WO2007110001A2 - Molécules chimériques fusionnées à la matriline-1, à usage thérapeutique - Google Patents

Molécules chimériques fusionnées à la matriline-1, à usage thérapeutique Download PDF

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WO2007110001A2
WO2007110001A2 PCT/CU2007/000012 CU2007000012W WO2007110001A2 WO 2007110001 A2 WO2007110001 A2 WO 2007110001A2 CU 2007000012 W CU2007000012 W CU 2007000012W WO 2007110001 A2 WO2007110001 A2 WO 2007110001A2
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protein
seq
amino acid
scfv
epo
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WO2007110001A3 (fr
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Rolando Paez Meireles
Glay Chinea Santiago
Diamile Gonzalez Roche
Jorge Víctor GAVILONDO COWLEY
Iraldo Bello Rivero
Anazuria MARTÍNEZ NUÑEZ
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Center for Genetic Engineering and Biotechnology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6435—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a connective tissue peptide, e.g. collagen, fibronectin or gelatin
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is related to the field of biotechnology, with the biological sciences and the pharmaceutical industry, and in particular with the modifications of molecules to improve their pharmacokinetics, increasing their average life time in blood and biological activity.
  • Previous Art The use of proteins and peptides for therapeutic use in humans has increased in recent years mainly due to: (1) discovery of new protein and peptide molecules, (2) better understanding of the mechanisms of action in vivo, ( 3) improvement in the protein expression systems and peptide synthesis and (4) improvement in the formulations or in the technologies of modification of molecules that allow to improve the pharmacodynamic and pharmacokinetic properties.
  • New drug release systems can be produced through a change in formulation (e.g. continuous release products or liposomes) or through an addition to the drug molecule ("PEGylation").
  • Injectable continuous release systems release drugs in a controlled and predetermined manner, and are especially suitable for drugs where it is important to avoid large fluctuations in plasma concentrations. Encapsulating a drug within a liposome can produce a prolonged half-life and a change in distribution to tissues with increased capillary permeability (eg, tumors, infected tissue). PEGylation provides a method for the modification of therapeutic proteins with the purpose of minimizing many of the limitations that are associated with these agents (eg poor stability, short half-life, immunogenicity) (Reddy, KR, 2000. Controlled-release, pegylation , liposomal formulations: New mechanisms in the delivery of injectable drugs. Ann. Pharmacother 34: 915-923).
  • EPOh is a 165 amino acid glycoprotein (Mikaye, T. et al. Purified of Human Erythropoietin. J. Biol. Chem. 252: 5558-5564, 1977). Its estimated molecular weight using sodium polyacrylamide-dodecyl sulfate gel ranges from 27,000 to 39,000 daltons (Mikaye, T. et al., Purification of Human Erythropoietin. J. Biol. Chem. 252: 5558-5564, 1977).
  • Other molecules to which different modified release technologies have been applied are Interferon alpha 2b rec.
  • This recombinant protein has been used with great success in the treatment of Hepatitis C.
  • a limitation of the treatment of Hepatitis C with IFN is that it has a half-life of 7 to 9 hours and is practically undetectable in blood at after 24 hours of administration
  • the fluctuations resulting from IFN exogenous during the course of treatment are considered to have a negative impact on the antiviral efficiency of the treatment.
  • IFN-Peg and microspheres have been developed (Reddy, KR, 2000. Controlled-release, pegylation, liposomal formulations: New mechanisms in the delivery of injectable drugs. Ann. Pharmacother. 34: 915-923; Diwan M, Park TG. 2003. Stabilization of recombinant interferon-alpha by pegylation for encapsulation in PLGA microspheres. Lnt J Pharm; 252: 111-122).
  • the object of this invention is the obtaining of chimeric biomolecules with superior characteristics in terms of the average life time and biological activity with respect to the non-chimeric protein.
  • the proteins of therapeutic interest bind with this objective to the carboxyl end of the Matriline 1 human cartilage matrix protein, fusion mediated by a flexible spacer of variable size.
  • the obtaining of several biomolecules is described by means of the fusion to each of a flexible amino acid spacer, and then the 45 amino acids corresponding to the carboxyl end of the Matriline 1 human cartilage matrix protein (Fig. 1, 2) .
  • the base biomolecule is associated in the form of trimers allowing the increase of its molecular size, which leads to an increase in the average life time in blood of the pharmaceutical product.
  • the resulting chimeric protein is formed by four globular domains (three corresponding to the base molecule and one corresponding to the helical trimer of the Matrilin 1 fragment.
  • the present invention comprises the use of any biomolecule of amino acid nature, such as proteins (Erythropoietin, GSCF, EGF, IFN, etc.), monoclonal antibodies, fragments of monoclonal antibodies, peptides fused to a certain flexible spacer followed by the Carboxyl end of the Matriline one.
  • proteins Erythropoietin, GSCF, EGF, IFN, etc.
  • monoclonal antibodies fragments of monoclonal antibodies, peptides fused to a certain flexible spacer followed by the Carboxyl end of the Matriline one.
  • the present invention comprises the use of any type of flexible spacer that allows the accessibility of this biomolecule to its respective receiver.
  • Figure 1 Schematic representation of the fusion biomolecule
  • Figure 4 General plasmid or pKGEPOI expression vector construction strategy
  • Figure 5 Map of human Erythropoietin expression vector in pKGEPOI eukaryotic cells
  • FIG. 6 Scheme of the synthetic genes MATN1s1, MATN1s2, MATN1s3 and
  • MATN1s4 designed to fuse the 3 'end of the rHuEPO gene.
  • Figure 8 Vectors pEPOMATN1, 1, pEPOMATN1, 2, pEPOMATNI, 3, pEPOMATN1, 4
  • Figure 9 Western blotting performed on samples from the culture supernatants of the CHO clone producing EPO-MATN 1, 1.
  • Lane 1 CHO cells without transfecting (Cntrl -)
  • Lane 2 Clone of the CHO line transfected with pKGEPOI (Cntrl +) and selected with MTX; Lanes 3 and 4: Clones 6 and 8 of CHO transfected with the plasmid pEPOMATNI, 1 and selected with MTX and Lane 5: transferable molecular mass standard (PMM).
  • PMM transferable molecular mass standard
  • Figure 10 SDS-PAGE electrophoresis of the EPO-MATN1.1 sample of the purification process.1- Molecular Weights Pattern, 2- Cell culture sample 3- G-25 elution, 4- Fraction not absorbed, 5- Washing with 20 mM Imidazole, 6- elution with 50 mM Imidazole, 7- elution with 100 mM Imidazole, 8- Sample control of Epo hu-r.
  • Figure 11 Study by HPLC gel filtration of EPO-MATN 1, 1 obtained by the procedure described and compared with the Epo hu-r.
  • Figure 12 A scheme of the main elements of the pJGm-25 vector used for the expression of the anti-CEA scFv in E. coli is presented.
  • pLac is the lactose promoter
  • SP is the signal peptide B
  • ApaLI and Notl are restriction sites between which the scFv anti-CEA is cloned
  • c-myc is the domain whose coding is recognized by the monoclone antibody! 9E10
  • 6-his is a domain that codes for 6 histidines with a view to the purification of the resulting protein by affinity chromatography by metal ions
  • Term is the terminator of transcription
  • Amp r is the ampicillin resistance gene.
  • Figure 13 Sequence and representative scheme of the composition by parts of the synthetic gene to digest and fuse the 3'end of the scFv anti-CEA in plasmid pJGm-25.
  • a synthetic band scheme includes, from left to right: additional bases prior to the restriction site Not 1 at the 5 'end (in green), site Not I (underlined), additional base T for correction of the frame of reading (in red), 33 bases corresponding to the binding or linker segment (in blue), 126 bases corresponding to the Matrilina 1 gene segment (in yellow), Not I site (underlined), additional bases subsequent to the restriction site Not I at the 3 'end (in green).
  • Figure 14 Representative scheme of the composition by parts of the scFv-sMATN1 chimeric fusion protein: scFv anti-CEA (white), linker (light blue), sMATNI (yellow), c-myc (black), 6 histidines (blue Dark).
  • EXAMPLE 1 Obtaining and characterizing the EPO-MATN1 protein.
  • EPO-MATNI Construction of the plasmid pEPOMATNI for final integration of the EPOMATN1 gene to the genome of Chinese Hamster Ovary Cells (CHO) and expression thereof of the fusion protein Erythropoietin-Fragment of the carboxyl end of the human Matrilin 1 protein (EPO-MATN1)
  • the polyadenylated RNA obtained was selected by oligo gel affinity chromatography dT cellulose.
  • Five micrograms of messenger RNA obtained by this route were used to synthesize complementary DNA (cDNA), an aliquot of which was used for amplification of the cDNA encoding human EPO by means of the polymerase chain reaction (PCR) (Randall, K et al., Science, 42394, 487-491, 1988).
  • PCR polymerase chain reaction
  • EPO-P1 SEQ ID No.2
  • EPO-P2 SEQ ID No.3
  • the PCR product was digested with EcoR I and Hind III and subcloned into the filament phage vectors M13mp18 and M13mp19. The correct sequence was verified by sequencing both chains of the DNA inserts by the method of dideoxynucleoside triphosphates (Sanger, S. et al., 1977. DNA sequencing with chainterminating inhibitors, "Proc. Nat. Acad. Sci. 74, pp.
  • FIG. 2 shows the general strategy of construction of the plasmid or expression vector pKGEPOI, this vector contains, in addition to the gene coding for the recombinant human erythropoietin ⁇ rHuEPO), the following regulatory elements; A transcription unit for selection in eukaryotic cells composed of the G418 resistance gene (neo) under the control of the Thymidine kinase (tk) promoter; an ampicillin resistance gene (Amp r ) for selection in Escherichia coli; an origin of replication derived from pBR322 for propagation in Escherichia coli; Ia C Enhancer Region and SV40 polyA region ( Figure 3).
  • the fusion genes called MATNIs (SEQ ID No.5), MATN1s2 (SEQ ID No.13), MATN1s3 (SEQ ID No.14), MATN1s4 (SEQ ID No.15) were constructed in the Department of Oligonucleotide Synthesis of the CIGB with the automatic system "Gene assembler Plus DNA synthesizer" of Pharmacia LKB (Uppsala, Sweden), during the design a Stu I internal restriction site located 21 bases from the 3 ' end of the hormone gene was taken into account ( previously cloned into the vector pKGEPOi) and this site was placed at the 5 ' end of the gene, then the 21 bases of the rHuEPO gene were located, then 33, 45, 48 or 60 bases (coding for a flexible spacer), and finally 126 bases corresponding to the 3 ' end of the human MATN1 gene, a stop codon and the recognition site for the Hind III enzyme.
  • Figure 6 shows schematically the synthetic genes MATN1s1 MATN1s2, MATN1s3 and MATN1s4 designed to fuse the 3 ' end of the rHuEPO gene.
  • the fusion genes were subcloned into the EcoR V site of the pZErO-2.1 vector of Invitrogen, obtaining the vectors pZMATNI, 2, 3 and 4 ( Figure 7), in which the correct sequence of the synthetic genes inserted, by automatic sequencing, with the use of the oligonucleotide primers M13Rev and M13Fw (from the Sequenase TM Sequence Kit version 2.0 of Amersham Int, UK), whose complementary sequences are in the plasmid pZErO-2.1 flanking the site of multiple cloning
  • the synthetic fragments corresponding to the MATN 1s1, MATN1s2, MATN1s3 and MATN1s4 genes were extracted from the vectors pZMATNI, pZMATN2, pZMATN3 and pZMATN4 respectively by digestion with the same enzymes (Stu 1 and Hind III).
  • the pure digested fragments were ligated with T4 ligase according to the conditions specified by the manufacturer.
  • the clones obtained were called pEPOMATN1, 1, pEPOMATN1, 2, pEPOMATN1, 3, pEPOMATNI, 4, Figure 8.
  • the sequences thereof were verified by automatic sequencing with the described oligonucleotides. For the amplification and expression of the EPOMATN1 chimeric genes.
  • the cell cell line was selected Chinese hamster ovary (CHO K1 DUX B1 1) d /? / r- (Urlab, G. and Chasin, LA (1980) Proa Nati Acad. Sci. USA, 77, 4216-4220) and the amplifiable selection marker DHFR to obtain a stable line producing the protein of interest.
  • the line of work was co-transfected with the plasmids pEPOMATNI and pED4 (Kaufman, R J., et al (1991) Nucleic Acids Res. 25, 19 (16): 4485-4490), which carries the coding gene for Ia dihydrofolate reductase (DHFR).
  • the cells that incorporated the genes of interest were selected in the presence of increasing concentrations of methotrexate.
  • a gene amplification was verified three times, accompanied by an increase in the expression of EPO up to approximately 33, 29, 30 and 31 pg / cell / day for clones E POMATN 1, 1, EPOMATN1.2, EPOMATN1.3 and EPOMATN1.4 respectively,.
  • the eluted sample was purified by metal interaction chromatography (IMAC).
  • the Chelating Sepharose FF matrix (Pharmacia) was used. 275 mL of the sample was applied to 1 mL of resin packed in a XK 16/20 column.
  • EPO-MATN 1, 1 was obtained with a purity greater than 95%, which eluted in the fractions corresponding to 50 mM and 100 mM Imidazole ( Figure 10).
  • EXAMPLE 2 Obtaining and characterizing a scFv-sMATNL fusion protein Fusion protein between a single chain antibody fragment (scFv) specific for the carcinoembryonic antigen (CEA), and a carboxyl end fragment of the human Matrilin 1 protein (sMATNI)
  • scFv single chain antibody fragment
  • CEA carcinoembryonic antigen
  • sMATNI carboxyl end fragment of the human Matrilin 1 protein
  • the plasmid which appears in Figure 12, has a Not I site for the insertion of new domains to the C-terminal end of the scFv and before the c-myc and 6 histidine domains.
  • the fusion gene to be inserted in the Not I site called sMATNI ( Figure 13, SEQ ID No.7) was built in the Department of Oligonucleotide Synthesis of the CIGB with the automatic system "Gene assembler Plus DNA synthesizer" of Pharmacia LKB ( Uppsala, Sweden). During the design it was considered to add a Not I site and a flexible spacer at the 5 'end of the gene, and the recognition site for the Not I enzyme at the 3' end of the gene. These modifications make possible the cloning in plasmid pJGm-25, so that Ia scFv-sMATN1 fusion protein is transcribed with the additional c-myc and 6 histidine domains Figure 14).
  • the synthetic sMATNI gene and plasmid pJGm-25 were digested with restriction enzyme Not I and ligated with T4 DNA Ligase (Promega). The products of the binding reactions were used for the transformation of competent E. coli (strain XL-1 Blue; Stratagene) by electroporation, and the transformed cells were plated in selective solid medium (LB agar, with 100 ⁇ g / mL ampicillin ) and were grown for 16 hours at 37 0 C.
  • the recombinant plasmids were selected after the purification of the plasmid DNA from several colonies (QIAGEN Min ⁇ Prep k ⁇ t), by means of digestion check with restriction enzymes that allow to detect the binding products expected, and the correct orientation of the sMATNI domain attached to the 3 'end of the anti-CEA scFv.
  • restriction analyzes bands of approximately 3.6 kb were obtained, corresponding to the linearized vector and approximately 750 bp for the scFv-sMATNl fusion coding gene
  • This assay revealed the expression of a protein of expected molecular weight (approx. 33 kDa), which was then evaluated by Western Blot using as a primary antibody a polyclonal antibody specific for the scFv obtained in rabbit, followed by goat anti-IgG antibodies of rabbit conjugated with horseradish peroxidase (Sigma).
  • the transfer of the proteins from the SDS-PAGE to Hybond C Extra nitrocellulose (Amersham Life Sciences) was carried out in a semi-dry transfer device (BioRad). In the development DAB (Sigma) was used as an insoluble substrate. It could be evidenced that the recombinant protein of the mentioned size was adequately identified by the specific antiserum.
  • a polyclonal antibody specific for the anti-CEA scFv obtained in rabbit was used, followed by goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (Sigma) for the detection of the binding of the fusion protein to CEA. After several washes, OPD (Sigma) and H 2 O 2 were used as chromogen and substrate for the development of the reactions, and a LabSystems Multiskan MS reader for the quantitative evaluation of them at 492 nm. In the test, the anti-CEA scFv was used as a positive control.
  • Plates were also coated with the following irrelevant antigens: 10 ⁇ g / mL bovine serum albumin (BSA, Sigma), 10 ⁇ g / mL ovalbumin, 10 ⁇ g / mL lysozyme, 10 ⁇ g / mL keyhole limpet haemocyanin (Sigma). Wells were included in all plates where only phosphate buffered saline was placed (PBS) without antigen (white). Absorbance values at least 4 times higher than those produced by the negative controls were considered positive.
  • BSA bovine serum albumin
  • PBS phosphate buffered saline
  • the recombinant bacteria obtained as described above were centrifuged and the periplasmic content was isolated by osmotic shock and brief sonication, and subsequently dialyzed for 72 hours in the coupling buffer (20 mM Tris-HCI, 1 M NaCI, 2OmM Imidazole, pH 7.0).
  • the bacterial periplasm preparations containing the scFv-sMATNI fusion protein were applied directly and independently to a Sepharose-IDA-Ni +2 matrix (lnVltrogen).
  • the gels were washed first with 10 times their volume using coupling buffer and then similarly with the wash buffer (20 mM Tris-HCI, 1 M NaCI, 150 mM Imidazole, pH 7.0) for Ia removal of contaminating proteins from E. coli. Elution of scFv-sMATN1 was performed with 2OmM Tris-HCI, 1 M NaCI, 250 mM Imidazole, pH 7.0).
  • the samples of the elution peaks were subjected to a denaturing 12% SDS-PAGE to confirm the presence of the protein of interest of approximately 33 kDa, and then analyzed by molecular sieve chromatography to determine the presence of multimers.
  • the purified anti-CEA scFv was used in a manner similar to that already described.
  • Superdex 200 Pulcoa
  • a conventional gel filtration process using an HPLC kit were used.
  • the column was first properly calibrated, with a set of proteins of known molecular weight.
  • the anti-CEA scFv was detected in a peak majority of approximately 27kDa and with a purity greater than 95%, all corresponding to a monomeric form.
  • the scFv-sMATN1 fusion protein appeared mostly with a size of approximately 9OkDa, which corresponds to an oligomeric form, very possibly trimeric according to its molecular weight. Its purity was also very high.
  • the absorbance was read at 492 nm in a LabSystems Multiskan MS device.
  • the values of optical density (OD) for each case were plotted on the axis of the ordinates (y), reflecting on the axis of the abscissa (x) the concentration expressed in ng / mL on a base 10 logarithm scale.
  • the DO 100 as the one to which the signal was kept to the maximum.
  • half of the DO 100 (DO 50) was calculated.
  • [Ab '] t is the concentration value of the sample corresponding to a value of OD 50 for the highest concentration of antigen to be compared and [Ab] t is the concentration value of the sample corresponding to an OD value 50 for the lower concentration of antigen to compare.
  • the six possible affinity determinations were made for the curves obtained, estimating the final Kaff as the average of these. Table II Affinity Constant Values
  • Kaff affinity constant, calculated as an average of six determinations ⁇ the standard deviation (in brackets).
  • Table Il reflects the Kaff values calculated for each of the variants tested.
  • the scFv-sMATN1 protein has a Kaff more than an order and a half of magnitude greater than that obtained for the anti-CEA scFv, which is only explained by arguing that the first molecule has increased its greed for the antigen by its new oligomeric character already identified by gel filtration.
  • EXAMPLE 3 Study of the specific activity of the hybrid proteins obtained by recombinant route bound to the MATN.
  • EXAMPLE 4 Study of the immunogenicity of hybrid proteins obtained by recombinant route linked to MATN1.
  • the procedure used for the determination of antibodies in the serum of treated patients consisted of a "Sandwich" ELISA.
  • the antibodies against the evaluated proteins contained in the samples bind in a first time to the protein for which the antibodies coupled to the solid phase are evaluated, the protein A coupled to the peroxidase enzyme recognizes the antibodies in a second time, allowing it acquiring the ability to transform a specific substrate that is indicative of the presence or not of such antibodies in the sample.
  • mice were immunized between 16 and 18 grams of weight and with an age between 30-38 weeks with each of the protein preparations described above, as well as with their counterparts without MATN 1:
  • the subcutaneous route was used for Immunization (Applied Biotechnology 2002; 19 (1): 15-18).
  • 1 microgram of recombinant protein or recombinant protein-MATN1 was administered to the mice for each immunization 3 times per week.
  • the sacrifices were always made after at least 3 days between the last administration and the blood collection. This was done with the objective of allowing the necessary time for the plasma clearance of the proteins, preventing them from being bound to the antibodies to be detected and masking them in the determination by the ELISA system.
  • mice were kept under controlled conditions of temperature (19 ⁇ 20 0 C) and humidity (55-65%), with ingestion of water and pelletized feed ab libitum. They were 15 days in acclimatization and subsequently distributed in 10 in boxes with the appropriate size.
  • the blood extractions were performed using the ocular dissection method, a simple and rapid final extraction method that allows a high volume of blood to be obtained before the coagulation of the same.
  • ELISAS of all samples collected from animals were performed according to the experimental design. In each time the mean ⁇ the interquartile range is shown of the logarithms of antibody titers. In the first week no antibodies are detected. No statistically significant differences were detected between recombinant protein variants and their counterparts with MATN 1 using the Mann-Whitney U test (see table V).
  • Table V Compared immunogenicity between recombinant protein variants and their homologs with MATN1.

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  • Oncology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des molécules chimériques fusionnées au peptide correspondant à l'extrémité carboxyle de la Matriline-1 humaine moyennant un espaceur flexible qui leur confère une durée de vie moyenne supérieure dans le sang et une activité spécifique supérieure par rapport aux molécules non fusionnées.
PCT/CU2007/000012 2006-03-29 2007-03-29 Molécules chimériques fusionnées à la matriline-1, à usage thérapeutique Ceased WO2007110001A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CU20060073 2006-03-29
CU2006-0073 2006-03-29

Publications (2)

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WO2007110001A2 true WO2007110001A2 (fr) 2007-10-04
WO2007110001A3 WO2007110001A3 (fr) 2007-11-15

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PCT/CU2007/000012 Ceased WO2007110001A2 (fr) 2006-03-29 2007-03-29 Molécules chimériques fusionnées à la matriline-1, à usage thérapeutique

Country Status (2)

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AR (1) AR060795A1 (fr)
WO (1) WO2007110001A2 (fr)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0635029A4 (fr) * 1992-04-10 1996-01-24 Gen Hospital Corp Proteine de matrice de cartilage et procedes d'utilisation.
US6900292B2 (en) * 2001-08-17 2005-05-31 Lee-Hwei K. Sun Fc fusion proteins of human erythropoietin with increased biological activities
CU23228A1 (en) * 2002-04-29 2007-09-26 Ct Ingenieria Genetica Biotech FRAGMENTS OF SPECIFIC ANTIBODIES FOR THE HUMAN CARCINOEMBRIONARY ANTIGEN (CEA) SEQUENCES OF ITS VARIABLE REGIONS AND VECTORS FOR THE MICROBIAL EXPRESSION OF THE SAME
US7081443B2 (en) * 2002-05-21 2006-07-25 Korea Advanced Institutes Of Science And Technology (Kaist) Chimeric comp-ang1 molecule
FR2856070A1 (fr) * 2003-06-13 2004-12-17 Bionexis Molecules pour le ciblage et l'assemblage in situ de proteines therapeutiques et leur utilisation

Also Published As

Publication number Publication date
AR060795A1 (es) 2008-07-16
WO2007110001A3 (fr) 2007-11-15

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