobin.
• This interface between the alpha chains and the beta chains is involved in the conformational transition responsible in part for the fixing and the release of oxygen, its reconstitution makes it possible to obtain oxygen transporters having satisfactory properties.
• polypeptide chains must have structures which bring them closer to the alpha type chains or the beta type chains.
• X will comprise one or more helical segments chosen from helical segments D, E and F of alpha or beta type chains, said segments may be linked together by non-helical segments or by single bonds.
• the globin V type polypeptide sequence will comprise one or more helical segments chosen from helical segments A, B and C of chains of alpha or beta type, said segments may be linked together by non-helical segments or by single bonds.
• the globin Z type polypeptide sequence will comprise one or more helical segments chosen from helical segments G and H of alpha or beta type chains, said segments may be linked together by non-helical segments or by single bonds.
• the helical segments play an important role in the structure of the normal tetramer and that this type of structure will have to be found in the synthetic tetramer, it is however possible to provide for the association of helical segments of different origin, that is to say either alpha type or beta type, as for the non-helical segments, they can be segments having the structure of their natural counterparts and / or be of different nature even if, moreover , the use of homogeneous sequences any alpha or any beta can be preferred.
• alpha, beta interface as it can be reconstituted by the chains sequenced according to the present invention is preferably the following:
• the different helical segments or not may also include mutations with respect to their natural counterparts, as will be described below.
• mutations preferably relate to the more stable beta-type subunit.
• the following mutations in the beta-type chain can be mentioned more particularly:
• NB only hemoglobins containing a single mutation were taken into account either in the ⁇ subunit or in the ⁇ subunit.
• the W and Y sequences must make it possible to reconstitute an alpha 1 beta 2 interface for HbA or the like which is involved in the transition R—> T. This is why the segments (W) and (Y ) will be similar to the C / CD and FG / G segments of the alpha, beta 2 interface of human hemoglobin, but it is possible to modify it if necessary by predicting certain point mutations.
• the essential elements involved in the structure of the alpha 1 beta 2 interface are as follows:
• polypeptide chains which are more particularly advantageous, mention should be made of the mixed alpha, beta chains, that is to say in particular the chains consisting of the N-terminal end of a beta-type chain of globin, and of the C-terminus of the globin alpha chain; or, conversely, a chain consisting of the N-terminal end of an alpha chain of globin and the C-terminal end of a beta-type chain of globin.
• the alpha-beta junction will preferably be located in the flexible area of the Theme pocket, that is to say in the EF segment.
• the most interesting polypeptide comprises the N-terminal end of the beta chain of globin fused to Tamino-acid 73 included in the C-terminal part of the alpha chain of globin from Tamino-acid 69 included.
• FIG. 8 This type of chimeric molecule associated in tetrameric form is represented in FIG. 8 in which the alpha 1 beta 2 interfaces reconstituted in the tetramer have been visualized.
• two polypeptide chains to be linked by a sequence, preferably peptide, located between the Z sequence of Tune and the V sequence of the other, the two chains are thus integral, which promotes the stability of the tetramer.
• the two chains can also be linked by crosslinking or by any type of biochemical interaction such as, for example, covalent bridging bonds (derivatives of diaspirin or of pyridoxal phosphate).
• the oxygen transporter according to the invention will include a heme structure; it may be a natural heme molecule or a metalioporphyrin in which the iron atom is replaced by another metal, in particular cobalt.
• the tetramer consists essentially of four identical polypeptides each having a beta-globin type structure. This type of tetramer called beta 4 will preferably be constituted by beta type chains comprising one of the mutations mentioned above.
• dimers It could, as has been described, be prepared by association of dimers, these dimers being formed by bonding the N and C ends of two subunits.
• the oxygen transporter according to the invention generally has a reduced affinity for oxygen compared to natural hemoglobin, preferably this affinity is reduced by at least 20% compared to that of natural hemoglobin.
• the transporter according to the present invention is preferably obtained by culture of a transformed organism producing said chain, it may be eukaryotic cells comprising a DNA sequence coding for said chain under the dependence of expression elements ensuring the expression of said sequence in said host cell.
• the host cell is preferably a eukaryotic cell and preferably a yeast and / or a higher eukaryotic cell or a transgenic animal.
• the expression system it may be either plasmids with autonomous replication, or piasmids allowing integration into the genome of the host cell.
• the coding sequences may be dependent on a heterologous but effective promoter in the host or on a homologous promoter, or even on a chromosomal promoter. It is also possible to provide systems ensuring, for example, the secretion of these polypeptide chains.
• tetramers is, most of the time, spontaneous, it can be favored according to the exact structure of the tetramer. by special conditions for dissolving, in particular under certain pH and / or ionic strength conditions.
• hemin group can be spontaneous, in particular in eukaryotes, and the tetramers obtained are "heminized" without there being any need for a specific step. Most of the time Theme will be added at a later stage.
• the present invention also relates to methods allowing the preparation of these polypeptide chains by techniques using the recombinant DNAs and the possible formation of the tetramer, as described above (Hoffman and ⁇ l, 1990; Nagai & Thogersen, 1984, 1987; Nagai and ⁇ l, 1985; Tame and ⁇ l, 1991; Wagenbach and ⁇ l, 1991).
• the present invention relates to the use of chains as a medicament, in particular for the transport of oxygen in combination with an acceptable support in the transfusion field.
• FIG. 1 shows schematically the nucleotide and amino acid sequence of the alpha chain
• FIG. 2 schematizes the nucleotide and amino acid sequence of the beta chain
• FIG. 3 shows schematically the spatial structure of the beta chain, with indication of the nomenclature of the propellers,
• FIG. 4 represents the diagram of the plasmid pATPrTet
• FIG. 5 represents the diagram of the plasmid pATPr cIIFX beta
• FIGS. 6 and 7 show diagrammatically the preparation of the vector pATPr cIIFX beta Gb
• FIG. 8 represents the diagram of the spatial structure of a dimer according to the invention, in particular the reconstruction of the alpha 1 beta 2 interface.
• FIG. 9 schematizes the stages of the construction of the vector pATPr Chim beta73-alpha69.
• the spatial configuration of the beta chain is represented in FIG. 3 with the nomenclature of the various segments which go from the NH 2 terminal end towards the COOH terminal end:
• NA A, AB, B, C, CE, E, EF, F, FG, G, GH, H and HC.
• the segments A, B, C, E, F, G and H have a substantially helical structure while the junction segments do not have such a structure.
• Amino acids are numbered either from the N-terminal end in ascending order, or by assigning an amino acid the designation of its helix and its rank in it.
• the 36th amino acid in the beta chain is Proline, it is beta 36 (C2) Pro, i.e. the second amino acid in Thélice C.
• the basic vector pAT Pr Tet has an origin of replication derived from pBR 322 and carries the gene for resistance to tetracycline. It allows the expression of heterologous proteins under the control of the Pr promoter of phage lambda. The activity of this promoter is repressed by the repressor CI (ailwash CI 8 57 , thermosensitive). The Pr promoter is blocked at 30 ° C. Raising the temperature to 42 ° C inactivates the repressor and induces transcription from the Pr promoter (FIG. 4).
• the E. coli CAG 1139 bacteria carrying the plasmid are cultured at 37 ° C. (1 L LB medium) until an OD at 600 nm of approximately 0.4 is obtained. Chloramphenicol is added to a final concentration of 170 ⁇ g / ml, and the incubation is continued at 37 ° C for 12-16 hours. The bacteria are lysed by treatment with a NaOH-SDS solution, and the plasmid DNA is purified from the bacterial pellet by precipitation with polyethylene giycol. The protocol used is very exactly that described by Sambrook et al (1989).
• the sequence coding for the fusion protein was obtained by the PCR technique from the double-stranded replicative form of phage M 13 mp10 (10 ng).
• the double-stranded replicative form is prepared according to the technique indicated above for the plasmid DNA, from an E. coli bacterial pellet, strain JM 101.
• a site recognized by SnaBI restriction tendonuclease is introduced at the end 5 'of the coding sequence.
• Xhol 25 units, 2 hours, 37 ° C
• It is precipitated with tethanol after extraction with phenol / chloroform.
• the precipitate is taken up in 10 ⁇ l of 10 mM Tris-HCl / 1 mM EDTA buffer.
• the insertion into the expression vector is carried out using a vector / insert ratio of 1/5 (i.e. 3 ⁇ l of the vector DNA solution and 3 ⁇ l of the insert solution).
• the mixture is incubated in the presence of T4 DNA ligase, 4 hours at room temperature.
• the reaction mixture is used to transform competent CAG 1139 bacteria (CaCl 2 treatment).
• the transformed bacteria selected by the resistance to tetracycline are tested for the expression of the fusion protein.
• the vector thus obtained pAT Pr cIIFX beta directs the synthesis of a fusion protein cIIFX beta globin containing:
• the E. coli CAG 1139 strain carrying the plasmid pAT Pr cIIFX beta is incubated at 30 ° C in M9 medium (Na 2 HPO 4 50 mM / KH 2 PO 4 20 mM / NaCI 8.5 mM / NH 4 Cl20 mM / MgSO ⁇ O.lmM) containing 2 g of glucose, 20 g of yeast extract and 0.5 g of vitamin B1 per liter) in the presence of tetracycline (10 mg per liter).
• M9 medium Na 2 HPO 4 50 mM / KH 2 PO 4 20 mM / NaCI 8.5 mM / NH 4 Cl20 mM / MgSO ⁇ O.lmM
• tetracycline 10 mg per liter
• the bacterial pellet (100 g wet weight) is thawed and suspended in 100 ml 50 mM Tris-HCl, pH 8/25% sucrose (w / v) / 1 mM EDTA.
• the bacteria are lysed by incubation in the presence of 200 mg of lysozyme (30 minutes in ice).
• MgCl 2 , MnCl 2 and DNase I are added so as to obtain respective final concentrations of 10 mM, 1 mM and 10 ⁇ g / ml.
• the protein precipitate is dissolved in a solution of guanidine-HCl 6M / Tris-AcOH 25 mM, pH 5.0 / EDTA 1 mM / dithiothreitol 1 mM.
• the solution is equilibrated in an 8M urea buffer / 25 mM Tris-AcOH, pH 5.0 / 1 mM EDTA / 1 mM dithiothreitol by filtration through a column of Sephadex G-25, then deposited on a column of CM-Sepharose (4 ⁇ 10 cm) balanced with the same buffer.
• the fusion protein is eluted using a linear gradient formed by 500 ml of 8M urea / 25 mM Tris-AcOH, pH 5.0 / EDTA 1 mM / dithiothreitol 1 mM and 500 ml of the same buffer supplemented with NaCl qsp a concentration of 0.2 M.
• the fusion protein is then purified by filtration on a column of Sephacryl S-200 (5 ⁇ 60 cm) in guanidine-5 M HCl / 50 mM Tris-HCl, pH 8.0 / 1 mM EDTA / 1 mM dithiothreitol.
• the protein-containing fractions are pooled, dialyzed against water and lyophilized, or dialyzed against the cleavage buffer to be immediately treated with activated factor X.
• the mutation is introduced into TADN using a synthetic oligonucleotide initiator of sequence dC AGG AGT CAG CAT CAC CCT ACC C.
• This sequence is complementary to that of cDNA coding for the beta subunit of globin framing the codon of Thistidine beta (NA2) .
• the triplet introduced CAT (instead of GTG) is altered so as to code for a methionine. Mutagenesis is performed using the replication of the phage M 13 into which is inserted cDNA coding for the beta chain of globin (Nagai & Thogersen, 1984), by the method of Nakamaye & Eckstein (19S6) recommended by Amersham (Kit Amersham).
• the product of the mutagenesis reaction is used to transform competent E. coli bacteria, strain JM 101 according to conventional methods (Nagai & Thogersen, 1987; Sambrook et al, 1989).
• the phages carrying the mutation are detected by T DNA sequencing (Sanger method, 1977).
• the complete sequence of cDNA coding for the fusion protein is verified in order to ensure the integrity of the coding sequence.
• the coding sequence containing the mutation is processed as described above to be inserted into the expression vector.
• the mutation is introduced into cDNA using Toligonuciéotide initiator dAGG AGT CAG GAT CAC CCT ACC C.
• the triplet GAT (for GTG) makes it possible to code for an isoieucine.
• the mutation is introduced into cDNA using Tohgonucleotide initiator dGGA CTC AAA GTA CCT CTG GGT.
• the triplet GTA (for GAA) makes it possible to code for a tyrosine.
• the construction coding for the beta-alpha globin chimera is constructed by PCR from two plasmids:
• Nsi 5 'GCA TTT AAT GCA TTG ATG CC 3'
• the fragment coding for CIIFX NH- (1-73) beta is amplified from the vector pAT Pr beta using the primers Nsi and Dinv.
• the conditions are as follows:
• the fragment coding for alpha (69-141) COOH of alpha globin is amplified from the plasmid alphalpJW101 (Wilson and ⁇ l, 1978) using primers D and Ch.
• the conditions are as follows: 5 ⁇ l PCR buffer
• a third amplification is carried out from the two purified fragments using the primers Nsi and Ch.
• the conditions are as follows:
• PCR product is precipitated with tethanol after extraction with phenol / chloroform, then purified. It is inserted into the plasmid pAT Pr Tet at the Nsi 1 and Xho 1 sites.
• This vector makes it possible to express a fusion protein comprising:
• FIG. 9 shows schematically the stages of construction.
• This plasmid is expressed in E. coli, strain CAG 1139 (Grossman et ⁇ l, 1983, Cell, 32, 1 51-159).
• This phenotype strain: F- thi leu thr LacY TonA SupE galK Lon 100 is transformed by the conventional method of treatment with CaCl 2 .
• the culture is carried out in LB medium in the presence of tetracycline 50 ⁇ g / ml at 30 ° C; when the OD reaches 0.8-1.2, the expression of the DNA protein is induced by passage at 42 ° C. After 2 hours, it is possible to demonstrate the synthesis of the chain (beta-alpha ) on SDS-PAGE gel stained with Coomassie blue by comparison with an untransformed CAG 1139 culture sample. It is estimated that the amount of beta-alpha chain reaches 20% of total proteins. The construction was sequenced to verify that there was no mutation during the successive PCRs. EXAMPLE 9 Functional Characterization of the Recombinant Hemoglobins
• the absorbance spectra are recorded and compared to the spectrum of native oxygenated THb A (Cary 219 spectrophotometer, Varian, Palo Alto, Ca, USA). These spectra make it possible to test the presence of methemoglobin or of hemichrome (Szebeni et al,
• the deoxygenation curves are recorded in solutions of Hb (60 to 100 ⁇ M in heme) in a 50 mM bisTris buffer, 100 mM NaCl, pH 7.2 at 25 ° C., 50 ⁇ M of EDTA and 20 ⁇ g / ml of cataiase are added to the buffer to limit the oxidation of hemoglobin.
• the concentration of NaCl, the pH can be modified to test the different properties of recombinant Hb (Bohr effect, anionic effectors).
• Other natural (2,3 diphosphoglycerate) or non-natural (IHP, clofibrate derivatives) effectors can be added to the solution at the start of the recording.
• the solution is first balanced under a gas mixture whose partial oxygen pressure is at least 350-450 mmHg to ensure an almost complete initial saturation of the hemoglobin.
• the solution is then slowly deoxygenated by admitting pure nitrogen into the measuring cuvette.
• the duration of the recording is 45 to 60 min to ensure the equilibrium conditions. These equilibrium conditions are demonstrated by the superposition of the deoxygenation and re-oxygenation curves.
• the amount of methemoglobin (oxidized Hb) formed during the recording is measured by spectrophotometry according to the method of Benippoh et ai (1973).
• the auto-oxidation speed of hemoglobin is measured by spectrophotometry of the solution (1 00 ⁇ M heme) incubated in a 100 mM Phosphate buffer at 37 ° C. as a function of time (0-> 24 h).
• the thermal stability is measured by incubation of the solutions (300 ⁇ M heme) at 65 ° C as a function of time (0—> 120 min).
• Photodissociation studies are performed using a 10-ns laser system (Quaritel YG 585) delivering 160 mJ at 532 nm.
• the energy of the photodissociation beam can be modified to study the conditions of partial photolysis.
• the samples are studied in cuvettes with 1 or 2 mm optical path.
• the detection beam at 436 nm is almost co-linear with the photodissociation beam.
• the intensity of transmitted light is detected by a photomultipiifier (lP28 Hamamatsu, Japan) and recorded on a digital oscilloscope (Lècroy 9400).
• the data is then transferred to an IBM microcomputer for calculations of absorbance changes and data analysis.
• Kinetic simulations are based on the two-state allosteric model.
• the curve recorded for native Hb A shows the existence of two rates of recombination of CO to hemoglobin: Tune is rapid and corresponds to recombination in the R state of high affinity for the ligand; the second is slower and corresponds to recombination in the T state of low affinity.
• Kinetic experiments make it possible to very quickly test the functionality of recombinant Hb with the minimum of sample.
• the chimeric subunits obtained according to the examples assemble in a tetramer which fixes the oxygen in a reversible manner practically without cooperativity but with an affinity slightly lower than that observed for native Hb A and thirty times lower to that of the ⁇ 4 tetramer.

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• 1992
  • 1992-03-18 FR FR9203224A patent/FR2688784B1/fr not_active Expired - Fee Related
• 1993
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