ES2549128T3 - Proteínas de fusión, usos de las mismas y procesos para producir las mismas - Google Patents

Proteínas de fusión, usos de las mismas y procesos para producir las mismas Download PDF

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ES2549128T3
ES2549128T3 ES07777164.0T ES07777164T ES2549128T3 ES 2549128 T3 ES2549128 T3 ES 2549128T3 ES 07777164 T ES07777164 T ES 07777164T ES 2549128 T3 ES2549128 T3 ES 2549128T3
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amino acids
consecutive amino
peptide linker
peptide
scfv fragment
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Yoram Reiter
Roy Noy
Kfir Oved
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Technion Research and Development Foundation Ltd
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Wood Science & Technology (AREA)
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  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract

Una proteína de fusión que comprende aminoácidos consecutivos que, comenzando en el extremo amino de la proteína, corresponden a aminoácidos consecutivos presentes en (i) un péptido restringido por CMH humano de citomegalovirus, (ii) un primer enlazador peptídico, (iii) una b-2 microglobulina humana, (iv) un segundo enlazador peptídico, (v) una cadena HLA-A2 de una molécula CMH de clase I humana, (vi) un tercer enlazador peptídico, (vii) una región variable de una cadena pesada de un fragmento scFv de un anticuerpo, y (viii) una región variable de una cadena ligera de tal fragmento scFv, en la que los aminoácidos consecutivos que corresponden a (vii) y (viii) se unen juntos directamente por un enlace peptídico o por aminoácidos consecutivos que corresponden a un cuarto enlazador peptídico, y el fragmento scFv se obtiene a partir de un anticuerpo que se une específicamente a mesotelina, en el que los aminoácidos consecutivos tienen la secuencia aminoacídica expuesta en la SEQ ID NO: 2.

Description

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descritos en el presente documento. Estos ejemplos no se deberán considerar como limitantes en ningún modo, ya que la invención se ponerse en práctica en formas similares, aún un poco diferentes. Sin embargo, estos ejemplos muestran a un experto en la técnica cómo poner en práctica diversas alternativas y realizaciones de la invención.
Generalmente, la nomenclatura usada en el presente documento y los procedimientos de laboratorio utilizados en la presente invención incluyen técnicas moleculares, bioquímicas, microbiológicas y de ADN recombinante. Dichas técnicas se explican completamente en la bibliografía. Véase, por ejemplo, "Molecular Cloning: A laboratory Manual" Sambrook y col., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel y col., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, Nueva York (1988); Watson y col., "Recombinant DNA", Scientific American Books, Nueva York; Birren y col. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, Nueva York (1998); metodologías como se expone en las Pat. de Estados Unidos Nº 4.666.828; 4.683.202; 4.801.531; 5.192.659 y 5.272.057; "Cell Biology: A Laboratory Handbook", Volúmenes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells -A Manual of Basic Technique" de Freshney, wiley-Liss, N. Y. (1994), Tercera Edición; "Current Protocols in Immunology" Volúmenes I-III Coligan J. E., ed. (1994); Stites y col. (eds), "Basic and Clinical Immunology" (8ª Edición), Appleton & Lange, Norwalk, CT (1994); Mishell y Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman y Co., Nueva York (1980); los inmunoensayos disponibles se describen extensamente en la bibliografía de patentes y científica, véase, por ejemplo, las Pat. de Estados Unidos Nº 3.791.932; 3.839.153; 3.850.752; 3.850.578; 3.853.987; 3.867.517; 3.879.262; 3.1901.654; 3.935.074; 3.984.533; 3.996.345; 4.034.074; 4.098.876; 4.879.219; 5.011.771 y 5.281.521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., y Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., y Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) y "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak y col., "Strategies for Protein Purification and Characterization -A Laboratory Course Manual" CSHL Press (1996); a lo largo de este documento se proporcionan otras referencias generales. Se cree que los procedimientos en el presente documento se conocen bien en la técnica y se proporcionan para la comodidad del lector.
Detalles Experimentales
Materiales y Métodos:
Clonación del Compuesto A
El scHLA-A2/SS1 (scFv) se construyó como se ha descrito previamente enlazando el extremo C de scHLA-A2 al extremo N del SS1 scFv a través de un enlazador corto ASGG (SEC ID NO: 4) (15). Para construir el scHLA-A2/SS1 (scFv) con un péptido restringido por CMH unido covalentemente, el péptido restringido por CMH se fusionó con el péptido enlazador GGGGSGGGGSGGGGSGGGGS (SEC ID NO: 6) al extremo N de la molécula de scHLA-A2/SS1 (scFv) por una reacción de extensión de solapamiento de PCR con los cebadores: 5'M1-5'GGAAGCGTTGGCGCATATGGGCATTCTGGGCTTCGTGTTTACC CTGGGCGGAGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGA TCCAGCGTACTCCAAAG3' (SEQ ID NO: 13) y 3'VLscSS1-5'GCAGTAAGGAATTCTCATTATTTTATTTCCAACTTTGT3' (SEC ID NO: 14). En el cebador 5'M1 se insertó una mutación silenciosa en la secuencia enlazadora, este cambio en la secuencia crea un sitio de restricción BamH1.
Los productos de PCR se subclonaron a un vector de clonación TA (pGEM-T Easy Vector, Promega™ Corporation, Madison, WI, Estados Unidos) y posteriormente a un vector de expresión basado en el promotor T7 (PRB) usando los sitios de restricción NdeI y EcoRI.
Para generar el Compuesto A, se usó M1/scHLA-A2/SS1 (scFv) como un modelo para la ligación con el cebador dsADN. El M1/scHLA-A2/SS1 (scFv) (en plásmido PRB) se digirió con NdeI y BamH1, y la fracción plasmídica se ligó al cebador dsADN que contenía la secuencia peptídica CMV y la extensión de la secuencia enlazadora 5'CMVcovLL (casete) : 5'TATGAACCTGGTGCCGATGGTCGCGACCGT TGGAGGTGGCGGTTCTGGCGGAGGAG3' (SEQ ID NO: 15) y 3'CMVcovLL (casete): 5'GATC CTCCTCCGCCAGAACCGCCACCTCCAACGGTCGCGACCATCGGCACCAGGTTCA3'(SEQ ID NO-16). El apareamiento de los cebadores (5'CMVcovLL (casete) y 3'CMVcovLL (casete)) se realizó incubando los cebadores a 95 ºC durante 2 min seguido de 1 h de incubación a temperatura ambiente. El producto de ligación se transformó a E-coli DH5α para amplificación plasmídica. El plásmido se purificó por el kit QIAGEN® miniprep (Qiagen®, Inc.,
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Claims (1)

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ES07777164.0T 2006-05-19 2007-05-17 Proteínas de fusión, usos de las mismas y procesos para producir las mismas Active ES2549128T3 (es)

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