ES2319586A1 - Method for obtaining anti-hdc antibodies and applications of same - Google Patents

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

Method of obtaining antibodies anti-hHDC and their applications.

Field of the Invention

The present invention provides a method for obtaining specific antibodies against histidine human decarboxylase (hHDC), as well as polynucleotides and polypeptides necessary to carry it out. Similarly, the uses of the aforementioned antibodies, as well as kits diagnosis of those who are also part of the present invention

State of the art

In mammalian cells, the active form of histidine decarboxylase (HDC) (E.C. 4.1.1.22, according to classification of the International Biochemistry Union) is a Homodimeric protein of approximately 110 kDa, very minor (less than 0.001% of the cellular protein content), whose activity It depends on pyridoxalphosphate or PLP. The HDC catalyzes the passage of L-histidine to histamine through decarboxylation of the first, and is expressed with activity appreciable in a small group of cell types: some cells of the central and peripheral nervous system, some leukocytes - basophils, mast cells, macrophages - digestive epithelia, and some types of neurons and cancer cells dedifferentiated.

Recent studies with mice that have the expression of histidine decarboxylase (knock-out mice) canceled (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. Comun. 305: 443-447; Fitzpartick et al . (2003) Proc. Natl. Acad. Sci. USA 100: 6027-6032). HDC is, therefore, involved in a multitude of inflammatory, cancerous processes and in various neuropathies.

It is well known that HDC, especially human HDC (hHDC), shows great instability both in vivo and in cell-free extracts, which makes 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-García 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.

Years later, Ohmori et al . (Ohmori et al . (1990) J. Biochem. 107: 834-839) purified the mouse HDC enzyme homogeneously from 9 x 10 10 P-815 mouse mastocytic cells collected from ascites fluid from 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.

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.

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 for different proteolytic systems (Moya-García et al . (2005) BioEssays 27: 57-63).

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).

To the inconveniences derived from Biochemical properties of HDC (instability, adhesion, etc.) there are to add that the cells that express the enzyme are found generally dispersed and in minority among other cell types histamine producers (bone marrow, blood, epithelia, stomach, brain). In the case of the hHDC, the difficulties of accessibility to fresh tissues, so obtaining antibodies against said enzyme becomes a company extremely arduous

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In the Spanish patent application ES 2 135 350 a method is developed to obtain antibodies anti-HDC against recombinant proteins, which they recognize the mouse HDC but they don't do it with the human version of The enzyme

The PCT application for publication number WO-98/30593, details the obtaining of antibodies specific polyclonal against hHDC that have been used in the development of cancer screening assay; said publication, details a method to obtain antibodies that react against hHDC regions other than those recognized by antibodies whose preparation describes the present application.

Finally, it is worth mentioning that in 1995 Kimio Yatsunami et al . (Yatsunami et al . (1995) J. Biol. Chem. 270 (51): 30813-30817) developed monoclonal antibodies (AcM) against hHDC using a conserved peptide as an immunogen 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.

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 hHDC in its active conformation to study the enzyme and analyze its participation in pathological processes in which it could be involved, 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.

Description of the invention Definitions

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 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 thereof, where said fragments comprise the variable regions Vh and / or Vl.

As indicated above, the sequence hHDC has been known for a decade, and as a result of studies carried out to carry out the present invention, are they have been able to deduce polypeptide fragments that the enzyme to maintain its activity; this fact has allowed to realize  bioinformatic analysis of comparison between sequences HDC rat and human codifiers, and predict which epitopes of the active protein possessed greater immunogenic potential for develop antibodies against hHDC. From this study, chose as immunogen the polypeptide consisting of the residues amino acids 492-506 (SEQ ID 2) of hHDC, encoded by the polynucleotide of SEQ ID 1, and a method for obtaining anti-hHDC antibodies and / or fragments thereof that react against the polypeptide selected.

In this sense, a first aspect of the invention relates to a polynucleotide, hereafter "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 includes fragments thereof, c) Sequence that differs from any of the sequences a or b due to the degeneration of the genetic code, or d) Sequences that share at least 80%, 90%, 95% or 98% homology with a, b or c.

A second aspect of the invention relates to with 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 it includes fragments of it, 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. 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 comprise but are not limited to: immunization techniques of non-human mammals, generation of hybridomas 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.

A fourth aspect of the invention relates to with 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 Vc and / or Vl variable regions.

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 Its use in therapy. Said medication may be used for 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 (ulcer gastric, Crohn's syndrome, etc.), infectious processes (salmonellosis, campylobacteriosis, enterotoxemia, etc.), different  types of cancer (colon cancer, myeloma, melanoma, leukemia blastocytic and mastocytic, lymphoma, etc.), etc.

A seventh aspect of the invention relates to with an analysis kit that includes the "antibodies of the invention "and / or fragments thereof. Similarly, the kit can include all those reagents necessary to carry out of the kit, this includes, without any type of limitation, the use of buffers, agents to prevent pollution, etc. On the other hand, the kit can also include all the supports and containers necessary for putting them in March and optimization.

Description of the figures

Figure 1. Immunoblot ("Western blot ") with the antibodies of the invention. In lanes 1 shows the detection of purified GST-1 / 152hHDC and in lanes 2 the detection of cell extracts is shown HMC-1 Streets 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 test, no signal of protein interaction with the H factor. Panel B is revealed with the AcM SIM 216-12.1 antibody (IgG2a), appearing in this case clear interaction bands antibody-protein

Figure 2. Immunoblot ("Western blot ") which shows the detection of immunoprecipitates, of the basophilic leukemia lines HMC-1 and KU-812F producing hHDC. He Immunorecognition was performed with the monoclonal antibody SIM 216-12.1. In both streets the detection is shown of hHDC, observing 3 major bands. The biggest of the bands corresponds to the expected size for the mature monomer of hHDC and the lower two with degraded forms of the protein.

Figure 3. Immunoblot ("Western blot ") of HEK-293 cell extracts transiently transfected with pcDNA3-1 / 512hHDC (lane 1), pcDNA3-1 / 512 (lane 2) or pEGFP (lane 3, negative control). HHDC detection for different extracts Cell phones are carried out with AcM SIM 216-12.1, finding bands clearly differentiated in lanes 1 and 2 corresponding antibody-hHDC interaction. The upper bands correspond to the expected size for the mature monomer of hHDC and those inferior to degraded forms of East.

Figure 4. Knowledge of hHDC in cells HEK-293 transfected with pcDNA3-1 / 512hHDC, which express transiently said protein, through the use of AcM SIM 216-12.1. Panel A shows the non-emission of fluorescence by non-transfected cells and panel B shows the fluorescence emission from the cytoplasm of cells Transfected

Detailed Explanation of the Invention

The following embodiments do not they intend to limit the invention but to illustrate it for its best understanding.

Example 1 Obtaining antibodies against hHDC 1.1. Immunization

The peptide chosen as immunogen (SEQ ID 2) comprising amino acids 492 to 506 of hHDC is coupled by the crosslinking reagent, sulfo-SMCC (Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate; Pierce, No 22322), to a carrier protein, preferably OVA, following the protocol indicated by the manufacturer.

For the immunization mammals are chosen not humans, preferably mice and more preferably females of the strain BALB / c. Non-human mammals are immunized via intraperitoneal to obtain polyclonal antibodies or monoclonal anti-hHDC. At the week of the second immunization, indentation is performed for the title determination and antibody reactivity present in the mouse serum. This determination can be carried out by different techniques well known in the state of the art, such as ELISA immunoassay ("Enzyme Linked Immuno Sorbent Asssay ") indirect against the conjugate invention peptide with another protein other than OVA. From these tests, the mouse with a serum of higher antibody titer is chosen as donor of B lymphocytes, to obtain antibodies monoclonal, or for obtaining polyclonal antibodies to from whey.

1.2. B lymphocyte fusion

B lymphocytes are fused with cells of myeloma, preferably myeloma cells Sp2 / 0-Ag-14 according to procedures described in Galfré and Milstein (1981) Method Enzymol 73: 3-47 and J. Goding (1980) J. Immunol. Methods 30: 285-308, in order to obtain producing hybrids of anti-hHDC antibodies.

1.3. Selection of producing hybridomas and trials of specificity

The selection of hybridomas producing antibodies of the invention is carried out by different techniques Preferably, the techniques chosen for the Realization of this selection are: a) ELISA, using as antigen the conjugate of the "peptide of the invention" with BSA, b) flow cytometry with line cells HMC-1, and c) immunoblot "Western blot" with cell extracts HMC-1 or fusion protein GTS-1 / 512hHDC. Finally, hybridomas monoclonal antibody secretors, in order to stabilize them, clone twice by dilution limit in medium liquid, isotype is determined, and stored in nitrogen liquid.

Through these tests different were chosen monoclonal antibodies, and from them the monoclonal antibody that, hereinafter, we will call as AcM SIM 216-12.1, which is used in the following essays.

1.3.a) Immunoblot specificity test ("Western-blot")

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 methods of 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 as the main source of messenger RNA of hHDC cells of the basophilic line HMC-1 . From E. Coli BL21 (DE3) pLysS cultures transformed with pGEX recombined with the 1 / 512hHDC fragment, in which the expression of the GTS-1 / 512hHDC fusion protein was induced by adding IPTG to the medium , bacterial extracts are prepared, and these are subjected to glutathione sepharose column affinity chromatography (Amersham Biosciences) to isolate the GTS-1 / 512hHDC protein.

GTS-1 / 512hHDC protein isolated and a cell extract of HMC-1 cells that express hHDC, underwent denaturing electrophoresis (SDS / PAGE) followed by transfer to nitrocellulose and analysis by "Western blot" with the antibodies of the invention and with a nonspecific antibody that does not recognize hHDC and serves as a control negative (Fig 1).

The result of the analysis (Panel A) shows that the nonspecific antibody does not show recognition signals or with hHDC (lane 1) or with GTS-1 / 512hHDC (lane 2); in Panel B, it is observed that the AcM SIM 216-12.1 detects both GTS-1 / 512hHDC and hHDC from HMC-1 cells, the latter with less intensity.

Example 2 Specificity of antibodies against hHDC 2.1. Immunoblot Assays ("Western-blot")

To corroborate the specificity of the AcM SIM 216-12.1 isolated against hHDC, we proceeded to Perform different tests.

Collection of immunoprecipitates: first place cell extracts were obtained from the lines HMC-1 and KU-182F, both producers of hHDC, with a lysis buffer called "lysis buffer II" of composition 20 mM Tris, 137 mM NaCl, 10% glycerol, Nonidet P40 1% and 2 mM EDTA.

For immunoprecipitation, a rabbit antiserum (called anti-GST-1 / 187hHDC) obtained immunizing, according to the protocol described in the patent application P9800019, with the fusion protein GST-1 / 187hHDC (called GST-1 / 187hHDC) isolated from the band an SDS-PAGE gel. Once the extracts are obtained cell phones and the antiserum, we proceeded to perform the immunoprecipitation as described below. For reduce nonspecific adsorption of the spheres of the Immunoadsorbent Protein A-Sepharose (Pharmacia) with which the antibodies of rabbits immunized with GST-1 / 187hHDC protein is incubated for 30 min at 4 ° C with a solution of BSA (15 mg / ml) in a lysis buffer (lysis buffer I) of composition 50 mM Hepes, 150 mM NaCl, EDTA 1 mM, 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 adsorption of Protein A-Sepharose spheres, 20 microliters of a suspension (50% v / v) thereof is incubated for 2 hours with 1 milliliter of serum anti-GST-1 / 187hHDC diluted 1: 500 in lysis buffer I. After incubation, the spheres of A-Sepharose protein with antibodies anti-GST-1 / 187hHDC set wash with lysis buffer I, and adjust to 50% (v / v).

A microlite of each of the extracts of HMC-1 and KU-182F cells (equivalent to 9 million cells) is incubated for 3 hours at 4 ° C with the A-Sepharose Protein spheres that have the antibody bound anti-GST-1 / 187hHDC. After successive washed, the spheres are collected by centrifugation and eluted with buffer containing 2.3% SDS; the denatured eluted ones SDS-PAGE gel electrophoresis separated followed by an electrotransference to nylon filters. Sayings filters are incubated with the AcM SIM 216-12.1 (dilution  1/2500) and the complexes formed by the AcM and the hHDC are located by a chemiluminescence reaction by incubating the filter with mouse anti-immunoglobulin conjugated with peroxidase (fig 2).

2.2. Immunorecognition Assay ("Western-blot")

To perform this assay cells are transfected HEK-293 with pcDNA 3 expression plasmid (Invitrogen) containing the sequence 1 / 512hHDC inserted under the cytomegalovirus (CMV) promoter control. The transfection is facilitates 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). The negative control is used an extract of HEK-293 cells transfected into parallel with the commercial expression plasmid pEGFP-N1 (Clontech) in its original form, which lacks of hHDC coding sequences.

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 filters nylon. Detection is carried out using as an antibody primary the AcM SIM 216-12.1 (dilution 1/2500), and as secondary antibody an anti-mouse antibody peroxidase conjugate (1/5000 dilution). The position and abundance of the bands is visualized by chemiluminescence (Fig 3). In the lane 1, which corresponds to the cell extract of cells transfected with the greatest amount of plasmid, a most intense main reaction band, corresponding to Mr intended for 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. 3rd street, no shows some band since the cells from which the  Extracts used only express the green protein plasmid fluorescent pEGGFP-N1.

2.3. Cellular immunofluorescence assays

Cells are transiently transfected HEK-293 with 1.5 micrograms of plasmid pcDNA3-hHDC1 / 512 as described in the example above, to express transiently the protein 1 / 152hHDC of HEK-293 cells. The cells are incubated in 24-well plates on object covers coated with polylysine 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. Then, the cells are washed with a solution of Isotonic phosphate-saline (PBS) and permeabilize with PBS / Triton X-100 0.2% for 5 minutes in ice. The preparation is blocked for 90 min at 4 ° C with PBS / 0.05% Tween-20 - 5% bovine fetal serum, and Subsequently, it is incubated with AcM SIM 216-12.1 (1: 500 dilution) for 45 minutes at 20 ° C. After successive washing with 0.05% PBS / Tween-20, the incubation preparation for 45 minutes with an anti-mouse antibody (1: 500 dilution) rhodamine marking (Jackson ImmunoResearch). Finally the Preparation is washed with 0.05% PBS / Tween-20 and visualized in the fluorescence microscope (Fig 4). The cells Transformed HEK-293 incubated with the antibody AcM SIM 216-216-12.1 issue fluorescence, unlike cells transfected with the plasmid control.

<110> CARLOS III HEALTH INSTITUTE and MALAGA UNIVERSITY

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Claims (15)

1. Polynucleotide, capable of encoding a polypeptide capable of generating antibodies or fragments of the same specific against hHDC, whose sequence is chosen from the group:

to.

Sequence comprising SEQ ID 1

b.

Sequence consisting of SEQ ID 1 or comprises fragments of this one.

C.

Sequence that differs from sequences a or b because to the degeneracy of the genetic code.

d.

Sequences that share at least 80%, 90%, 95% or 98% homology with any of the sequences previous.

2. Polypeptide according to claim 1, capable of generating specific antibodies against hHDC and whose sequence is chosen from the group:

to.

Sequence comprising SEQ ID 2

b.

Sequence consisting of SEQ ID 2 or comprises fragments of this one.

C.

Sequence that shares at least 80%, 90%, 95% or 98% homology sequence a or b.

3. Antibodies or fragments thereof specific against hHDC, wherein said antibodies specifically interact against any of the polypeptides of claim 2. 4. Use of any of the polypeptides according to claim 2 for the generation of antibodies or fragments of the same specific against hHDC. 5. Use according to claim 4 wherein antibodies or fragments thereof are obtained by techniques of hybridoma generation. 6. Use according to claim 4 wherein antibodies or fragments thereof are obtained by the "phage display" technology. 7. Use according to claim 4 wherein antibodies or fragments thereof are obtained by Immunization of non-human mammals. 8. Use of antibodies or fragments of themselves according to claim 3, for the preparation of a medicine. 9. Use of antibodies or fragments of themselves according to claim 3, for the preparation of a medication for the treatment of neurological degeneration. 10. Use of antibodies or fragments of themselves according to claim 3, for the preparation of a medication for the treatment of anaphylaxis and / or processes allergic 11. Use of antibodies or fragments of themselves according to claim 3, for the preparation of a medication for the treatment of epithelial degeneration digestive 12. Use of antibodies or fragments of themselves according to claim 3, for the preparation of a medication for the treatment for the treatment of different types of cancer. 13. Use of antibodies or fragments of themselves according to claim 3, for the preparation of a medicine for the treatment of infectious processes. 14. Use of the antibodies or fragments thereof according to claim 3 for the in vitro diagnosis of diseases. 15. Diagnostic kit comprising the use of antibodies or fragments thereof according to the claim 3.