WO2007093663A1 - Method for obtaining anti-hdc antibodies and applications of same - Google Patents
Method for obtaining anti-hdc antibodies and applications of same Download PDFInfo
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- WO2007093663A1 WO2007093663A1 PCT/ES2007/070032 ES2007070032W WO2007093663A1 WO 2007093663 A1 WO2007093663 A1 WO 2007093663A1 ES 2007070032 W ES2007070032 W ES 2007070032W WO 2007093663 A1 WO2007093663 A1 WO 2007093663A1
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- antibodies
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
-
- 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
Definitions
- the present invention provides a method for obtaining specific antibodies against human histidine decarboxylase (hHDC), as well as the polynucleotides and polypeptides necessary to carry it out.
- hHDC histidine decarboxylase
- the uses of the aforementioned antibodies, as well as the diagnostic kits of which they are part, are also the subject of the present invention.
- HDC histidine decarboxylase
- HDC especially human HDC (hHDC)
- hHDC human HDC
- the only possibility that exists to structurally characterize the enzyme is based on generating three-dimensional models through bioinformatic approaches (Moya-Garcia et al. BioEssays (2005) 27: 57-63).
- the native protein was not obtained from mammalian tissues until 1984, (Taguchi et al. (1984) J. Biochem, 259: 5214-5221), when it was partially purified after eight purification steps obtaining a total of 940 micrograms of HDC from 400 g of fetal rat liver.
- isolated cDNAs encoding HDC give rise to a very unstable primary peptide between 74 and 77 KDa, which is present in a very minor percentage in histamine producing cells.
- This primary peptide undergoes a partial proteolysis process, from which the 53-57 KDa native HDC monomer that is part of the active protein is generated.
- Smaller peptides that are usually detected in mammalian cell extracts, are products of the digestion of the active enzyme that has a very short half-life (2-4 hours) and is a substrate of different proteolytic systems (Moya-Garc ⁇ a et al. (2005) BioEssays 27: 57-63).
- 270 51): 30813-30817) developed monoclonal antibodies (AcM) against hHDC using as an immunogen a peptide conserved in the HDC sequences of human (residues 218-232), mouse (residues 225-239), and rat (residues 221-235). Said AcM only recognized HDC in a denatured state.
- Antibody throughout the description, this term will refer to both monoclonal and polyclonal antibodies.
- Fragments of the polynucleotide throughout the description, this term will refer to all those fragments of SEQ ID 1 capable of encoding a polypeptide for the generation of specific antibodies against hHDC.
- Fragments of the polypeptide throughout the description this term will refer to all those fragments of SEQ ID 2 capable of producing specific antibodies against hHDC.
- Antibody fragments throughout the description, this term will refer to all those antibody fragments capable of interacting with SEQ ID 2 and fragments of the same, where said fragments comprise the variable regions Vh and / or Vl.
- the hHDC sequence has been known for a decade, and as a result of the studies carried out to carry out the present invention, it has been possible to deduce polypeptide fragments that the enzyme has to conserve in order to maintain its activity; This fact has allowed us to perform bioinformatic analyzes of comparison between the coding sequences of rat and human HDC, and to predict which epitopes of the active protein had the highest immunogenic potential to develop antibodies against hHDC.
- polypeptide consisting of amino acid residues 492-506 (SEQ ID 2) of hHDC, encoded by the polynucleotide of SEQ ID 1, was chosen as an immunogen, and a method for obtaining anti-hHDC antibodies was developed and / or fragments thereof that react against the selected polypeptide.
- a first aspect of the invention relates to a polynucleotide, hereinafter "polynucleotide of the invention", capable of encoding a polypeptide capable of specific antibodies against hHDC, where the sequence of said polynucleotide is chosen from the group: a) Sequence comprising SEQ ID 1, b) Sequence consisting of SEQ ID 1 or comprising fragments thereof, c) Sequence that differs from any of the a or b sequences due to the degeneracy of the genetic code, od) Sequences that share at least 80%, 90%, 95% or 98% homology with a, bo c.
- a second aspect of the invention relates to a polypeptide, hereinafter "polypeptide of the invention", capable of generating specific antibodies against hHDC, where the sequence of said polypeptide is chosen from the group: a) Sequence comprising SEQ ID 2, b) Sequence consisting of SEQ ID 2 or comprising fragments thereof, c) Sequence that shares at least 80%, 90%, 95% or 98% homology with the sequence a or b.
- a third aspect of the invention relates to a method, hereinafter "method of the invention", for obtaining antibodies and / or fragments thereof against hHDC, which comprises the use of the polypeptide of the invention and / or fragments of the same.
- Said polypeptide and its fragments can be obtained by chemical synthesis and / or bacterial transformation with the polynucleotide of the invention or fragments thereof.
- said transformation comprises the binding by recombinant DNA techniques of the polynucleotide of the invention or fragments thereof to vectors that comprise but are not limited to plasmids, cosmids, lambda phage derivatives, phagemids, among others.
- the method of obtaining antibodies and / or fragments thereof is based on techniques that They include but are not limited to: immunization techniques for non-human mammals, hybridoma generation by cell fusion, "phage display” methods, etc.
- it comprises the use of the polypeptide of the invention or fragments thereof in combination with at least one carrier protein, which comprises but is not limited to chicken egg albumin (OVA), albumin. bovine serum (BSA), hemocyanin of the California limpet Megatura cranulatus (KLH, Pierce), etc.
- carrier protein comprises but is not limited to chicken egg albumin (OVA), albumin. bovine serum (BSA), hemocyanin of the California limpet Megatura cranulatus (KLH, Pierce), etc.
- a fourth aspect of the invention relates to antibodies, hereinafter "antibodies of the invention", and / or fragments thereof specific against hHDC that interact specifically with any of the polypeptides of the invention or fragments thereof.
- said antibody fragments comprise the variable regions Vc and / or Vl.
- a fifth aspect of the invention is related to the use of the antibodies of the invention and / or fragments thereof for in vitro and / or in vivo diagnosis.
- a sixth aspect of the invention is related to the use of the antibodies of the invention and / or fragments thereof for the manufacture of a medicament for use in therapy.
- Said medication It can be used for the treatment of diseases, which include but are not limited to pathologies related to neurological degeneration (Alzheimer's, Parkinson's, memory disorders), anaphylaxis and allergic processes, degeneration of digestive epithelia (gastric ulcer, Crohn's syndrome, etc.), infectious processes (salmonellosis, campylobacteriosis, enterotoxemia, etc.), different types of cancer (colon cancer, myeloma, melanoma, blastocytic and mastocytic leukemia, lymphoma, etc), etc.
- diseases include but are not limited to pathologies related to neurological degeneration (Alzheimer's, Parkinson's, memory disorders), anaphylaxis and allergic processes, degeneration of digestive epithelia (gastric ulcer, Crohn's syndrome, etc.), infectious processes (
- a seventh aspect of the invention relates to an analysis kit comprising the "antibodies of the invention" and / or fragments thereof.
- the kit can include all those reagents necessary to carry out the set-up of the kit, this includes, without any limitation, the use of buffers, agents to prevent contamination, etc.
- the kit can also include all the supports and containers necessary for commissioning and optimization.
- FIG. 1 Immunoblot ("Western blot") with the antibodies of the invention.
- lanes 1 the detection of purified GST-l / 152hHDC is shown and in lanes 2 the detection of extracts of HMC-1 cells.
- Lanes 3 corresponds to the pattern of molecular weights.
- panel A the development was carried out with a rabbit anti-factor H monoclonal antibody (IgG2a), which is used as a negative control of the assay, with no signal showing interaction of the protein with the factor H.
- Panel B is revealed with the AcM antibody SIM 216-12.1
- FIG. 1 Immunoblot (“Western blot") showing the detection of immunoprecipitates of the basophilic leukemic lines HMC-I and KU-812F producing hHDC. Immunorecognition was performed with the monoclonal antibody SIM 216-12.1. In both streets the detection of hHDC is shown, observing 3 major bands. The largest of the bands corresponds to the expected size for the mature hHDC monomer and the lower two with the degraded forms of the protein.
- FIG. 3 Western blot of HEK-293 cell extracts transiently transfected with pcDNA3-l / 512hHDC (lane 1), pcDNA3-l / 512 (lane 2) or pEGFP (lane 3, negative control).
- the hHDC detection for the different cell extracts is carried out with AcM SIM 216-12.1, with clearly differentiated bands in lanes 1 and 2 corresponding to the antibody-hHDC interaction.
- the upper bands correspond with the expected size for the mature hHDC monomer and those smaller than its degraded forms.
- FIG. 4 Immuno-monitoring of hHDC in HEK-293 cells transfected with pcDNA3-1 / 512hHDC, which expressly express said protein, using AcM SIM 216-12.1.
- Panel A shows the non-emission of fluorescence by non-transfected cells and panel B shows the emission of fluorescence from the cytoplasm of transfected cells.
- the peptide chosen as an immunogen (SEQ ID 2) comprising amino acids 492 to 506 of hHDC is coupled by the cross-linking reagent, sulfo-SMCC (SuIfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1- carboxylate; Pierce, No 22322), to a carrier protein, preferably OVA, following the protocol indicated by the manufacturer.
- SEQ ID 2 The cross-linking reagent, sulfo-SMCC (SuIfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1- carboxylate; Pierce, No 22322)
- non-human mammals are chosen, preferably mice and more preferably females of the BALB / c strain.
- Non-human mammals are immunized intraperitoneally to obtain polyclonal or monoclonal anti-hHDC antibodies.
- One week after the second immunization bleeding is performed to determine the titre and reactivity of antibodies present in the mouse serum. This determination can be carried out by different techniques well known in the state of the art, such as indirect ELISA ("Enzyme Linked Immuno Sorbent Asssay”) immunoassay against the peptide of the invention conjugated to another protein other than OVA. From these tests, the mouse with a serum of greater antibody titre is chosen as a donor of B lymphocytes, for obtaining monoclonal antibodies, or for obtaining polyclonal antibodies from the serum.
- indirect ELISA Enzyme Linked Immuno Sorbent Asssay
- B lymphocytes are fused with myeloma cells, preferably Sp2 / 0-Ag-14 myeloma cells according to the procedures described in Galfré and Milstein (1981) Method Enzymol 73: 3-47 and J. Goding (1980) J. Immuno1 . Methods 30: 285-308, in order to obtain hybrids producing anti-hHDC antibodies.
- hybridomas producing the antibodies of the invention is carried out by different techniques.
- the techniques chosen for the realization of this selection are: a) ELISA, using the conjugate of the "peptide of the invention” with BSA as an antigen, b) cytometry of flow with cells of the HMC-I line, and c) Western blotting with HMC-I cell extracts or the GTS-l / 512hHDC fusion protein.
- the monoclonal antibody secreting hybridomas, in order to stabilize them are cloned twice by limit dilution in liquid medium, the isotype is determined, and stored in liquid nitrogen.
- a recombinant plasmid was obtained by fusing the 512 residues of the hHDC protein, which we will call 1 / 512hHDC, with a vector - preferably the Pharmacia pGEX vector - which allows mass production of the 512 amino acid recombinant fragment of the hHDC;
- the fusion protein expressed from said plasmid will be called GTS-1 / 512hHDC.
- the procedures for extraction, manipulation, in vitro recombination of DNA and bacterial transformation for the mass obtaining of the recombinant product are described in the Spanish patent application 9800019, using HMC-I basophilic line cells as the main source of hHDC messenger RNA. . From cultures of E.
- the isolated GTS-l / 512hHDC protein and a cellular extract of HMC-I cells expressing hHDC were subjected to denaturing electrophoresis (SDS / PAGE) followed by transfer to nitrocellulose and "Western blot" analysis with the antibodies of the invention and with a nonspecific antibody that does not recognize hHDC and serves as a negative control (FIG 1).
- lysis buffer II 20 mM Tris composition, 137 mM NaCl, 10% glycerol, 1% Nonidet P40 and 2mM EDTA.
- anti-GST-l / 187hHDC a rabbit antiserum
- GST-l / 187hHDC the fusion protein isolated from the band of an SDS-PAGE gel.
- lysis buffer I 50 mM Hepes composition, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20 , Glycerol 10%, pH 7.5; after incubation, the spheres are washed three times with lysis buffer I without BSA.
- a microlite of each of the HMC-I and KU-182F cell extracts (equivalent to 9 million cells) is incubated for 3 hours at 4 ° C with the spheres of Protein A-Sepharose that have the anti-GST-l / 187hHDC antibody bound. After successive washing, the spheres are collected by centrifugation and eluted with buffer containing 2.3% SDS; Denatured eluates are separated by SDS-PAGE gel electrophoresis followed by electrotransfer to nylon filters.
- Said filters are incubated with the AcM SIM 216-12.1 (1/2500 dilution) and the complexes formed by the AcM and the hHDC are located by a chemiluminescence reaction by incubating the filter with peroxidase-conjugated mouse antiimunoglobulin (FIG 2) .
- FOG 2 peroxidase-conjugated mouse antiimunoglobulin
- HEK-293 cells are transfected with the expression plasmid pcDNA 3 (Invitrogen) containing the sequence l / 512hHDC inserted under the control of the cytomegalovirus (CMV) promoter. Transfection is facilitated using the FuGENE (Roche) system as well as different amounts of expression plasmids (FIG. 3: 1.5 micrograms, lane 1, and 1 microgram, lane 2).
- FOG. 3 1.5 micrograms, lane 1, and 1 microgram, lane 2
- the AcM SIM 216-12.1 is incubated with the extract of transfected HEK-293 cells, on the third day of transfection.
- the precipitates formed are separated by electrophoresis and transferred to nylon filters.
- Detection is carried out using as primary antibody AcM SIM 216-12.1 (dilution 1/2500), and as secondary antibody an anti-mouse antibody conjugated to peroxidase (dilution 1/5000).
- the position and abundance of the bands is visualized by chemiluminescence (FIG 3).
- lane 1 which corresponds to the cell extract of cells transfected with the greatest amount of plasmid, a more intense main reaction band is observed, corresponding to the Mr planned for the hHDC protein, and another smaller band that probably represents degradation products of said protein.
- HEK-293 cells are transiently transfected with 1.5 micrograms of plasmid pcDNA3-hHDCl / 512 as described in the previous example, to transiently express the protein l / 152hHDC protein from the cells
- HEK-293 The cells are incubated in 24-well plates on polylysine coated object covers for 8 hours. After this time, the culture medium is removed and the cells are fixed with 3.7% formaldehyde for 30 minutes on ice. The cells are then washed with an isotonic phosphate-saline solution (PBS) and permeabilized with PBS / Triton X-IOO 0.2% for 5 minutes on ice. The preparation is blocked for 90 min at 4 ° C with
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Abstract
The invention relates to a polynucleotide that can encode a polypeptide capable of generating antibodies, or specific fragments thereof, against HDC, having a sequence that is selected from group: a. a sequence comprising SEQ ID 1; b. a sequence comprising SEQ ID 1 or fragments of same; c. a sequence that differs from sequences a and b owing to the degeneration of the genetic code; and d. a sequence with at least 80%, 90%, 95% or 98% homology with any of the aforementioned sequences.
Description
MÉTODO DE OBTENCIÓN DE ANTICUERPOS ANTI-hHDC Y APLICACIONES DE LOS MISMOSMETHOD OF OBTAINING ANTI-hHDC ANTIBODIES AND APPLICATIONS OF THE SAME
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La presente invención proporciona un método para la obtención de anticuerpos especificos frente a la histidina descarboxilasa humana (hHDC) , asi como los polinucleótidos y polipéptidos necesarios para llevarlo a cabo. Del mismo modo, los usos de los mencionados anticuerpos, asi como los kits de diagnostico de los que formen parte también son objeto de la presente invención .The present invention provides a method for obtaining specific antibodies against human histidine decarboxylase (hHDC), as well as the polynucleotides and polypeptides necessary to carry it out. Similarly, the uses of the aforementioned antibodies, as well as the diagnostic kits of which they are part, are also the subject of the present invention.
ESTADO DE LA TÉCNICASTATE OF THE TECHNIQUE
En las células de mamiferos, la forma activa de la histidina descarboxilasa (HDC) (E. C. 4.1.1.22, según la clasificación de la Unión Internacional de Bioquimica) es una proteina homodimérica de aproximadamente 110 kDa, muy minoritaria (menos de 0.001% del contenido proteico celular) , cuya actividad depende de piridoxal- fosfato o PLP. La HDC cataliza el paso de L-histidina a histamina mediante la descarboxilación de la primera, y se expresa con actividad apreciable en un reducido grupo de tipos celulares: algunas células del sistema nervioso central y periférico, algunos leucocitos basófilos, mastocitos, macrófagos -, epitelios digestivos, y algunos tipos de neuronas y células cancerosas desdiferenciadas.
Estudios recientes con ratones que tienen anulada la expresión de la histidina descarboxilasa (ratones knock-out) (Hiroshi Ohtsu et al. 2003, Biochemical and Biophysical Research Communications 305 (2003) 443-447 New functions of histamine found in histidine decarboxylase) indican que la histamina está implicada en procesos tales como la maduración de los mastocitos, la reabsorción de la medula ósea, la transmisión sináptica de los tipos neuronales, y la progresión e invasión de tejidos cancerosos (Schneider et al (2002) Trends Immunol . 23: 255-263; Ohtsu & Watanebe (2002) Biochem. Biophys . Res. Común. 305: 443-447; Fitzpartick et al. (2003) Proc. Nati. Acad. Sci. USA 100: 6027- 6032) . La HDC está, por lo tanto, implicada en multitud de procesos inflamatorios, cancerosos y en diversas neuropatias .In mammalian cells, the active form of histidine decarboxylase (HDC) (EC 4.1.1.22, according to the International Biochemistry Union classification) is a homodimeric protein of approximately 110 kDa, very minor (less than 0.001% of the content cellular protein), whose activity depends on pyridoxalphosphate or PLP. HDC catalyzes the passage of L-histidine to histamine by decarboxylation of the first, and is expressed with appreciable activity in a small group of cell types: some cells of the central and peripheral nervous system, some basophilic leukocytes, mast cells, macrophages -, digestive epithelia, and some types of neurons and dedifferentiated cancer cells. Recent studies with mice that have the histidine decarboxylase expression (knock-out mice) voided (Hiroshi Ohtsu et al. 2003, Biochemical and Biophysical Research Communications 305 (2003) 443-447 New functions of histamine found in histidine decarboxylase) indicate that Histamine is involved in processes such as mast cell maturation, bone marrow resorption, synaptic transmission of neuronal types, and progression and invasion of cancerous tissues (Schneider et al (2002) Trends Immunol. 23: 255 -263; Ohtsu & Watanebe (2002) Biochem. Biophys. Res. Common. 305: 443-447; Fitzpartick et al. (2003) Proc. Nati. Acad. Sci. USA 100: 6027-6032). HDC is, therefore, involved in a multitude of inflammatory, cancerous processes and in various neuropathies.
Es bien sabido que la HDC, especialmente la HDC humana (hHDC) , muestra una gran inestabilidad tanto in vivo como en extractos libres de células, lo cual dificulta considerablemente su purificación y caracterización. Hasta el momento, la única posibilidad que existe para caracterizar estructuralmente la enzima se basa en generar modelos tridimensionales mediante aproximaciones bioinformáticas (Moya-Garcia et al. BioEssays (2005) 27: 57-63) . La proteina nativa no se obtuvo a partir de tejidos de mamiferos hasta 1984, (Taguchi et al. (1984) J. Biochem, 259: 5214-5221), cuando se purificó parcialmente tras ocho pasos de purificación
obteniéndose un total de 940 microgramos de HDC a partir de 400 g de higado fetal de rata.It is well known that HDC, especially human HDC (hHDC), shows great instability both in vivo and in cell-free extracts, which makes its purification and characterization considerably difficult. So far, the only possibility that exists to structurally characterize the enzyme is based on generating three-dimensional models through bioinformatic approaches (Moya-Garcia et al. BioEssays (2005) 27: 57-63). The native protein was not obtained from mammalian tissues until 1984, (Taguchi et al. (1984) J. Biochem, 259: 5214-5221), when it was partially purified after eight purification steps obtaining a total of 940 micrograms of HDC from 400 g of fetal rat liver.
Años mas tarde, Ohmori y colaboradores (Ohmori et al. (1990) J. Biochem. 107: 834-839) purificaron a homogeneidad la enzima HDC de ratón a partir de 9 x 1010 células mastociticas de ratón P-815 recogidas del liquido ascitico de 250 ratones previamente inoculados con 4,5 millones de células/ratón. En este caso, y tras nueve pasos de purificación, sólo se obtuvieron 15 microgramos de HDC.Years later, Ohmori et al. (Ohmori et al. (1990) J. Biochem. 107: 834-839) homogeneously purified the mouse HDC enzyme from 9 x 10 10 P-815 mouse mastocytic cells collected from the liquid ascites of 250 mice previously inoculated with 4.5 million cells / mouse. In this case, and after nine purification steps, only 15 micrograms of HDC were obtained.
En 1991, Zahnow y colaboradores (Zahnow et al. (1991) DNA Seq. 1: 395-400) obtuvieron la secuencia del mensajero de la hHDC, y en 1994, Yatsunami y colaboradores (Yatsunami et al. (1994) J. Biol . Chem. 269: 1554-1559) aislaron 24 Kb del gen humano localizado en el cromosoma 15.In 1991, Zahnow et al. (Zahnow et al. (1991) DNA Seq. 1: 395-400) obtained the hHDC messenger sequence, and in 1994, Yatsunami et al. (Yatsunami et al. (1994) J. Biol Chem. 269: 1554-1559) isolated 24 Kb of the human gene located on chromosome 15.
En mamiferos, los cDNAs aislados que codifican para HDC dan lugar a un péptido primario de entre 74 y 77 KDa muy inestable, que esta presente en un porcentaje muy minoritario en las células productoras de histamina. Este péptido primario sufre un proceso de proteólisis parcial, a partir del cual se genera el monómero nativo de HDC de 53-57 KDa que forma parte de la proteina activa. Péptidos de menor tamaño que suelen detectarse en extractos de células de mamiferos, son productos de la digestión de la enzima activa que posee una vida media muy corta (2-4 horas) y es sustrato de distintos
sistemas proteolíticos (Moya-García et al. (2005) BioEssays 27: 57-63).In mammals, isolated cDNAs encoding HDC give rise to a very unstable primary peptide between 74 and 77 KDa, which is present in a very minor percentage in histamine producing cells. This primary peptide undergoes a partial proteolysis process, from which the 53-57 KDa native HDC monomer that is part of the active protein is generated. Smaller peptides that are usually detected in mammalian cell extracts, are products of the digestion of the active enzyme that has a very short half-life (2-4 hours) and is a substrate of different proteolytic systems (Moya-García et al. (2005) BioEssays 27: 57-63).
Se sabe que el producto primario de la traducción de la HDC no es activo, ya que el extremo carboxilo impide la recepción del sustrato, por lo que la enzima debe perder, al menos, 5-10 KDa de su extremo carboxilo para poder ser activa. Con versiones recombinantes del enzima purificada, la máxima actividad se obtiene con HDC de mamíferos que conservan su secuencia primaria desde el residuo 69 al 515, y poseen un peso molecular aproximado de 50 KDa (Engel et al. (1996) Biochem. J. 320: 365-368; Olmo et al. (2000) Eur. J. Biochem. 267: 1527-1531; Fleming et al. (2004) Biochem. J. 381: 769- 778) .It is known that the primary product of the translation of the HDC is not active, since the carboxyl end prevents the reception of the substrate, so the enzyme must lose at least 5-10 KDa from its carboxyl end in order to be active . With recombinant versions of the purified enzyme, the maximum activity is obtained with HDC of mammals that retain their primary sequence from residue 69 to 515, and have an approximate molecular weight of 50 KDa (Engel et al. (1996) Biochem. J. 320 : 365-368; Olmo et al. (2000) Eur. J. Biochem. 267: 1527-1531; Fleming et al. (2004) Biochem. J. 381: 769-778).
A los inconvenientes derivados de las propiedades bioquímicas de HDC (inestabilidad, adhesión, etc.) hay que añadir que las células que expresan la enzima se encuentran generalmente dispersas y en minoría entre otros tipos celulares productores de histamina (médula ósea, sangre, epitelios, estómago, cerebro) . En el caso de la hHDC, se suman las dificultades de accesibilidad a tejidos frescos, por lo que la obtención de anticuerpos frente a dicha enzima se convierte en una empresa extremadamente ardua.To the disadvantages derived from the biochemical properties of HDC (instability, adhesion, etc.) it should be added that the cells that express the enzyme are generally dispersed and in minority among other histamine-producing cell types (bone marrow, blood, epithelia, stomach, brain) In the case of hHDC, the difficulties of accessibility to fresh tissues are added, so that obtaining antibodies against said enzyme becomes an extremely arduous company.
En la solicitud de patente española ES 2 135 350 se desarrolla un método para obtener anticuerpos anti-HDC frente a proteínas recombinantes, que reconocen la HDC
de ratón pero no lo hacen con la versión humana de la enzima .In Spanish patent application ES 2 135 350 a method is developed to obtain anti-HDC antibodies against recombinant proteins, which recognize HDC mouse but do not do it with the human version of the enzyme.
La solicitud de PCT de número de publicación WO- 98/30593, detalla la obtención de anticuerpos policlonales especificos contra hHDC que han sido utilizados en el desarrollo de ensayo de detección de cáncer; dicha publicación, detalla un método para obtener anticuerpos que reaccionan frente a regiones de hHDC distintas a las que son reconocidas por los anticuerpos cuya obtención describe la presente solicitud.The PCT application of publication number WO-98/30593, details the obtaining of specific polyclonal antibodies against hHDC that have been used in the development of cancer screening assay; said publication details a method for obtaining antibodies that react against regions of hHDC other than those recognized by the antibodies whose preparation describes the present application.
Por último, cabe mencionar que en 1995 Kimio Yatsunami y colaboradores (Yatsunami et al. (1995) J. Biol . Chem.Finally, it should be mentioned that in 1995 Kimio Yatsunami et al. (Yatsunami et al. (1995) J. Biol. Chem.
270 (51) : 30813-30817) desarrollaron anticuerpos monoclonales (AcM) frente a hHDC usando como inmunógeno un péptido conservado en las secuencias de HDC de humano (residuos 218-232), ratón (residuos 225-239), y rata (residuos 221-235) . Dicho AcM sólo reconocia HDC en estado desnaturalizado.270 (51): 30813-30817) developed monoclonal antibodies (AcM) against hHDC using as an immunogen a peptide conserved in the HDC sequences of human (residues 218-232), mouse (residues 225-239), and rat (residues 221-235). Said AcM only recognized HDC in a denatured state.
Los anticuerpos anti-HDC comerciales, en general, sólo detectan HDC de rata (ES 2 135 350, Dartsch et al. (1999) Histochem J. 31: 507-14), y si reconocen a la hHDC, lo hacen en su estado desnaturalizado. Por estas razones, es imprescindible poseer AcM que reconozcan a la hHDC en su conformación activa para estudiar al enzima y analizar su participación en procesos patológicos en los que pudiera estar implicada, tales
como degeneraciones neurológicas (esquizofrenia, trastornos de la memoria, etc.), procesos alérgicos, degeneración de epitelios digestivos (ulcera gástrica, sindrome de Crohn, etc) , algunos tipos de cáncer (cáncer de colon, leucemias blastociticas y mastocitica) , etc.Commercial anti-HDC antibodies, in general, only detect rat HDC (ES 2 135 350, Dartsch et al. (1999) Histochem J. 31: 507-14), and if they recognize hHDC, they do so in their state denatured. For these reasons, it is essential to have AcM that recognize the hHDC in its active conformation to study the enzyme and analyze its participation in pathological processes in which it could be involved, such such as neurological degenerations (schizophrenia, memory disorders, etc.), allergic processes, degeneration of digestive epithelia (gastric ulcer, Crohn's syndrome, etc.), some types of cancer (colon cancer, blastocytic and mastocytic leukemia), etc.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
Definiciones .Definitions .
Anticuerpo: a lo largo de la descripción, este término hará referencia tanto a anticuerpos monoclonales como policlonales .Antibody: throughout the description, this term will refer to both monoclonal and polyclonal antibodies.
Fragmentos del polinucleótido : a lo largo de la descripción, este término hará referencia todos aquellos fragmentos de SEQ ID 1 capaces de codificar para un polipéptido para la generación de anticuerpos especificos frente a hHDC .Fragments of the polynucleotide: throughout the description, this term will refer to all those fragments of SEQ ID 1 capable of encoding a polypeptide for the generation of specific antibodies against hHDC.
Fragmentos del polipéptido: a lo largo de la descripción este término, hará referencia todos aquellos fragmentos de SEQ ID 2 capaces de producir anticuerpos especificos frente a hHDC.Fragments of the polypeptide: throughout the description this term will refer to all those fragments of SEQ ID 2 capable of producing specific antibodies against hHDC.
Fragmentos de anticuerpos: a lo largo de la descripción, este término hará referencia a todos aquellos fragmentos de anticuerpos capaces interaccionar frente a SEQ ID 2 y fragmentos de la
misma, donde dichos fragmentos comprenden las regiones variables Vh y/o Vl.Antibody fragments: throughout the description, this term will refer to all those antibody fragments capable of interacting with SEQ ID 2 and fragments of the same, where said fragments comprise the variable regions Vh and / or Vl.
Como se ha indicado anteriormente, la secuencia de hHDC se conoce desde hace una década, y como resultado de los estudios realizados para llevar a cabo la presente invención, se han podido deducir fragmentos polipeptidicos que ha de conservar la enzima para mantener su actividad; este hecho ha permitido realizar análisis bioinformáticos de comparación entre las secuencias codificantes de HDC de rata y humana, y predecir qué epitopos de la proteina activa poseian mayor potencial inmunogénico para desarrollar anticuerpos frente a hHDC. A partir de este estudio se eligió como inmunógeno el polipéptido constituido por los residuos aminoacidicos 492-506 (SEQ ID 2) de hHDC, codificado por el polinucleótido de SEQ ID 1, y se desarrolló un método para la obtención de anticuerpos anti-hHDC y/o fragmentos de los mismos que reaccionan frente al polipéptido seleccionado.As indicated above, the hHDC sequence has been known for a decade, and as a result of the studies carried out to carry out the present invention, it has been possible to deduce polypeptide fragments that the enzyme has to conserve in order to maintain its activity; This fact has allowed us to perform bioinformatic analyzes of comparison between the coding sequences of rat and human HDC, and to predict which epitopes of the active protein had the highest immunogenic potential to develop antibodies against hHDC. From this study, the polypeptide consisting of amino acid residues 492-506 (SEQ ID 2) of hHDC, encoded by the polynucleotide of SEQ ID 1, was chosen as an immunogen, and a method for obtaining anti-hHDC antibodies was developed and / or fragments thereof that react against the selected polypeptide.
En este sentido, un primer aspecto de la invención se relaciona un polinucleótido, en adelante "polinucleótido de la invención", capaz de codificar un polipéptido capaz de anticuerpos especificos frente a hHDC, donde la secuencia de dicho polinucleótido es elegida del grupo: a) Secuencia que comprende SEQ ID 1, b) Secuencia que consiste en SEQ ID 1 o comprende fragmentos de ésta, c) Secuencia que difiera de cualquiera de las secuencias a o b debido a la
degeneración del código genético, o d) Secuencias que compartan al menos un 80%, 90%, 95% ó 98% de homologia con a, b o c.In this sense, a first aspect of the invention relates to a polynucleotide, hereinafter "polynucleotide of the invention", capable of encoding a polypeptide capable of specific antibodies against hHDC, where the sequence of said polynucleotide is chosen from the group: a) Sequence comprising SEQ ID 1, b) Sequence consisting of SEQ ID 1 or comprising fragments thereof, c) Sequence that differs from any of the a or b sequences due to the degeneracy of the genetic code, od) Sequences that share at least 80%, 90%, 95% or 98% homology with a, bo c.
Un segundo aspecto de la invención se relaciona con un polipéptido, en adelante "polipéptido de la invención", capaz de generar anticuerpos especificos frente a hHDC, donde la secuencia de dicho polipéptido es elegida del grupo: a) Secuencia que comprende SEQ ID 2, b) Secuencia que consiste en SEQ ID 2 o comprende fragmentos de ésta, c) Secuencia que comparta al menos un 80%, 90%, 95% ó 98% de homologia con la secuencia a o b.A second aspect of the invention relates to a polypeptide, hereinafter "polypeptide of the invention", capable of generating specific antibodies against hHDC, where the sequence of said polypeptide is chosen from the group: a) Sequence comprising SEQ ID 2, b) Sequence consisting of SEQ ID 2 or comprising fragments thereof, c) Sequence that shares at least 80%, 90%, 95% or 98% homology with the sequence a or b.
Un tercer aspecto de la invención se relaciona con un método, en adelante "método de la invención", para la obtención de anticuerpos y/o fragmentos de los mismos frente a hHDC, que comprende la utilización del polipéptido de la invención y/o fragmentos del mismo. Dicho polipéptido y sus fragmentos pueden ser obtenidos mediante sintesis quimica y/o transformación bacteriana con el polinucleótido de la invención o fragmentos del mismo. Preferentemente, dicha transformación comprende la unión mediante técnicas de ADN recombinante del polinucleótido de la invención o fragmentos del mismo a vectores que comprenden pero no se limitan a plásmidos, cósmidos, derivados del fago lambda, fagémidos, entre otros. En una realización preferida de este aspecto de la invención el método de obtención de anticuerpos y/o fragmentos de los mismos está basado en técnicas que
comprenden pero no se limitan a: técnicas de inmunización de mamiferos no humanos, generación de hibridomas por fusión celular, métodos de "phage display", etc. En una realización más preferida de este aspecto de la invención éste comprende el uso del polipéptido de la invención o fragmentos del mismo en combinación con al menos una proteina transportadora, que comprende pero no se limita a albúmina de huevo de pollo (OVA) , albúmina sérica bovina (BSA) , hemocianina de la lapa californiana Megatura cranulatus (KLH, Pierce) , etc .A third aspect of the invention relates to a method, hereinafter "method of the invention", for obtaining antibodies and / or fragments thereof against hHDC, which comprises the use of the polypeptide of the invention and / or fragments of the same. Said polypeptide and its fragments can be obtained by chemical synthesis and / or bacterial transformation with the polynucleotide of the invention or fragments thereof. Preferably, said transformation comprises the binding by recombinant DNA techniques of the polynucleotide of the invention or fragments thereof to vectors that comprise but are not limited to plasmids, cosmids, lambda phage derivatives, phagemids, among others. In a preferred embodiment of this aspect of the invention the method of obtaining antibodies and / or fragments thereof is based on techniques that They include but are not limited to: immunization techniques for non-human mammals, hybridoma generation by cell fusion, "phage display" methods, etc. In a more preferred embodiment of this aspect of the invention it comprises the use of the polypeptide of the invention or fragments thereof in combination with at least one carrier protein, which comprises but is not limited to chicken egg albumin (OVA), albumin. bovine serum (BSA), hemocyanin of the California limpet Megatura cranulatus (KLH, Pierce), etc.
Un cuarto aspecto de la invención se relaciona con anticuerpos, en adelante "anticuerpos de la invención", y/o fragmentos de los mismos especificos frente a hHDC que interaccionan especificamente frente a cualquiera de los polipéptidos de la invención o fragmentos de los mismos. Preferentemente, dichos fragmentos de anticuerpos comprenden las regiones variables Vc y/o Vl.A fourth aspect of the invention relates to antibodies, hereinafter "antibodies of the invention", and / or fragments thereof specific against hHDC that interact specifically with any of the polypeptides of the invention or fragments thereof. Preferably, said antibody fragments comprise the variable regions Vc and / or Vl.
Un quinto aspecto de la invención está relacionado con el uso de los anticuerpos de la invención y/o fragmentos de los mismos para el diagnóstico in vitro y/o in vivo.A fifth aspect of the invention is related to the use of the antibodies of the invention and / or fragments thereof for in vitro and / or in vivo diagnosis.
Un sexto aspecto de la invención está relacionado con el uso de los anticuerpos de la invención y/o fragmentos de los mismos para la fabricación de un medicamento para su uso en terapia. Dicho medicamento
puede ser empleado para el tratamiento de enfermedades, que comprenden pero no se limitan a patologias relacionadas con la degeneración neurológica (Alzheimer, Parkinson, trastornos de la memoria) , anafilaxis y procesos alérgicos, degeneración de epitelios digestivos (úlcera gástrica, sindrome de Crohn, etc) , procesos infecciosos (salmonelosis, campilobacteriosis, enterotoxemia, etc.), diferentes tipos de cáncer (cáncer de colon, mieloma, melanoma, leucemias blastociticas y mastocitica, linfoma, etc) , etc .A sixth aspect of the invention is related to the use of the antibodies of the invention and / or fragments thereof for the manufacture of a medicament for use in therapy. Said medication It can be used for the treatment of diseases, which include but are not limited to pathologies related to neurological degeneration (Alzheimer's, Parkinson's, memory disorders), anaphylaxis and allergic processes, degeneration of digestive epithelia (gastric ulcer, Crohn's syndrome, etc.), infectious processes (salmonellosis, campylobacteriosis, enterotoxemia, etc.), different types of cancer (colon cancer, myeloma, melanoma, blastocytic and mastocytic leukemia, lymphoma, etc), etc.
Un séptimo aspecto de la invención se relaciona con un kit de análisis que comprenda los "anticuerpos de la invención" y/o fragmentos de los mismos. Del mismo modo, el kit puede incluir todos aquellos reactivos necesarios para llevar a cabo la puesta a punto del kit, esto incluye, sin ningún tipo de limitación, el uso de tampones, agentes para prevenir la contaminación, etc. Por otro lado, el kit también puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización.A seventh aspect of the invention relates to an analysis kit comprising the "antibodies of the invention" and / or fragments thereof. In the same way, the kit can include all those reagents necessary to carry out the set-up of the kit, this includes, without any limitation, the use of buffers, agents to prevent contamination, etc. On the other hand, the kit can also include all the supports and containers necessary for commissioning and optimization.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
Figura 1. Inmunotransferencia ("Western blot") con los anticuerpos de la invención. En las calles 1 se muestra la detección de GST-l/152hHDC purificada y en las calles 2 se muestra la detección de extractos de
células HMC-1. Las calles 3 se corresponde con el patrón de pesos moleculares . En el panel A, el revelado fue realizado con un anticuerpo monoclonal antifactor H de conejo (IgG2a) , que es empleado como control negativo del ensayo, no apareciendo señal de interacción de la proteina con el antifactor H. El panel B es revelado con el anticuerpo AcM SIM 216-12.1Figure 1. Immunoblot ("Western blot") with the antibodies of the invention. In lanes 1 the detection of purified GST-l / 152hHDC is shown and in lanes 2 the detection of extracts of HMC-1 cells. Lanes 3 corresponds to the pattern of molecular weights. In panel A, the development was carried out with a rabbit anti-factor H monoclonal antibody (IgG2a), which is used as a negative control of the assay, with no signal showing interaction of the protein with the factor H. Panel B is revealed with the AcM antibody SIM 216-12.1
(IgG2a) , apareciendo en este caso claras bandas de interacción anticuerpo-proteina.(IgG2a), appearing in this case clear antibody-protein interaction bands.
Figura 2. Inmunotransferencia ("Western blot") que muestra la detección de inmunoprecipitados, de las lineas de leucémicas basófilas HMC-I y KU-812F productoras de hHDC . El inmunoreconocimiento se realizó con el anticuerpo monoclonal SIM 216-12.1. En ambas calles se muestra la detección de hHDC, observándose 3 bandas mayoritarias . La mayor de las bandas se corresponde con el tamaño esperado para el monómero maduro de hHDC y las dos inferiores con las formas degradadas de la proteina.Figure 2. Immunoblot ("Western blot") showing the detection of immunoprecipitates of the basophilic leukemic lines HMC-I and KU-812F producing hHDC. Immunorecognition was performed with the monoclonal antibody SIM 216-12.1. In both streets the detection of hHDC is shown, observing 3 major bands. The largest of the bands corresponds to the expected size for the mature hHDC monomer and the lower two with the degraded forms of the protein.
Figura 3. Inmunotransferencia ("Western blot") de extractos celulares de HEK-293 transfectados transitoriamente con pcDNA3-l/512hHDC (calle 1) , pcDNA3-l/512 (calle 2) o pEGFP (calle 3, control negativo) . La detección hHDC para los diferentes extractos celulares es llevada a cabo con AcM SIM 216- 12.1, encontrándose bandas claramente diferenciadas en las calles 1 y 2 correspondientes la interacción anticuerpo-hHDC . Las bandas superiores se corresponden
con el tamaño esperado para el monómero maduro de hHDC y las inferiores a las formas degradadas de éste.Figure 3. Western blot of HEK-293 cell extracts transiently transfected with pcDNA3-l / 512hHDC (lane 1), pcDNA3-l / 512 (lane 2) or pEGFP (lane 3, negative control). The hHDC detection for the different cell extracts is carried out with AcM SIM 216-12.1, with clearly differentiated bands in lanes 1 and 2 corresponding to the antibody-hHDC interaction. The upper bands correspond with the expected size for the mature hHDC monomer and those smaller than its degraded forms.
Figura 4. Inmunocitoreconocimiento de hHDC en células HEK-293 transfectadas con pcDNA3-l/512hHDC, que expresan transitoriamente dicha proteina, mediante el empleo de AcM SIM 216-12.1. El panel A muestra la no emisión de fluorescencia por células no transfectadas y el panel B muestra la emisión de fluorescencia procedente del citoplasma de células transfectadas.Figure 4. Immuno-monitoring of hHDC in HEK-293 cells transfected with pcDNA3-1 / 512hHDC, which expressly express said protein, using AcM SIM 216-12.1. Panel A shows the non-emission of fluorescence by non-transfected cells and panel B shows the emission of fluorescence from the cytoplasm of transfected cells.
EXPLICACIÓN DETALLADA DE LA INVENCIÓNDETAILED EXPLANATION OF THE INVENTION
Los siguientes ejemplos de realización no pretenden limitar la invención sino ilustrarla para su mejor comprensión.The following exemplary embodiments are not intended to limit the invention but to illustrate it for better understanding.
Ejemplo 1. Obtención de anticuerpos frente a hHDCExample 1. Obtaining antibodies against hHDC
1.1. Inmunización1.1. Immunization
El péptido elegido como inmunógeno (SEQ ID 2) que comprende los aminoácidos 492 a 506 de hHDC se acopla mediante el reactivo entrecruzante, sulfo-SMCC (SuIfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1- carboxylate; Pierce, No 22322), a una proteina portadora, preferentemente OVA, siguiendo el protocolo indicado por el fabricante.The peptide chosen as an immunogen (SEQ ID 2) comprising amino acids 492 to 506 of hHDC is coupled by the cross-linking reagent, sulfo-SMCC (SuIfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1- carboxylate; Pierce, No 22322), to a carrier protein, preferably OVA, following the protocol indicated by the manufacturer.
Para la inmunización se eligen mamiferos no humanos, preferentemente ratones y más preferentemente hembras de la cepa BALB/c. Los mamiferos no humanos son inmunizados via intraperitoneal para la obtención de
anticuerpos policlonales o monoclonales anti-hHDC. A la semana de la segunda inmunización, se realiza una sangria para la determinación titulo y reactividad de anticuerpos presente en el suero del ratón. Esta determinación puede ser llevada a cabo mediante diferentes técnicas sobradamente conocidas en el estado de la técnica, tales como inmunoensayo tipo ELISA ("Enzyme Linked Immuno Sorbent Asssay") indirecto frente al péptido de la invención conjugado con otra proteina distinta de OVA. A partir de estos ensayos, el ratón con un suero de mayor titulo de anticuerpos es elegido como donante de linfocitos B, para la obtención de anticuerpos monoclonales, o para la obtención de anticuerpos policlonales a partir del suero.For immunization non-human mammals are chosen, preferably mice and more preferably females of the BALB / c strain. Non-human mammals are immunized intraperitoneally to obtain polyclonal or monoclonal anti-hHDC antibodies. One week after the second immunization, bleeding is performed to determine the titre and reactivity of antibodies present in the mouse serum. This determination can be carried out by different techniques well known in the state of the art, such as indirect ELISA ("Enzyme Linked Immuno Sorbent Asssay") immunoassay against the peptide of the invention conjugated to another protein other than OVA. From these tests, the mouse with a serum of greater antibody titre is chosen as a donor of B lymphocytes, for obtaining monoclonal antibodies, or for obtaining polyclonal antibodies from the serum.
1.2. Fusión de linfocitos B .1.2. B lymphocyte fusion.
Los linfocitos B son fusionados con células de mieloma, preferentemente las células de mieloma Sp2/0-Ag-14 según los procedimientos descritos en Galfré y Milstein (1981) Method Enzymol 73:3-47 y J. Goding (1980) J. Immuno1. Methods 30: 285-308, con el fin de obtener hibridos productores de anticuerpos anti-hHDC.B lymphocytes are fused with myeloma cells, preferably Sp2 / 0-Ag-14 myeloma cells according to the procedures described in Galfré and Milstein (1981) Method Enzymol 73: 3-47 and J. Goding (1980) J. Immuno1 . Methods 30: 285-308, in order to obtain hybrids producing anti-hHDC antibodies.
1.3. Selección de Hibridomas productores y ensayos de especificidad.1.3. Selection of producing hybridomas and specificity tests.
La selección de hibridomas productores de los anticuerpos de la invención se lleva a cabo mediante diferentes técnicas. Preferentemente, las técnicas elegidas para la realización de esta selección son: a) ELISA, empleando como antigeno el conjugado del "péptido de la invención" con BSA, b) citometria de
flujo con células de la línea HMC-I, y c) inmunotransferencia "Western-blot" con extractos de células HMC-I o de la proteína de fusión GTS-l/512hHDC . Finalmente, los hibridomas secretores de anticuerpos monoclonales, con el fin de estabilizarlos, se clonan dos veces por dilución límite en medio líquido, se determina el isotipo, y se almacenan en nitrógeno líquido .The selection of hybridomas producing the antibodies of the invention is carried out by different techniques. Preferably, the techniques chosen for the realization of this selection are: a) ELISA, using the conjugate of the "peptide of the invention" with BSA as an antigen, b) cytometry of flow with cells of the HMC-I line, and c) Western blotting with HMC-I cell extracts or the GTS-l / 512hHDC fusion protein. Finally, the monoclonal antibody secreting hybridomas, in order to stabilize them, are cloned twice by limit dilution in liquid medium, the isotype is determined, and stored in liquid nitrogen.
Mediante estas pruebas se eligieron diferentes anticuerpos monoclonales, y a partir de los mismos se seleccionó el anticuerpo monoclonal que, en adelante, denominaremos como AcM SIM 216-12.1, que es el utilizado en los siguientes ensayos.By means of these tests different monoclonal antibodies were chosen, and from them the monoclonal antibody was selected which, hereinafter, we will call as AcM SIM 216-12.1, which is the one used in the following tests.
1.3.a) Ensayo de especificidad por Inmunotransferencia pWestern-blot")1.3.a) pWestern-blot Immunoblot specificity test ")
En primer lugar se obtuvo un plásmido recombinante fusionando los 512 residuos de la proteína hHDC, que denominaremos l/512hHDC, con un vector - preferentemente el vector pGEX de Pharmacia - que permite la obtención en masa del fragmento recombinante de 512 aminoácidos de la hHDC; la proteína de fusión expresada a partir de dicho plásmido se denominará GTS- l/512hHDC. Los procedimientos de extracción, manipulación, recombinación in vitro de ADN y transformación bacteriana para la obtención en masa del producto recombinante son descritos en la solicitud de patente española 9800019, empleándose como fuente principal de ARN mensajero de hHDC células de la línea basófila HMC-I. A partir de los cultivos de E. CoIi BL21 (DE3) pLysS transformados con pGEX recombinado con el fragmento de l/512hHDC, en los que se había inducido
la expresión de la proteína de fusión GTS-l/512hHDC por adición de IPTG al medio, se preparan extractos bacterianos, y éstos se someten a cromatografía de afinidad en columna de glutation-sepharosa (Amersham Biosciences) para aislar la proteína GTS-l/512hHDC .First, a recombinant plasmid was obtained by fusing the 512 residues of the hHDC protein, which we will call 1 / 512hHDC, with a vector - preferably the Pharmacia pGEX vector - which allows mass production of the 512 amino acid recombinant fragment of the hHDC; The fusion protein expressed from said plasmid will be called GTS-1 / 512hHDC. The procedures for extraction, manipulation, in vitro recombination of DNA and bacterial transformation for the mass obtaining of the recombinant product are described in the Spanish patent application 9800019, using HMC-I basophilic line cells as the main source of hHDC messenger RNA. . From cultures of E. CoIi BL21 (DE3) pLysS transformed with pGEX recombined with the fragment of 1 / 512hHDC, in which it had been induced Expression of the GTS-l / 512hHDC fusion protein by the addition of IPTG to the medium, bacterial extracts are prepared, and these are subjected to glutathione sepharose column affinity chromatography (Amersham Biosciences) to isolate the GTS-l / protein. 512hHDC
La proteína GTS-l/512hHDC aislada y un extracto celular de células HMC-I que expresan hHDC, se sometieron a electroforesis desnaturalizante (SDS/PAGE) seguida de transferencia a nitrocelulosa y análisis por "Western blot" con los anticuerpos de la invención y con un anticuerpo inespecífico que no reconoce a hHDC y sirve de control negativo (FIG 1) .The isolated GTS-l / 512hHDC protein and a cellular extract of HMC-I cells expressing hHDC were subjected to denaturing electrophoresis (SDS / PAGE) followed by transfer to nitrocellulose and "Western blot" analysis with the antibodies of the invention and with a nonspecific antibody that does not recognize hHDC and serves as a negative control (FIG 1).
El resultado del análisis (Panel A) muestra que el anticuerpo inespecífico no muestra señales de reconocimiento ni con hHDC (calle 1) ni con GTS- l/512hHDC (calle 2); en el Panel B, se observa que el AcM SIM 216-12.1 detecta tanto GTS-l/512hHDC como hHDC procedente de las células HMC-I, ésta última con menor intensidad.The result of the analysis (Panel A) shows that the nonspecific antibody does not show recognition signals either with hHDC (lane 1) or with GTS-1 / 512hHDC (lane 2); In Panel B, it can be seen that the SIM 216-12.1 AcM detects both GTS-l / 512hHDC and hHDC from the HMC-I cells, the latter with less intensity.
Ejemplo 2. Especificidad de los anticuerpos frente a hHDC.Example 2. Specificity of antibodies against hHDC.
2.1. Ensayos de Inmunotransferencia ("Western-blot")2.1. Immunoblotting assays ("Western blot")
Para corroborar la especificidad del AcM SIM 216-12.1 aislado frente hHDC, se procedió a realizar distintas pruebas .To corroborate the specificity of the AcM SIM 216-12.1 isolated against hHDC, different tests were carried out.
Recolección de inmunoprecipitados : en primer lugar se obtuvieron extractos celulares de las líneas HMC-I y KU-182F, ambas productoras de hHDC, con un tampón de
lisis denominado "tampón de lisis II" de composición Tris 20 mM, NaCl 137 mM, glicerol 10%, Nonidet P40 1% y EDTA 2mM.Collection of immunoprecipitates: first, cell extracts of the HMC-I and KU-182F lines, both producing hHDC, were obtained with a buffer of lysis called "lysis buffer II" of 20 mM Tris composition, 137 mM NaCl, 10% glycerol, 1% Nonidet P40 and 2mM EDTA.
Para la inmunoprecipitación, se empleó un antisuero de conejo (denominado anti-GST-l/187hHDC) obtenido inmunizando, según el protocolo descrito en la solicitud de patente P9800019, con la proteina de fusión GST-l/187hHDC (denominada GST-l/187hHDC) aislada de la banda de un gel de SDS-PAGE. Una vez obtenidos los extractos celulares y el antisuero, se procedió a realizar la inmunoprecipitación tal y como se describe a continuación. Para reducir la adsorción inespecifica de las esferas del inmunoadsorbente Proteina A- Sepharosa (Pharmacia) con la que se aislan los anticuerpos de los conejos inmunizados con la proteina de GST-l/187hHDC, se incuba durante 30 min a 4° C con una solución de BSA (15 mg/ml) en un tampón de lisis (tampón de lisis I) de composición Hepes 50 mM, NaCl 150 mM, EDTA 1 mM, EGTA 2,5 mM, DTT 1 mM, Tween-20 0,1%, Glicerol 10%, ph 7,5; tras la incubación, las esferas se lavan tres veces con el tampón de lisis I sin BSA. Reducida la adsorción de las esferas de Proteina A-Sepharosa, 20 microlitros de una suspensión (50% v/v) de las mismas se incuban durante 2 horas con 1 mililitro de suero anti-GST-l/187hHDC diluido 1: 500 en tampón de lisis I. Finalizada la incubación, las esferas de proteina A-Sepharosa con los anticuerpos anti-GST-l/187hHDC fijados se lavan con tampón de lisis I, y se ajustan al 50% (v/v) .For immunoprecipitation, a rabbit antiserum (called anti-GST-l / 187hHDC) was obtained by immunizing, according to the protocol described in patent application P9800019, with the fusion protein GST-l / 187hHDC (called GST-l / 187hHDC) isolated from the band of an SDS-PAGE gel. Once the cell extracts and the antiserum were obtained, the immunoprecipitation was performed as described below. To reduce the nonspecific adsorption of the spheres of the immunoadsorbent Protein A-Sepharose (Pharmacia) with which antibodies from rabbits immunized with the GST-1 / 187hHDC protein are isolated, incubate for 30 min at 4 ° C with a solution of BSA (15 mg / ml) in a lysis buffer (lysis buffer I) of 50 mM Hepes composition, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20 , Glycerol 10%, pH 7.5; after incubation, the spheres are washed three times with lysis buffer I without BSA. Reduced the adsorption of Protein A-Sepharose spheres, 20 microliters of a suspension (50% v / v) of them are incubated for 2 hours with 1 milliliter of anti-GST-l / 187hHDC serum diluted 1: 500 in buffer of lysis I. After incubation, the A-Sepharosa protein spheres with the fixed anti-GST-l / 187hHDC antibodies are washed with lysis buffer I, and adjusted to 50% (v / v).
Un microlito de cada uno de los extractos de células HMC-I y KU-182F (equivalente a 9 millones de células) se incuba durante 3 horas a 4 °C con la esferas de
Proteína A-Sepharosa que tienen unido el anticuerpo anti-GST-l/187hHDC . Tras sucesivos lavados, las esferas se recogen por centrifugación y se eluyen con tampón de conteniendo 2,3% SDS; los eluídos desnaturalizados se separan por electroforesis en gel de SDS-PAGE seguida de una electrotranferencia a filtros de nylon. Dichos filtros se incuban con el AcM SIM 216-12.1 (dilución 1/2500) y los complejos formados por el AcM y la hHDC se localizan mediante una reacción de quimiolumi- niscencia incubando el filtro con antiimunoglobulina de ratón conjugada con peroxidasa (FIG 2) .A microlite of each of the HMC-I and KU-182F cell extracts (equivalent to 9 million cells) is incubated for 3 hours at 4 ° C with the spheres of Protein A-Sepharose that have the anti-GST-l / 187hHDC antibody bound. After successive washing, the spheres are collected by centrifugation and eluted with buffer containing 2.3% SDS; Denatured eluates are separated by SDS-PAGE gel electrophoresis followed by electrotransfer to nylon filters. Said filters are incubated with the AcM SIM 216-12.1 (1/2500 dilution) and the complexes formed by the AcM and the hHDC are located by a chemiluminescence reaction by incubating the filter with peroxidase-conjugated mouse antiimunoglobulin (FIG 2) .
2.2. Ensayo de ϊnmunorreconocimiento ("Western-blot")2.2. Immunorecognition assay ("Western-blot")
Para realizar este ensayo se transfectan células HEK- 293 con el plásmido de expresión pcDNA 3 (Invitrogen) que contiene la secuencia l/512hHDC insertada bajo el control del promotor de citomegalovirus (CMV) . La transfección se facilita utilizando el sistema FuGENE (Roche) así como distintas cantidades de plásmidos de expresión (FIG. 3: 1,5 microgramos, calle 1, y 1 microgramo, calle 2) . Como control negativo se emplea un extracto de células HEK-293 transfectadas en paralelo con el plásmido de expresión comercial pEGFP- Nl (Clontech) en su forma original, que carece de secuencias codificantes de hHDC.To perform this assay, HEK-293 cells are transfected with the expression plasmid pcDNA 3 (Invitrogen) containing the sequence l / 512hHDC inserted under the control of the cytomegalovirus (CMV) promoter. Transfection is facilitated using the FuGENE (Roche) system as well as different amounts of expression plasmids (FIG. 3: 1.5 micrograms, lane 1, and 1 microgram, lane 2). As a negative control, an extract of HEK-293 cells transfected in parallel with the commercial expression plasmid pEGFP-N1 (Clontech) in its original form, lacking hHDC coding sequences, is used.
El AcM SIM 216-12.1 se incuba con el extracto de células HEK-293 transfectadas, al tercer día de la transfección. Los precipitados formados se separan mediante electroforesis y se transfieren a filtros de nylon. La detección se lleva a cabo utilizando como anticuerpo primario el AcM SIM 216-12.1 (dilución 1/2500) , y como anticuerpo secundario un anticuerpo anti-ratón conjugado a peroxidasa (dilución 1/5000) .
La posición y abundancia de las bandas se visualiza por quimioluminiscencia (FIG 3) . En la calle 1, que corresponde al extracto celular de células transfectadas con la mayor cantidad de plásmido, se observa una banda de reacción principal más intensa, que corresponde a la Mr prevista para la proteina hHDC, y otra banda de menor tamaño que probablemente representa productos de degradación de dicha proteina. Cuando la transfección se realiza a partir de un microgramo de plásmido (calle 2) la intensidad de las bandas es débil, y apenas se detecta la banda de degradación. La calle 3, no muestra banda alguna puesto que las células de las que proceden los extractos empleados únicamente expresan la proteina verde fluorescente del plásmido pEGGFP-Nl .The AcM SIM 216-12.1 is incubated with the extract of transfected HEK-293 cells, on the third day of transfection. The precipitates formed are separated by electrophoresis and transferred to nylon filters. Detection is carried out using as primary antibody AcM SIM 216-12.1 (dilution 1/2500), and as secondary antibody an anti-mouse antibody conjugated to peroxidase (dilution 1/5000). The position and abundance of the bands is visualized by chemiluminescence (FIG 3). In lane 1, which corresponds to the cell extract of cells transfected with the greatest amount of plasmid, a more intense main reaction band is observed, corresponding to the Mr planned for the hHDC protein, and another smaller band that probably represents degradation products of said protein. When the transfection is performed from a plasmid microgram (lane 2) the intensity of the bands is weak, and the degradation band is barely detected. Lane 3 shows no band since the cells from which the extracts used only express the green fluorescent protein of plasmid pEGGFP-Nl.
2.3. Ensayos de inmunofluorescencia celular2.3. Cellular immunofluorescence assays
Se transfectan transitoriamente células HEK-293 con 1,5 microgramos de plásmido pcDNA3-hHDCl/512 según se describe en el ejemplo anterior, para expresar de manera transitoria la proteina l/152hHDC de las célulasHEK-293 cells are transiently transfected with 1.5 micrograms of plasmid pcDNA3-hHDCl / 512 as described in the previous example, to transiently express the protein l / 152hHDC protein from the cells
HEK-293. Las células se incuban en placas de 24 pocilios sobre cubre-objetos recubiertos con polilisina durante 8 horas. Transcurrido este tiempo, se retira el medio de cultivo y las células se fijan con formaldehido al 3,7% durante 30 minutos en hielo. A continuación, las células se lavan con una solución de fosfato-salino isotónico (PBS) y se permeabilizan con PBS/Tritón X-IOO 0,2% durante 5 minutos en hielo. La preparación se bloquea durante 90 min a 4° C conHEK-293. The cells are incubated in 24-well plates on polylysine coated object covers for 8 hours. After this time, the culture medium is removed and the cells are fixed with 3.7% formaldehyde for 30 minutes on ice. The cells are then washed with an isotonic phosphate-saline solution (PBS) and permeabilized with PBS / Triton X-IOO 0.2% for 5 minutes on ice. The preparation is blocked for 90 min at 4 ° C with
PBS/Tween-20 al 0,05%-suero fetal bovino al 5%, y subsecuentemente, se incuba con el AcM SIM 216-12.1PBS / 0.05% Tween-20 - 5% bovine fetal serum, and subsequently incubated with AcM SIM 216-12.1
(dilución 1: 500) durante 45 minutos a 20° C. Tras
lavados sucesivos con PBS/Tween-20 al 0,05%, la preparación de incuba durante 45 minutos con un anticuerpo antiratón (dilución 1:500) marcado con rhodamina (Jackson ImmunoResearch) . Finalmente la preparación se lava con PBS/Tween-20 al 0,05% y se visualiza en el microscopio de fluorescencia (FIG 4) . Las células HEK-293 transformadas incubadas con el anticuerpo AcM SIM 216-216-12.1 emiten fluorescencia, a diferencia de las células transfectadas con el plásmido control .
(1: 500 dilution) for 45 minutes at 20 ° C. After successive washes with 0.05% PBS / Tween-20, the preparation is incubated for 45 minutes with an anti-mouse antibody (1: 500 dilution) labeled with rhodamine (Jackson ImmunoResearch). Finally, the preparation is washed with 0.05% PBS / Tween-20 and visualized in the fluorescence microscope (FIG 4). Transformed HEK-293 cells incubated with the AcM SIM 216-216-12.1 antibody emit fluorescence, unlike cells transfected with the control plasmid.
Claims
1. Polinucleótido, capaz de codificar un polinpeptido capaz de generar de anticuerpos o fragmentos de los mismos especificos frente a hHDC, cuya secuencia es elegida del grupo:1. Polynucleotide, capable of encoding a polynpeptide capable of generating antibodies or fragments of the same against hHDC, whose sequence is chosen from the group:
a. Secuencia que comprende SEQ ID 1 b. Secuencia que consiste en SEQ ID 1 o comprende fragmentos de ésta. c. Secuencia que difiera de las secuencias a o b debido a la degeneración del código genético. d. Secuencias que compartan al menos un 80%, 90%, 95% ó 98% de homologia con cualquiera de las secuencias anteriores .to. Sequence comprising SEQ ID 1 b. Sequence consisting of SEQ ID 1 or comprises fragments thereof. C. Sequence that differs from sequences a or b due to the degeneracy of the genetic code. d. Sequences that share at least 80%, 90%, 95% or 98% homology with any of the above sequences.
2. Polipéptido, según la reivindicación 1, capaz de generar anticuerpos especificos frente a hHDC y cuya secuencia es elegida del grupo:2. Polypeptide according to claim 1, capable of generating specific antibodies against hHDC and whose sequence is chosen from the group:
a. Secuencia que comprende SEQ ID 2 b. Secuencia que consiste en SEQ ID 2 o comprende fragmentos de ésta. c. Secuencia que comparta al menos un 80%, 90%, 95% ó 98% de homologia la secuencia a o b.to. Sequence comprising SEQ ID 2 b. Sequence consisting of SEQ ID 2 or comprises fragments thereof. C. Sequence that shares at least 80%, 90%, 95% or 98% homology sequence a or b.
3. Anticuerpos o fragmentos de los mismos especificos frente a hHDC, en donde dichos anticuerpos interaccionan especificamente frente cualquiera de los polipéptidos de la reivindicación 2. 3. Antibodies or fragments thereof specific against hHDC, wherein said antibodies interact specifically against any of the polypeptides of claim 2.
4. Uso de cualquiera de los polipéptidos según la reivindicación 2 para la generación de anticuerpos o fragmentos de los mismos especificos frente a hHDC .4. Use of any of the polypeptides according to claim 2 for the generation of antibodies or fragments thereof specific against hHDC.
5. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por técnicas de generación de hibridomas .5. Use according to claim 4 wherein the antibodies or fragments thereof are obtained by hybridoma generation techniques.
6. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por la tecnologia de "phage display".6. Use according to claim 4 wherein the antibodies or fragments thereof are obtained by the "phage display" technology.
7. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por inmunización de mamiferos no humanos.7. Use according to claim 4 wherein the antibodies or fragments thereof are obtained by immunization of non-human mammals.
8. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento .8. Use of the antibodies or fragments thereof according to claim 3, for the preparation of a medicament.
9. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de la degeneración neurológica.9. Use of the antibodies or fragments thereof according to claim 3, for the preparation of a medicament for the treatment of neurological degeneration.
10. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de la anafilaxis y/o procesos alérgicos .10. Use of the antibodies or fragments thereof according to claim 3, for the preparation of a medicament for the treatment of anaphylaxis and / or allergic processes.
11. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento la degeneración de epitelios digestivos . 11. Use of the antibodies or fragments thereof according to claim 3, for the preparation of a medicament for the treatment of degeneration of digestive epithelia.
12. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento para el tratamiento de diferentes tipos de cáncer.12. Use of the antibodies or fragments thereof according to claim 3, for the preparation of a medicament for the treatment for the treatment of different types of cancer.
13. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de procesos infecciosos .13. Use of the antibodies or fragments thereof according to claim 3, for the preparation of a medicament for the treatment of infectious processes.
14. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3 para el diagnóstico in vitro de enfermedades .14. Use of the antibodies or fragments thereof according to claim 3 for the in vitro diagnosis of diseases.
15. Kit de diagnóstico que comprende la utilización de anticuerpos o fragmentos de los mismos según la reivindicación 3. 15. Diagnostic kit comprising the use of antibodies or fragments thereof according to claim 3.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200600378A ES2319586B1 (en) | 2006-02-17 | 2006-02-17 | METHOD OF OBTAINING ANTI-HHDC ANTIBODIES AND APPLICATIONS OF THE SAME. |
| ESP200600378 | 2006-02-17 |
Publications (1)
| Publication Number | Publication Date |
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| WO2007093663A1 true WO2007093663A1 (en) | 2007-08-23 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/ES2007/070032 WO2007093663A1 (en) | 2006-02-17 | 2007-02-16 | Method for obtaining anti-hdc antibodies and applications of same |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2319586B1 (en) |
| WO (1) | WO2007093663A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998030593A2 (en) * | 1997-01-06 | 1998-07-16 | Promega Corporation | Assay of histidine decarboxylase to detect cancer |
-
2006
- 2006-02-17 ES ES200600378A patent/ES2319586B1/en not_active Expired - Fee Related
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2007
- 2007-02-16 WO PCT/ES2007/070032 patent/WO2007093663A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998030593A2 (en) * | 1997-01-06 | 1998-07-16 | Promega Corporation | Assay of histidine decarboxylase to detect cancer |
Non-Patent Citations (3)
| Title |
|---|
| FLEMING V. ET AL.: "Mapping of catalytically important residues in the rat L-histidine decarboxilase enzyme using bioinformatic and site-dirested mutagenesis approaches", BIOCHEM. J., vol. 379, 2004, pages 253 - 261 * |
| POLLARD H. ET AL.: "Monoclonal antibody against L-histidine decarboxilase for localization of histaminergic cells", NEUROSCIENCE LETTERS, vol. 54, no. 1, 1985, pages 53 - 58, XP002073362 * |
| YATSUNAMI K. ET AL.: "Comparative studies of human recombinant 74 and 54 kDa L-histidine decarboxilases", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 22, 1995, pages 30813 - 30817, XP002073361 * |
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| Publication number | Publication date |
|---|---|
| ES2319586B1 (en) | 2010-02-10 |
| ES2319586A1 (en) | 2009-05-08 |
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