ITMI20072083A1 - MONOCLONAL RABBIT ANTIBODY (MAB CV152) ANTI-INDOLEAMINE 2,3-DIOXYGENASIS (IDO) - Google Patents

Description

Patent application for industrial invention entitled:

MONOCLONAL RABBIT ANTIBODY (MAB CV152) ANTI-INDOLEAMINE 2,3-DIOXYGENASE (IDO).

FIELD OF THE INVENTION

The present invention relates to a rabbit monoclonal antibody, for use as an immunodiagnostic and research reagent.

FRONT TECHNIQUE

The IDO antigen (indoleamine 2,3-dioxygenase) is an intracellular enzyme involved in the complex system of regulation of the cell-mediated immune response. It is a 42 KDa monomeric hemoprotein, expressed at the level of various districts such as the brain, intestine, lungs, placenta and main lymphoid organs. The importance of the immunoregulatory role of IDO was defined following the discovery of the maternal-fetal tolerance mechanism, which may probably concern the phenomenon of immune tolerance in general. The enzyme plays a key role in the catabolism of tryptophan, the so-called kynurenine pathway. It catabolizes a limiting step: the conversion of the amino acid into N-formylquinurenine by breaking the pyrrole ring of tryptophan. The product of this reaction becomes a substrate for the subsequent reactions that lead to the formation of kynurenine, anthranilic acid and the corresponding 3-hydroxylated derivatives, quinolinic acid, and NAD <+>, an enzymatic cofactor that contributes to cellular energy metabolism. Tryptophan is an essential amino acid: therefore, its catabolism by IDO can exert a cytostatic effect against various cells, including some microbes and neoplastic cells. Alongside its antimicrobial and anticancer effects, IDO is an important immunoregulatory molecule. This action was particularly evident in accessory immune cells, such as dendritic cells (CD), where the highest levels of expression of this enzyme were found. CDs are mainly responsible for the stimulation of T lymphocytes. In CDs, IDO is induced by the action of IFN-γ (gamma-type interferon). The regulation of the immune system, due to the activity of this enzyme, essentially derives from the modulation of the amount of tryptophan and the synthesis of kynurenins. An increase in the active enzyme at the CD level causes a reduction in tryptophan both intracellular and in the surrounding environment; the consequence is the blockage of the cell cycle of leukocytes.

The action of the enzyme is also linked to the formation of the products of catabolism. Both anthranilic acid and quinolinic acid play a role in modulating the proliferation of T lymphocytes; moreover, kynurenins interact directly with T cells, activating their apoptotic pathway, thus causing a reduction in the activities they perform. It is therefore evident that the IDO enzyme plays a fundamental role in maintaining immune homeostasis and regulating peripheral tolerance. However, there are still many unclear aspects not only of the immunology of this enzyme but also of how its catalytic activity is regulated once expressed. Anti-IDO antibodies are currently commercially available, for example the anti-IDO monoclonal antibody, clone 10.1, (Upstate, cat. 05-840) and the rabbit polyclonal antibody (Chemicon, cat. AB9900).

These antibodies are generally used in Western blots.

SUMMARY

The present invention relates to a rabbit anti-IDO hybridoma (indoleamine 2,3-dioxygenase) deposited on 25/9/2007, at the CBA of Genoa with n ° PD7004 and the anti-IDO rabbit monoclonal antibodies obtained from it (Mab cv152). The antibody is characterized by the fact that it recognizes the indoleamine 2,3-dioxygenase enzyme in both native and denatured form, preferably murine (Acc N ° NP_032350). In particular, it recognizes the epitopes present on at least two of the following peptides: IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and IDO-C: CRGTGGTNPMTFLRSVKDTTEK (SEQ ID NO: 3) derived from the murine IDO protein sequence, even more preferably in combination with the epitopes present on the IDO-A peptide: CKPSKKKPTDGDKSEEPSNVES (SEQ ID NO: 1).

The invention also relates to a composition comprising at least one of the following IDO-A peptides: CKPSKKKPTDGDKSEEPSNVES (SEQ ID NO: 1), IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and IDO-C: CRGTGGTNPMTFLKQ NO: ) or the analogs derived from the human IDO sequence, for use as immunogens, preferably after conjugation with a suitable carrier protein, such as for example KLH (Keyhole limpet hemocyanin), in combination with suitable adjuvants.

All the marked or otherwise modified forms, even covalently, of this antibody, as well as the purified, lyophilized and / or frozen forms, also fall within the present invention, provided that the antibody maintains its specificity.

The invention also relates to a process for the preparation and purification of the antibody starting from the hybridoma, which is amplified in a suitable growth medium and in conditions of optimal cell density for the purpose. The medium conditioned by the hybridoma is recovered, loaded on an affinity column, for example with protein-A sepharose for antibody purification.

DESCRIPTION OF THE FIGURES

Figure 1: IDO antigenicity profile. From the analysis of the protrusion index it appears that the last 70 amino acid residues (indicated by the arrow) have the most pronounced immunogenic characteristics. Figure 2: Antibody titre of one of the rabbits immunized with the conjugated peptides evaluated by ELISA test. The results relate to one of the two immunized rabbits. The antibody titer is higher in immune samples even at higher dilutions; the difference between the antibody titers of the immune and basal samples allows us to positively evaluate the effectiveness of the immunization of rabbits.

Figure 3: Results obtained by Western Blot performed with immunized rabbit antiserum (Pre-screening phase). The 42 KDa molecular weight band corresponds to the IDO enzyme recognized by the specific antibodies present in the antisera of immunized rabbits. Lane HTR: cells transfected with the empty vector or with the cDNA encoding for IDO with the gene construct described in example 3.

Figure 4: Western Blot performed on different culture supernatants of the single clones of the hybridoma cv152. HTR: non-transfected cells; HTR-IDO: transfected cells; DC: Dendritic Cells; DC-IFN-γ: dendritic cells stimulated with interferon-γ.

Figure 5: Western Blot of the purification of the monoclonal antibody cv152 using an affinity column. The lysates of HTR and HTR-IDO cells were separated electrophoretically, blotted on nitrocellulose and experimented with: 1: culture supernatant of the cv152 clone before purification; 2; flow through; 3: purified Mab cv 152 (concentration 2 μg / ml); 4: Purified Mab cv 152 (concentration 4 μg / ml).

Figure 6: Western-blot with purified Mab cv152. Dendritic cell (DC) lysates activated or not activated with IFN-γ were used.

Figure 7. Immunoprecipitation. The test was performed on lysates of HTR cells not transfected, or transfected with the gene encoding for IDO that express it in a 42 KDa band. It is noted that the antibody recognizes the protein and precipitates it. Arrows: H, Heavy Chain of the antibody used in the immunoprecipitation; IDO, specific band of the immunoprecipitated enzyme.

Figure 8. Immunofluorescence with the antibody Mab cv152 conjugated to Alexa Fluor 488 (A20181 Molecular Probes), on non-transfected HTR cells, or transfected with the gene encoding for IDO, which is highlighted as bright (green) cytoplasmic granules.

Figure 9. Comparative Western Blotting. The anti-IDO monoclonal antibody, clone 10.1, (Upstate, cat. 05-840), the rabbit polyclonal antibody (Chemicon, cat. AB9900) and Mab cv152, all at a concentration of 2μg / mL, on non-HTR cells transfected, and transfected with the gene encoding for IDO.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an anti-IDO (indoleamine 2,3-dioxygenase) hybridoma of rabbit deposited on 25/9/2007, at the CBA of Genoa with n ° PD7004, and the monoclonal antibodies derived therefrom. Hybridoma was obtained by fusion of splenocytes, derived from rabbits immunized with a mixture of peptides derived from the 350-405 region of the mouse IDO enzyme residues 350-405, and rabbit plasmacytoma cells (H Spieker-Polet et al. , 1995, PNAS USA 92: 9348-9352) in order to obtain hybrid cells that maintain the growth characteristics of tumor cells and the synthesis of antibodies typical of B lymphocytes. The hybridomas were prepared following the fusion protocol described in the same article.

The hybridoma grows in continuous culture and in suspension, preferably in RPMI medium containing glucose, L-glutamine, Hepes, MEM Amino acids, MEM Non-essential amino acids, MEM Vitamins, sodium pyruvate, sodium bicarbonate, 2-Mercaptoethanol, Gentalyn, also containing 10-15% FCS. The karyotype is an intraspecific fusion and the antibody isotype has been characterized as a rabbit IgG. To obtain the antibody of the invention, a mixture of three peptides was used, IDO-A: CKPSKKKPTDGDKSEEPSNVES (SEQ ID NO: 1), IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and IDO-C: CRGTRSGTKDPMTT in quantity equimolar, conjugated to the carrier protein KLH, in combination with suitable adjuvants (preferably FIA and FCA), according to a particularly efficient protocol, which provides for a first intradermal immunization with 150 µg of antigen, conjugated with KLH, with the addition of FCA (Freund's complete adjuvant ) for each animal, and then 2 successive intramuscular immunizations, spaced 2 weeks apart, with lower amounts of conjugated antigen (50 pg), with the addition of FIA (Incomplete Freund's Adjuvant) for each immunization, per animal.

days steps Amount of FCA antigen / adjuvant

1 ml

0 I immunization 150 μg / ml FCA

14 II immunization 50 μg / ml FIA

28 III immunization 50 μg / ml FIA The antibody produced by the hybridoma selected from a certain number of hybridomas, recognizes the indoleamine 2,3-dioxygenase (IDO) enzyme, both in native and denatured form. It recognizes the IDO protein, of molecular weight 42KDa by SDS-PAGE, both in Western-blot and in immunoprecipitation, as well as in immunofluorescence. This implies that the immune response induced by the mixture of the three peptides mimics the immune response induced by the protein in its native and three-dimensionally conformed form.

The antibody, a rabbit IgG, preferably recognizes the murine enzyme, of sequence as in Acc N ° NP_032350, both in recombinant form, expressed for example in mast cell tumor (HTR) starting from the cDNA sequence, and the protein natural murine, produced in dendritic cells, such as those extracted from the mouse spleen in which it can be overexpressed after incubation with interferonγ, allowing the visualization of a specific band of 42 KDa (MW corresponding to the IDO protein, determined by denaturing SDS-PAGE) in lysates of both cellular preparations.

Regarding antigenic specificity, it is useful to remember that the murine protein is obviously not immunogenic in the mouse, while it is in the rabbit. This represents one of the advantages of using the rabbit system for the preparation of hybridomas, compared to the classic mouse system, in particular for this protein. The rabbit system in fact allows to have antibodies with remarkable specificity and affinity even towards murine antigens. In this case this is particularly useful as the system of study of the molecular mechanisms in which indoleamine 2,3-dioxygenase is involved (regulation of the cell-mediated immune response and probably in the phenomenon of immune tolerance) is for now only murine.

The antibody purified from the hybridoma of the invention by affinity chromatography for example on Protein A-Sepharose, due to its characteristics of affinity, specificity and stability, makes possible all immunodiagnostic applications, such as immunoprecipitation and immunofluorescence (also by direct labeling of the antibody with fluorochrome) which are not possible or which do not give comparable results with antibodies known in the art.

These properties are therefore unexpected with respect to known antibodies, both mouse monoclonal and rabbit polyclonal.

There are also advantageous features that the hybridoma can be amplified in a practically unlimited way over time and that it constitutes an inexhaustible source of antibody, by simple amplification in culture, under conditions known to the skilled in the art. The conditioned medium containing the antibody in high concentration can be stored and frozen allowing purification of semi-industrial quantities of antibody starting from different pools of conditioned medium. Therefore, the conditioned supernatant obtained by growing the hybridoma in the growth medium and in the conditions of cell density most suitable for the fermentation purpose, as well as the anti-IDO rabbit antibody purified from this medium, falls within the scope of the present invention.

The results obtained, in terms of antibody properties, are attributable not only to the immunization protocol but above all to the choice of the immunogen and in particular to the selection of the IDO-A, B and C peptides in the most hydrophilic region of the IDO protein.

According to a further embodiment, the invention therefore relates to a composition comprising at least one, or preferably two (IDO-B and IDO-C), even more preferably all three, the following peptides: IDO-A of amino acid sequence CKPSKKKPTDGDKSEEPSNVES (SEQ ID NO : 1), IDO-B, CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and IDO-C, CRGTGGTNPMTFLRSVKDTTEK (SEQ ID NO: 3) as well as the homologs derived from the human IDO sequence (Acc. No. NP_002155) for use as immunogens, preferably in equimolar quantities, optionally in combination with suitable adjuvants.

The immunogenic composition, preferably administered with adjuvants known in the art, such as complete or incomplete Freund's, used in the immunization protocol described above, made it possible to obtain an antibody with peculiar characteristics and therefore fully falls within the scope of the present invention.

The tests carried out, better detailed in the experimental part, allow the use of the cv152 antibody to be extended to any immunoassay, including immunoprecipitation, Western-blot, ELISA and immunofluorescence. This represents an advantage over currently available reagents, which are generally to be used only in Western-blot.

The immunoassay can be designed in combination with other standard reagents or proteins or even as a competitive assay. The invention also comprises a kit in which the cv152 monoclonal antibody, preferably in purified form, is present, in a hermetically sealed tube or vial, or in lyophilized form, or resuspended in a suitable buffer, this buffer optionally comprising stabilizing agents and / or bacteriostatic agents. In the kit, the antibody is associated with secondary reagents and dependent on the assay in which it is used, for example with optionally labeled secondary antibodies to rabbit IgG, or with suitable buffers for the dilution of the antibody or with reagents for the colorimetric development of the immunological reaction, together with a package insert of the conditions of use.

The antibody according to the present invention also has the advantage of being able to be used directly in the marking with fluorochromes, resulting stable to such treatments.

Preferably, fluorochromes such as: fluorescein, phycoerythrin, phycocyanin or other commercial fluorochromes known in the art are used as labeling molecules, such as AlexaFluor 488 (Molecular Probes A-20181). Other markers, for example radioactive substances, such as <125> l, or enzymatic markers (such as HRP, β galactosidase, alkaline phosphatase etc.) capable of generating a chromogenic reaction (chromogenic enzymes), can however be used. According to this aspect, therefore, all the marked forms of this antibody, or in any case modified without altering the specific binding properties, also fall within the invention.

The characteristics of the antibody, in particular the fact that it recognizes the murine protein in its three-dimensional conformation, allow its use in co-immunoprecipitation studies where the formation of immune complexes including the proteins that functionally interact with the IDO protein can be evaluated. its native and / or active form and possibly their dosage. It is currently known that IDO can exist in both active and inactive form, but the regulatory mechanisms are still obscure. Coimmunoprecipitation tests aimed at identifying the association between IDO and other enzymes such as methyl transferases, kinases, phosphorylases and others, could reveal how and in response to which stimuli the protein acquires the active form. The results of these studies on IDO would make the molecular mechanisms through which the kynurenin pathway is modulated by post-translational modifications of the enzyme more understandable.

According to a further aspect, the invention relates to a process for the preparation and purification of the antibody starting from the hybridoma deposited on 25/9/2007, at the CBA of Genoa, with n ° PD7004, in which the hybridoma is amplified in a suitable medium , and in conditions of optimal cellular density and the conditioned medium is recovered, possibly filtered or deprived of cellular debris and then loaded on an affinity column, for example with protein A sepharose for the purification of the antibody on a laboratory scale.

The availability of this antibody in semi-industrial quantities would make it possible to standardize quantitative assays on an international scale, which is highly desirable for an enzyme such as IDO, which is involved in immune tolerance regulation mechanisms and which also has antimicrobial and antimicrobial activity. antitumor.

EXPERIMENTAL PART

Example 1. Preparation and immunization of animals with the peptides derived from the IDO protein.

Animals

White New Zealand rabbits were supplied by Charles River, Calco, Italy. The animals used were housed under standard conditions for two weeks (humidity 65 ± 5%, temperature 22 ± 1 ° C, 12 hours of light alternating with 12 hours of darkness) before immunization.

Preparation of the peptides

The entire amino acid sequence of the murine IDO enzyme, equal to 407 amino acids (GenBank deposit NP_032350) was analyzed using a special multimedia program (OMIGA 2.0) to identify the most hydrophilic portions of the molecule. Within the most hydrophilic region, according to the analysis of the program (see Figure 1), some peptide regions were identified, previously used as immunogens in ten different immunizations. Based on the preliminary results of ELISA and western blotting tests of specific antisera, it was decided to sacrifice the animal whose serum had a high antibody titer and a highly specific response in the Western Blotting test. The animal selected for the purification of splenocytes had been immunized with an equimolar mixture of the following peptides: IDO-A: CKPSKKKPTDGDKSEEPSNVES (SEQ IDNO: 1)

IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ IDNO: 2)

IDO-C: CRGTGGTNPMTFLRSVKDTTEK (SEQ IDNO: 3)

The peptides were included in the 350-405 region of the mouse protein. The peptides were synthesized by A&A Lab, California, USA.

The peptides are incomplete immunogens, and have therefore been made reactive by conjugation with a suitable carrier protein. Following the Pierce protocol (Imject Maleimide Activated mcKLH Kit), the peptides were conjugated with KLH (Keyhole limpet hemocyanin) as the carrier protein.

Immunization

The conjugated peptides were used to induce the production of specific antibodies in the rabbit. In particular, an equimolar mixture of all three peptide sequences was used, administered with known adjuvants (FCA: Freund's complete adjuvant (Sigma F5881) and FIA: Freund's incomplete adjuvant (Sigma F5506). Two White New Zealand rabbits, immunized according to the protocol outlined in Table 1.

Table 1: Immunization protocol

* FCA: Freund's complete adjuvant, ** FIA: Freund's incomplete adjuvant

The plasma of each rabbit was then evaluated by ELISA, as described in the following example.

Example 2. Verification of antibody production in immunized animals by ELISA assay.

The Elisa test (enzyme-linked immunosorbent assay) allows to easily and relatively quickly detect the presence of substances with antigenic properties, or specific antibodies for these substances. In the specific case it was used to verify the production of antibodies in immunized rabbits, for the peptides IDO-A, IDO-B, IDO-C.

For this purpose, the same non-conjugated peptides were used as antigens, so as to allow the dosage of only antibodies directed against the hapten and not those produced by the animal against the carrier protein KLH. After taking the blood sample and preparing the antiserum from / from the rabbit, the test is carried out as follows:

- the mixture of the three peptides (example 1) at the final concentration of 1 μΜ each, was solubilized in glycine buffer (20 mM glycine, 30 mM NaCI, pH 9.2) and adsorbed on an ELISA plate (50 μL / well), incubating over-night at 4 ° C;

- the wells of the plate were saturated with a 10% solution of BSA (Bovine Serum Albumine) in PBS (100 μL / well) and incubated for 2 hours at 37 ° C in a humidified atmosphere. This phase serves to block the non-specific sites so that the antibody binds selectively to the peptide;

- 50 μL / well of each sample, the anti-IDO antibodies, were plated and incubated for 2 hours at 37 ° C in a humidified atmosphere;

- a secondary biotinylated anti-rabbit antibody (50 μL / well) was added and incubated for 1 hour at 37 ° C in a humidified atmosphere. This antibody was able to recognize the Fc fragment of rabbit immunoglobulins, thus allowing to reveal only those adhered to the plate by means of I DO peptides. The secondary antibody was used at a dilution of 1: 1000 in PBS, 0.5% BSA;

- the Streptavidin-Peroxidase enzyme (100 μL / well) was added to reveal the secondary antibody and incubated for 30 minutes at 37 ° C in a humidified atmosphere, and then the specific binding was revealed colorimetrically by adding to the various wells ( 100 μL / well) hydrogen peroxide (H202, substrate of Streptavidin Peroxidase) and the chroniogen ABTS (2,2-Azino-Bis acid, 3-Ethylbenzothiazolin-6-sulphonic acid, Sigma). The spectrophotometric reading was carried out at two different wavelengths: 405 nm, which allows the reading of the samples, and 620 nm, which allows to determine the absorption of the plastic of which the plate is made (White). Each step of the assay was preceded by washing the wells which allows to eliminate the components added in excess, not bound. The washing solution consists of PBS, Tween 200.05%.

The results obtained are displayed in Figure 2, in which it is shown that the antibody titer is higher in the immune samples than in the basal ones, even at higher dilutions, confirming the effectiveness of the immunization protocol.

Example 3. Verification of the specificity of rabbit antibodies and / or antisera by Western-blot.

In order to verify and study the specificity of anti-IDO antibodies, 2 evaluation systems were developed: one based on HTR cells (a clone of murine mastocytoma p1HTR, described by T.F. Gajewski, The Journal of Immunology, 1996, 156 : 2909-2917) which express the antigen in recombinant form, the other based on mouse dendritic cells, which naturally express this enzyme and in which overexpression can be induced by stimulation with interferon-γ.

Cloning and transfection of DNA encoding IDO in HTR cells.

The murine nucleotide sequence encoding for IDO (NP 032350) was introduced by cloning into the pEF-bos expression vector containing the resistance to puromycin (Mizushima S., Nagata S. (1990). Nucleic Acids Res. 18 (17): 5322). Plasmid DNA purified from the transformed and selected bacteria was transfected into HTR-type murine myeloma cells by a highly efficient electroporation technique. This technique consists in subjecting the cells to a short-term electric shock, in order to generate the pores in the plasma membrane through which the introduction of exogenous DNA can take place. As a negative control, the same HTR cells were transfected with the pefBos expression vector not containing the gene encoding the IDO enzyme (HTR).

Preparation of dendritic cells (DC)

Dendritic cells were prepared from mouse spleens using the Nycodenz density gradient purification method (Sigma D2158). These cells were incubated overnight in a thermostat with fresh medium in the presence or not of IFN-γ at a concentration of 200 ng / ml. The DCs, less adherent than other cells, were then recovered from the supernatant.

The cells were washed twice with PBS and centrifuged to remove the culture medium. Samples containing proteins were lysed and denatured with sample buffer (glycerol 10%, Sodium Dodecyl Sulphate 2%, Tris-HCI 62.5 mM pH 6.8, β-mercaptoethanol 5%). Before loading them on the gel, the samples were boiled for 5 minutes, incubated on ice for 2 minutes and microcentrifuged at high speed, then loaded onto the gel wells together with the Protein Ladder standard, (Fermentas SM 0661) containing the reference proteins by weight. molecular (PM) known, so as to have an indication of the PM of the separated proteins.

Western blot was performed using both transfected cells (HTR and HTR-IDO) and dendritic cells (DC), according to the protocol described in Fallarino, F., et al., J Immunol. 2004, 173: 3748-3754. Development was carried out by chemiluminescence.

Figure 3 shows the results of a Western blot test performed on the lysates of cells not transfected and transfected with Ido nucleotide sequence. (HTR and HTR-IDO) The polyclonal antibody derived from the serum of immunized rabbit binds the IDO protein at 42 KDa as can be seen only in transfected cells. There are also many non-specific bands in both cell subsets, due to the nature of the non-purified polyclonal antibody. This test was performed to verify if the peptide sequences used for immunization could induce a response towards the whole protein. It should be noted that only immunization with the three peptides together: IDO A, B and C gave antibodies capable of recognizing the whole protein, giving a positive result both in Western-blot and in ELISA.

Example 4. Preparation of rabbit hybridomas and selection of single clones.

Following the positivity of both the Western-blot and the Elisa test, the animal in which the antibody response had been best was selected, to be sacrificed for cell fusion.

Cell fusion

For the fusion, the splenocytes deriving from the immunized rabbit and the rabbit plasmacytoma cells 240E1 cells (H Speker-Polet et al., 1995, PNAS USA 92: 9348-9352) were used so as to obtain hybrid cells that maintain the characteristics of tumor cell growth and antibody synthesis typical of B lymphocytes. Cells were prepared following the described fusion protocol.

The fusion was carried out at 37 ° C in a serum free medium containing 50% of PEG 4000 (polyethylene glycol), prepared by melting PEG 4000 before use at 68 ° C, to which was added the medium without serum, preheated to 68 ° C, and mixed. The cells were fused in the medium at a ratio of 2 splenocytes: 1 plasmacytoma cell to obtain a cell suspension.

The cell suspension was then centrifuged at 1200 rpm for 8 minutes at RT, the supernatant was removed and the cells resuspended by adding slowly and continuously stirring medium containing 15% FCS; the cells were then plated at a concentration of 2 x 10 <5> /0.5 ml in 26 48-well plates, 800 μl per well.

Then, after 3 days, 500 μΙ were replaced in each well with fresh RPMI ++ medium (RPMI, L-glutamine, Hepes, MEM Amino acids, MEM non-essential amino acids, MEM Vitamins, Sodium bicarbonate, 2-ME, Gentalyn) 15% FCS, HAT 2X (hypoxanthine / aminopterine / thymidine concentrated twice) medium containing elements necessary for the selection of resistant hybridomas, since the growth of unfused cells is inhibited.

The culture medium was replaced every 4-5 days with RPMI medium <++> 15% FCS, HAT 1X. The identification of the different growing hybridomas was performed using the two screening tests developed (ELISA and Western Blot tests), to check for the presence of IDO-specific antibodies. All the hybridomas that tested positive for both tests were expanded to a concentration of about 5 x 10 <6> cells, such as to allow freezing in Freezing Medium (8% DIVIDED, 92% fetal bovine serum) before at -80 ° C and then in liquid nitrogen.

By selecting the supernatants of about 800 hybridomas, isolated clonally to obtain a stable clone, with ELISA and Western Blot (carried out as described in example 3) the best hybridomas were identified for the subsequent phases. An example of the Western-blot assay, carried out with the supernatant of the hybridoma selected for expansion and purification, and using IDO expressed both in recombinant and native form, is shown in Figure 4 (Figure 4).

Clonal isolation of the hybridomas was carried out by diluting the cell suspension resulting from cell fusion to a concentration of 0.5 cells / well, therefore:

- the fusion partners used for the fusion (240E1 cells) were also used as a feeder, ie as a support and as a supplier of nutrients for the clones in formation. These cells were plated at a concentration of 5 x 10 <4> cells / 100 μL in RPMI ++ medium, HAT 1X (6 96-well plates).

- the hybridoma cells to be cloned were centrifuged at 1700 rpm for 7 minutes, then washed with RPMI ++ medium without HAT and diluted to a concentration of 0.5 cells / 100 μL and added to the 6 previously prepared plates (100 μL / well);

- the plates were incubated in a thermostat at 37 ° C and after 3 days HAT was added in order to select the growing clones by eliminating the feeder cells no longer necessary.

- the clones were periodically checked and amplified in order to perform the ELISA and Western Blot tests, in order to determine the specificity and affinity of the antibody.

- This procedure was performed once again (two sequential subclonings) for the isolation of the cell clone. The clone identified with the initials cv152 was selected, which was then expanded and from whose supernatant the rabbit monoclonal antibody of the present invention, characterized as an IgG, was purified.

Example 5. Purification of Mab cv152.

The monoclonal antibody was purified after growth and expansion of the hybridoma, replacing the culture supernatant with fresh medium every 4 days, in flasks of increasing volume, for repeated expansion cycles. Hybridoma and supernatant were also frozen and thawed and the conditioned medium was stored to a total volume of 2 liters for purification.

Purification was performed at 4 ° C, using the A-sepharose protein (Sigma P3391), which is capable of binding the Fc portion of the antibody molecules, according to the manufacturer's instructions.

The culture supernatant recovered from the flasks was centrifuged at 3000 rpm for 15 minutes, filtered with a 0.4 μm filter and balanced at the same pH of the column to facilitate the subsequent binding between the antibody and protein A; the resin was activated with a wash with buffer A (NaH2P040.02M and NaCI 0.15 M) and the culture supernatant loaded into the column, the eluate was collected to check the binding capacity of the resin and a possible presence of unbound antibody ; the column is washed with buffer A, in order to remove the molecules not specifically bound to the resin (flow through) \ the antibody was eluted with a solution at pH 3, buffer B (Na2HPO40.2 M and citric acid 0.1 M). Fractions of 1 ml were collected, quickly neutralized with a solution at pH 9 (Tris / HCI 1 M), to avoid possible degradation of the antibody to acid pH.

The antibody was then dosed by measuring the optical density at 280 nm of the various collected fractions; those that appeared to be more concentrated were pooled and dialyzed against 1X PBS, according to the resin manufacturer's instructions.

The purified antibody (Mab cv152) obtained from the cv152 clone, having a concentration of 0.982 mg / ml, was frozen at -20 ° C in aliquots and subsequently used.

A new Western Blot test was performed on an aliquot of purified Mab cv152, using lysates of HTR cells, transfected and not transfected with IDO. As shown in Figure 5, tests were performed using the supernatant in full (pre-column), the eluate of the column (flow through) and the purified antibody at 2 different concentrations, in TBS-Tween, 1% BSA. The data in Figure 5 confirm that the antibody is completely purified from the culture medium using the affinity column used.

Example 6. Characterization of the cv152 antibody (purified) by Western blotting, immunoprecipitation and immunofluorescence.

In order to verify that the antibody recognized not only the protein expressed in recombinant form, but also that naturally produced by dendritic cells, Mab cv152 was tested on lysates of DC cells, stimulated or not with IFN-γ, which in such cell types, induces the overexpression of IDO.

To this end, dendritic cells were prepared from mouse spleens using the density gradient purification method with Nycodenz (Sigma, D2158). These cells were incubated overnight in a thermostat with fresh medium in the presence or not of IFN-γ at a concentration of 200 ng / ml. The DCs, less adherent than other cells, were recovered from the supernatant and used.

The cells were washed twice with PBS and centrifuged to remove the culture medium. Samples containing the proteins were lysed and denatured with sampie buffer (glycerol 10%, Sodium Dodecyl Sulphate 2%, Tris-HCI 62.5 mM pH 6.8, β-mercaptoethanol 5%). Before loading them on the gel, the samples were boiled for 5 minutes, incubated on ice for 2 minutes and microcentrifuged at high speed; then they were loaded onto the gel wells together with the Protein Ladder standard, containing the reference proteins of known molecular weight (MW).

To verify that the cv152 antibody was not only specific for the antigen produced in recombinant form, but also recognized that produced naturally by dendritic cells, the western-blot was also carried out on DC extracts, in a similar way to what has already been described in Example 3. The purified antibody was used at a concentration of 2μg / mL in TBS-Tween, 1% BSA, detected with the HRP-labeled secondary antibody. The results are shown in Figure 6 where the presence of a specific band corresponding to the IDO protein is detected, and a band with a molecular weight of about 50KDa which could correspond to the glycosylated form of the enzyme.

Immunoprecipitation with Mab purified from clone cv152 was performed on Usati of HTR cells not transfected and transfected with the gene encoding for IDO, according to the protocol outlined as follows:

- cells were washed twice with 1X PBS, lysed with RIPA (Tris-HCI 50mM, pH 7.4, NP40 1%, NaCI 150mM, EDTA 1mM, Deoxycholic acid 0.25%, Sigma D6750) IP <*> (Roche protease inhibitor cocktail 1836153, Na3V0410 μΜ, Sigma S6508), 200μL / sample, and placed on ice for 15 minutes; finally centrifuged at 4 ° C for 15 minutes at 12000 rpm. The purified antibody was added to the cell lysate (HTR and HTR-IDO) (4μg / sample) and incubated at 4 ° C for 2 hours under stirring. Protein A-agarose (Vector SP0050), 40μL / sample was added and the incubation proceeded at 4 ° C for 1 hour under stirring. The immunoprecipitate (HTR) and the immunoprecipitate HTR-IDO were washed 3 times with RIPA IP <*>, 2 times with PBS and then loaded on gel for electrophoretic separation followed by the normal Western Blotting procedure described above.

The results are presented in figure 7. The interesting fact that can be seen is that the cv152 antibody is able to recognize and immunoprecipitate the IDO protein as seen in the right lane. This binding is specific as it is absent in the lane in which the immunoprecipitate of the isate of the non-IDO-expressing HTR cells (HTR) has been loaded. This data is of fundamental importance since at the moment the monoclonal or polyclonal antibodies on the market can only be used in western blot (data refer to the package leaflets of commercial antibodies). This indicates that the cv152 antibody is able to recognize the IDO protein in denatured form, but also in its three-dimensional structure. The purified cv152 antibody was also labeled with an AlexaFluor 488 dye (Molecular Probes A-20181). In short: 10μL of a 1M sodium bicarbonate solution (final solution 1/10 = 0.1M) was added to 100μg of antibody. The solution was added to the AF488 dye, mixing gently and incubating for 1 hour at room temperature under stirring, in the dark. The reagent was purified by column chromatography, then centrifuged and eluted. It was then used in immunofluorescence to verify that, even when labeled, it was able to bind the IDO protein at the cytoplasmic level. In particular, HTR and HTR-IDO cells were used. As can be seen from Figure 8, there is a specific binding of the antibody cv152 to the IDO protein in the cells expressing this enzyme. Instead, there is only a background staining in the cells used as a negative control (HTR) was carried out according to the following steps.

The immunofluorescence assay was performed as described below. The slides were degreased and treated with 0.01 mg / ml polylysine. Transfected or non-transfected HTR cells with IDO were added, and then fixed and permeabilized with PFA 4% (Formalin 10% Sigma HT50-1-1) pH 7-7.6 and PBS TRITON X-1000.1% respectively. (Sigma). After saturation of the non-specific bonds, the labeled antibody was added, diluted 1/20 in 2% PBS BSA. A drop of DAPI (FLUKA 32670) 2 μg / mL was added and then the slide incubated 45 seconds RT in the dark, then washed with PBS, fixed with Mowiol and viewed under the fluorescence microscope.

The results obtained are described in Figure 8, which shows that the antibody can be labeled without altering its specificity.

Example 7. Comparison of the characteristics of the cv152 antibody with those of commercial antibodies.

In Figure 9 a Western-blot experiment carried out as described in Example 3. A murine monoclonal antibody (Upstate cat. 05-840) a rabbit polyclonal antibody (Chemicon, cat. AB9900) and the Mab according to the invention were used at the same concentration (2μg / mL in TBS tween 20, 1% BSA).

The antibodies were used, comparatively, on extracts of HTR cells transfected with IDO or not.

From the intensity and specificity of the bands, it can be seen that the cv152 antibody is both more efficient and more specific towards the band corresponding to IDO (42 KDa).

Sequences listing:

IDO-A: CKPSKKKPTDGDKSEEPSNVES (SEQ ID NO: 1), IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) IDO-C: CRGTGGTNPMTFLRSVKDTTEK (SEQ ID NO: 3).

Claims (17)

CLAIMS 1. Hybridoma of rabbit anti-LDO (indoleamine 2,3-dioxygenase) filed on 25/9/2007, at the CBA of Genoa with n ° PD7004. 2. Rabbit anti-IDO monoclonal antibody obtained from the hybridoma according to claim 1. 3. Antibody according to claim 2 characterized by the fact of recognizing the indoleamine 2,3-dioxygenase enzyme in both native and denatured form. 4. Antibody according to claim 3 characterized in that it recognizes the whole IDO protein (42 KDa by SDS-PAGE). 5. Antibody according to claim 4 where the IDO protein is murine. 6. Antibody according to claims 3-5 characterized in that it recognizes at least one of the epitopes included in the peptides: IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and IDO-C: CRGTGGTNPMTFLRSVKDTTEK (SEQ ID NO: 3) both when present on the whole protein and in a peptide form. 7. Antibody according to claims 2-6 conjugated with a tracer. 8. Antibody according to claim 7 wherein said tracer is selected from: a fluorochrome, biotin or a chromogenic enzyme. 9. Process for preparing the monoclonal antibody according to claims 2-6 in which the hybridoma according to claim 1 is used. 10. Use of the antibody according to claims 2-8 in immunoassays. Use according to claim 10 where such immunoassays are selected from the group consisting of: immunoprecipitation, Western blot, ELISA, immunofluorescence. 12. Kit comprising the anti-IDO rabbit antibody according to claims 2 - 8 for the determination of the presence of the IDO enzyme, the dosage and / or characterization of the protein. 13. Kit according to claim 12 where the antibody is in lyophilized form, or resuspended in a buffer solution, optionally also frozen, in natural or marked form. 14. Use of the antibody according to claims 2-8 to identify intracellular interactions of the IDO enzyme. 15. Composition comprising at least one of the following IDO-A peptides: CKPSKKKPTDGDKSEEPSNVES (SEQ ID NO: 1), IDO-B: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and IDO-C: CRGT GGTN PMTFLRSVKD NOTEK () IDO-C: CRGT GGTN PMTFLRSVKD NOTEK () as an immunogen. 16. Composition according to claim 15 comprising the peptides IDO-B and IDO-C, respectively of sequence: CKSEEPSNVESRGTGGTNPMT (SEQ ID NO: 2) and CRGTGGTNPMTFLRSVKDTTEK (SEQ ID NO: 3). 17. Use of the composition according to claims 15-16 for the preparation of an immunogen for the IDO protein, optionally in combination with suitable carrier proteins.