MXPA97003191A - Antibodies against allogenic and yxenogenic proteins, their use in diagnosis and therapy and methods for the determination of myths - Google Patents

Abstract

The present invention relates to a method for the immunological detection of natural antibodies that recognize a protein that can be extracted from mammalian organs recognized by the monoclonal antibodies secreted by the hybridomas deposited in the ECACC under the accession numbers 930806103 or 930806104 and present in approximately 93% of human solid tumors, characterized in that it comprises the detection of an immunocomplex between natural antibodies and optionally labeled proteins.

Description

ANTIBODIES AGAINST ALGAE AND XENOGENIC PROTEINS. ITS USE IN DIAGNOSTICS- AND THERAPY AND METHODS FOR. THE DETERMINATION OF THEMSELVES DESCRIPTION OF THE INVENTION The present invention relates to diagnostic methods for the detection of natural antibodies that recognize proteins that can be extracted from mammalian organs, having a molecular weight of approximately 14 Kda units endowed with antitumor activity when administered with xenogenic species. The invention also relates to natural antibodies in purified form and to their use in diagnostics and therapy. O92 / 10197 describes protein substances obtainable by extraction of mammalian organs, particularly the liver, with perchloric acid and hypertonic saline solutions. One of said substances having a molecular weight of approximately 14 Kda units has been isolated and partially sequenced (Italian Patent Application No. MI 94001469) and it turns out that it is the main protein responsible for the biological properties observed, namely antitumor activity and its ability to increase, when administered to animals of a different species from REF: 24561 which was extracted, antibodies capable of recognizing tumor antigens. The Italian Patent Application also describes two monoclonal antibodies, secreted by the hybridomas deposited in the European Collection of Animal Cell Cultures (ECACC), Portón Do n, Salisbury, UK under the numbers 930806103 and 930806104 that recognize common epitopes for tumor antigens. and for the 14 Kda liver protein of CABRA (hereinafter referred to as UK 114) used as the antigen in the hybridoma preparation. It has now been found that natural antibodies that recognize both allogeneic and xenogeneic proteins that can be extracted from organs (particularly liver) endowed with the properties described above and present in approximately 93% of solid human tumors, circulate in mammalian serum . Antibodies, normally present in low titer (<1/100) in the serum of healthy animals or humans, can be stimulated by injecting the corresponding antigen, causing an extraordinary increase in titer and capacity in the sera to produce cytotoxic effects against the cells cancerous Regardless of the possible interpretations of the results observed and the significance of the presence of the natural anti-UK 114 antibodies, the interpretations of which are out of place for the validity of the present application, the antibodies and their detections are useful for of diagnosis, prognosis and therapy. In a first embodiment, the invention therefore relates to a method for the immunological detection of natural anti-UK 114 antibodies. The invention also relates to purified anti-UK 114 antibodies and their use in diagnosis, therapy and immunocytochemistry. The methods for the detection of natural antibodies are of the conventional type and can be based on direct, competitive reactions or reactions of the "sandwich" type. The protocols can be used either as solid supports or immunoprecipitation techniques. The antibodies and / or antigens of the type UK 114 used in the immunoassays will be properly labeled with the enzymes (for example, in ELISA techniques) or with radioactive or fluorescent compounds, dyes or pigments, etc. The immunocomplex signal can optionally be augmented by the biotin-avidin system. The choice of the configuration of the method and the relative materials is within the skill of an average technician. It has also been found that anti-UK 114 antibodies present in the serum of patients treated with the proteins described in WO92 / 10197 or with purified UK 114 are endowed with cytotoxic and cytolytic activity in vitro and in vivo against human tumor cells. The invention, in another embodiment, also provides a cytotoxic and cytotoxic composition comprising monoclonal or polyclonal antibodies or hyperimmune sera, produced by the immunization of animals or humans with proteins that can be extracted from goat liver with perchloric acid and recognized by secreted monoclonal antibodies by the hybridomas deposited in the ECACC under the accession numbers 930806103 or 930806104. In vivo, the cytotoxic activity reaches a peak of a few days after the administration of the antigen (UK 114 or UK 101) and decreases slowly in subsequent weeks. The titer of anti-UK 114 antibodies of the IgG type, on the other hand, increases slowly over time as evaluated by an ELISA method. It can be assumed, therefore, that the first peak of cytotoxicity is due to the IgM type antibodies and / or other serum components not yet identified. In any case, the importance of the methods that allow the determination of antibodies and / or cytotoxicity is evident, in such a way that the therapy can be adequately monitored. The administration of the antibodies, either natural or monoclonal, according to the invention, can be carried out parenterally at doses sufficient to achieve a cytotoxic effect. Suitable administration methods are substantially identical with those already known for the administration of vaccines, antibodies or hyperimmune sera. This invention and the following examples 2-4 provide another guide for the therapeutic and diagnostic applications of the antibodies described in the foregoing. The following Examples further illustrate the invention.

Example 1 2.5% SDS and 5% mercaptoethanol are added to 100 μl of goat liver perchloric extract (WO92 / 10197; UK 101). The samples are heated at 100 ° C for 5 minutes and 4 μl is analyzed on SDS-PAGE using the PHAST device SYSTEM (Pharmacia) with gradient polyacrylamide gel 8/25 (Pharmacia). Alternatively, homogeneous gels even at high density can be used. The separated proteins are transferred electrophoretically using the Phast-Transfer apparatus (Pharmacia). The nitrocellulose membrane (0.22 μ) is pre-balanced in Tris buffer (25 mM) glycine (192 mM) to which 20% methanol was added. The test was performed at 5V x 20 minutes (I A / cm2). Then the membrane is incubated, after one pass of saturation, to avoid nonspecific binding, with the serum under examination.

The binding of the antibody optionally present was detected with a second anti-human antibody A3b, suitably labeled for colorimetric, radioimmunometric or chemiluminescent methods.

Example 2 Immunosorbent assays bound to an enzyme (ELISA) for the detection of antibodies to UK 114. The following buffers were used: (A) 50 mM sodium bicarbonate, pH 8.5, (B) 50 mM sodium phosphate, pH of 7.2, (C) 50 M sodium phosphate, pH 7.4, 0.1% (w / v) NaN3, (D) 40 mM potassium phosphate, pH 7.4, 150 mM NaCl, 0.02% (w / v) NaN3 , 0.05% (v / v) Tween 20, (E) 50 mM sodium phosphate, pH 7.4, 0.1% (w / v) NaN3, 0.1% (w / v) BSA, (F) sodium phosphate 50 mM, pH 7.4, 150 mM NaCl, 0.1% (w / v) NaN3, 0.5% (w / v) BSA without protease, 0.05% (v / v) Tween 20, 0.5% (v / v) of normal goat. 3.5 ml of UK 114 (3.5 mg) in buffer A are mixed with 9 μl of NHS-LC Biotin (S.p.A., Milan) at 2 mg / ml in dimethyl sulfoxide. The mixture is incubated at 22 ° C for 3 hours and dialyzed for 24 hours at 4 ° C against several changes of buffer B in a Spectrapor 6 dialysis tube (exclusion of 3500 D units). The microtitre wells are coated with 100 μl of BSA-biotin (5 μg / l in C buffer) for 24 hours at 4 ° C, then washed and treated with 100 μl of streptavidin (5 μg / ml in buffer E). After a second incubation overnight at 4 ° C, the wells are washed and treated with 100 μl of UK 114-biotin at a different concentration (2, 1 and 0.5 μg / ml in buffer E) for 24 hours at 4 hours. ° C. After washing, the wells are blocked. Wells uncoated with antigen are prepared following the same procedure, except that the third stage of incubation using buffer E instead of UK 114-biotin solution. The washing step is carried out by adding 300 μl / well of buffer D three times. Blocking is accomplished by adding 300 μl of buffer C containing 10% sucrose and different blocking agents: 1% and 5% bovine serum albumin, 1% glycine, 1% BSA added with 3% polyvinylpyrrolidone, % normal goat serum. After 30 minutes at room temperature, the blocking solution is discarded and the wells are dried in a vacuum chamber and then stored rolled up in polyethylene and a thin sheet of aluminum at 4 ° C until use.

ELISA procedure Samples of serum and negative control are diluted 1:21 with buffer F before being tested. 100 μl of samples and standard solutions (1, 2, 4 and 8 U / ml in buffer F) are added to the coated wells and incubated for 1 hour at room temperature on a horizontal shaker (400-500 rpm ). The wells are then washed four times with 300 μl of buffer D, after which 100 μl of a 1: 3000 solution of goat anti-human IgG horse radish peroxidase-labeled antibody, dissolved in the Stabilgen Shock Absorber, is added. After 1 hour of incubation at room temperature on the horizontal shaker (400-500 rpm), the plates are washed and developed using 100 μl of Blus Star Solution (H202 / TMB). After incubation at room temperature (18-24 ° C) for 15 minutes, the reaction is stopped by adding 100 μl of 1 M H2SO4 and the optical density (OD) is read using a double wavelength photometer at 450 nm and 630 nm as the reference wavelength. Samples with OD greater than 8 U / ml standard are further diluted before being tested. The concentration of the sample is calculated from the standard curve.

Title Determination in Patients Supporting Immunotherapy The evaluation of the anti-UK 114 antibody titer, which is carried out in patients who support therapy for UK 101 show an antibody response greater than the background value in 28% of cancer patients of breast and 46% of patients with colon cancer just after 15 days of administration of UK 101 (4 subcutaneous injections). After 40 days from the start of UK 101 therapy, the increase in anti-UK 114 antibody titer can be detected in 63% and 97% of patients with colon and breast cancer, respectively (Figures 1 and 2) .

Example 3 Cytotoxic effect of the anti-UK antibody 114 on tumor cell lines. The tumor cell lines KATO-III (gastric carcinoma) and NICH (small cell lung carcinoma), respectively positive and negative for surface staining by indirect immunofluorescence with the monoclonal antibody secreted from the hybridoma P3D1DII deposited in ECACC under the number 930806103 and rabbit anti-UK 114 polyclonal antibody (rAb), are used for cytotoxic studies. Cytotoxicity is evaluated as the percentage of 51Cr release of cell lines incubated for variable periods of time with different concentration of the monoclonal antibody mAb P3D1DII, anti-UK 114 Ab Rb or with a monoclonal or polyclonal antibody unrelated as the control, in the presence of a homologous or heterologous complement source (guinea pig) or in the presence of purified human monocytes. It was found that both mAb antibodies P3D1DII and anti-UK 114 rAb but not the control-uncoupled antibodies induced in KATO-III a complement-dependent cytotoxicity, which was absent when the heat-inactivated or C9-deficient sera were used as a complement source. The cytotoxicity was restored when the deficient serum of C9 was reconstituted with purified C9. In addition, the anti-UK 114 Rb Ab, but not the unrelated Rb Ab induced antibody-dependent cellular cytotoxicity (ADCC), when KATO-III was used as the target. The preabsorption of the anti-UK-114 antibody with the purified antigen, eliminated the cytotoxicity induced by the antibody. In contrast, no complement cytotoxicity or ADCC was observed for NICH. These results suggest that anti-UK 114 antibodies have a potential complement-dependent cytotoxicity and antibody-cell for tumor cell lines with surface expression of the UK-114 antigen.

Example 4 The in vivo cytotoxicity of anti-UK 114 antibodies in nude mice xenografted with human tumor cell lines. The nude mice are stimulated s.c. with 1 x 106 human colon adenocarcinoma cells HT29. 8 hours later, received an injection at the tumor site of either 0.2 ml of anti-UK 114 rabbit hyperimmune serum (titre 1/12) produced by immunization of rabbits with UK 114. The treatment was repeated on day 7, 11, 14, 18, 21, 25 after the stimulus. Another group of mice was treated similarly with sera from tumor patients who responded clinically to the administration of UK-114 and the antibody produced anti-UK 114 Ab. A control group received only the vehicle. The treated groups showed a statistically significant increase in life extension and decrease in new tumors. The latency of the tumor was also extraordinarily prolonged in the treated groups compared to the control group. By way of example, the results obtained with the rabbit antiserum and with the human serum are reported in the following Table.

It is stated in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (4)

1. A method for the immunological detection of natural antibodies that recognize a protein that can be extracted from mammalian organs recognized by the monoclonal antibodies secreted by the hybridomas deposited in the ECACC under accession numbers 930806103 or 930806104 and present in approximately 93% of human solid tumors, characterized in that it comprises the detection of an immunocomplex between the natural antibodies and the optionally labeled proteins.

2. The method according to claim 1, characterized in that the immunocomplex is detected with a second labeled anti-spies antibody.

3. Natural antibodies that recognize proteins that can be extracted from mammalian organs, recognized by the monoclonal antibodies secreted by the hybridomas deposited in the ECACC under accession numbers 930806103 and 930806104 and present in approximately 93% of human solid tumors.

4. The cytotoxic compositions characterized in that they comprise antibodies or hyperin unes sera, produced by the immunization of animals or humans with proteins that can be extracted from goat liver with perchloric acid and recognized by the monoclonal antibodies secreted by the hybridomas deposited in the ECACC under the numbers of access 930806103 or 930806104.