IE911285A1 - Monoclonal antibodies against PP4, processes for the¹preparation thereof and the use thereof - Google Patents

Abstract

The invention relates to monoclonal antibodies specifically directed against the protein PP4, to hybridoma cell lines producing such antibodies, to a method for the production of such antibodies and to the use thereof for purifying PP4 or for diagnosis.

Description

BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 019 - Ma 830 Dr. Ha/Sd Description Monoclonal antibodies against PP4, processes for the preparation thereof and the use thereof The invention relates to monoclonal antibodies specifically directed against the protein PP4, to hybridoma cell lines producing such antibodies, and to the use thereof.

The isolation and physicochemical characterization of 5 placental protein 4 (PP4) was carried out for the first time by Bohn (EP 0123 307). This protein was also found subsequently by other groups of researchers, who did not initially recognize its identity and called it annexin V, lipocortin V or VAC alpha. Sequencing of the correspond10 ing cDNA (Grundmann et al. (1988) Proc. Natl. Acad. Sci. 85. 3708-3712) showed that PP4 belongs to a family of proteins which are now called lipocortins or annexins.

The annexins play a crucial regulatory part in the release of mediators of inflammation. It is possible by administering annexins to inhibit the release of mediators of inflammation.

The annexins are valuable therapeutics for coagulation disturbances such as disseminated intravascular coagulation (DIC).

Human placenta, which is available in adequate quantity, represents a suitable source for obtaining PP4. The established processes for purifying PP4 from natural sources are, however, elaborate and associated with losses of PP4. A similar statement applies to genetically engineered PP4 which can be obtained in biologically active form from cultures of E. coli or animal cells. - 2 There is thus a need for a rapid purification process which is associated with minimal losses.

The use of monoclonal antibodies for purification purposes has already been described and established for some other proteins. The conditions and the agents which can be used to elute again the protein which is bound to immobilized monoclonal antibodies are crucial for the yield and the native properties of the purified protein. It is often possible to achieve dissociation of monoclonal antibody and protein only by extreme pH values or chaotropic agents, which frequently leads to denaturation of the protein. This is why not any monoclonal antibody is suitable for rapid and non-deleterious purification.

The object of the invention was therefore to develop monoclonal antibodies against PP4 which, when bound to suitable supports, permit non-deleterious and efficient purification of PP4 from various starting materials.

The invention relates to monoclonal antibodies against PP4, to a process for the preparation thereof and the use thereof, preferably for the purification or diagnosis of PP4 .

Surprisingly, monoclonal antibodies which bind the annexin PP4 only in the presence of divalent cations, preferably calcium, have been found. Solutions which do not contain calcium or contain chelating agents are able to desorb PP4 in biologically active form and with high yield and purity from the antibody. Such monoclonal antibodies, bound to suitable support materials, are very suitable for purifying PP4 from human cells or organs as well as for the purification of a recombinant PP4 from cultures of E. coli or animal cells. Such antibodies can likewise be used for diagnostic purposes.

As has been shown, PP4 can be regarded as a marker for certain diseases. Normally, PP4 is detectable only in very low concentrations (0-10 ng/ml) in human body fluids such as, for example, plasma. The concentration of this protein is significantly increased in certain diseases, for example some tumors, and can be used as a parameter for rapid diagnosis. This includes both the quantitative determination of PP4 with the aid of monoclonal antibodies in body fluids, for example using a radioimmunoassay or enzyme immunoassay, and the qualitative or quantitative determination on cell surfaces, in tissue extracts or on histological specimens, such as tissue sections.

Antibodies suitable for these purposes can be prepared in the following manners Obtaining antibody-producing cells Mammals, for example BALB/c mice, are immunized by subcutaneous injection of an emulsion of 50 ^g of PP4 (purified from human placenta as described in Example 1) in complete Freund's adjuvant (day 1). On each of days 28 and 56, 50 μς of PP4, emulsified in incomplete Freund's adjuvant, are likewise injected subcutaneously. This is followed on day 92 by an intraperitoneal injection of 100 μg of PP4 in 0.5 ml of physiological saline. On day 95 lymphocytes are obtained by mechanical disintegration of the spleen.

Fusion of lymphocytes with myeloma cells Hybridoma cells are obtained by standard processes (Kdhler and Milstein, 1975; Nature 256:495-497): for example, the myeloma cell line SP2/0-Agl4 is cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). For a fusion to generate hybridoma cells, typically the spleen cells from one mouse (about 108) are mixed with 5 x 107 myeloma cells, washed in serum-free DMEM and spun down. After the supernatant has been completely removed, 0.5 ml of a 50% strength solution of polyethylene glycol 4000 in DMEM are added dropwise over the course of 1 minute to the cell pellet. The suspension is incubated at 37 °C for 90 seconds and then diluted by addition of 7.5 ml of DMEM over a period of 5 minutes. After incubation at room temperature for 10 minutes, DMEM is added to make up to 40 ml, and the cells are spun down. After the supernatant has been aspirated off, the cells are resuspended in DMEM containing 20% FCS and dispensed on 6 microtiter plates (200 pi per cavity). Hybridoma cells are selected by adding 13.6 mg/ml hypoxanthine, 0.18 mg/ml aminopterin and 3.9 mg/ml thymidine (HAT medium). The medium is replaced by fresh one at intervals of 3 - 4 days and, after 10 days, HAT medium is replaced by HT medium.

Biotinylation of PP4 Biotinylated PP4 is required for the assay for PP4specific antibodies. This can be prepared in the following ways PP4 is dialyzed against 0.1 M NaHCO3, pH 8.0. To the solution is added 0.1 mg of D-biotin N-hydroxysuccinimide ester (0.1% strength solution in dimethyl sulfoxide) per milligram of protein. The reaction mixture is incubated at room temperature for 4 h and then dialyzed against 0.01 M Na2HPOA, 0.01 M NaH2PO4, pH 7.2, 0.15 M NaCl.

Assay for PP4 antibodies 14 days after the fusion, the cell culture supernatants of the fused cells are assayed for antibodies against PP4 using an enzyme immunoassay: Polystyrene microtest plates are incubated with 0.5 pg/ml goat anti-mouse IgG in 0.1 M NaHC03, pH 9.6 (24 h, 4°C). Subsequently, cell culture supernatants are applied (2 h, 37 °C), followed by incubation with biotinylated PP4 (1 pg/ml). This is followed by incubation at 37“C for 30 min with a complex of avidin and biotinylated peroxidase (1 ^g/ml each). The substrate used is a solution of 0.1% (weight/volume) 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonate) and 0.012% (volume/volume) H202 in 0.1 M citric acid, 0.1 M Na2HP0A, pH 4.5. After incubation at 37 °C for 30 min, the absorption at 405 nm is measured. Washing between the individual incubation steps was carried out with 0.01 M Na2HPO4, 0.01 M NaH2POA, pH 7.2/ 0.15 M NaCl, 0.05% Tween 20 (PBS/Tween). All the reagents are diluted in 0.02 M Tris/HCl, pH 7.4, 0.15 M NaCl, 0.02 M CaCl2, 2% bovine serum albumin (TBS/BSA). The enzyme immunoassay is carried out in parallel using the same cell culture supernatants, but replacing the calcium chloride in the dilution buffer by 0.02 M EDTA. Antibodies which bind PP4 only in the presence of CaCl2 but not of EDTA are selected. These are particularly suitable for purifying PP4 by immunoaffinity chromatography .

Cloning of antibody-producing cell lines Cell lines which show a positive reaction in the assay for PP4 antibodies in the presence of EDTA but not in the presence of calcium are cloned by the limiting dilution method. For this, about 60 cells in 20 ml Df-JEIi containing 20% FCS and 5% human endothelial culture supernatant (Costar) are distributed over the 96 cavities of a cell culture plate. Single clones are identified under the microscope and assayed for antibody production. The cloning is repeated twice.

Purification of monoclonal antibodies For the production of antibodies, clonal cell lines are transferred into roller bottles and cultivated in Iscove's modified Dulbecco's medium. Cell supernatant is obtained by centrifugation and concentrated about 10 times by ultrafiltration. The concentrate is passed through protein A-RSepharose CL-4B (Pharmacia), and bound IgG is eluted with 0.2 M glycine/HCl, pH 3.0. The protein-containing fractions are dialyzed against 0.1 M citrate, pH 6.5, and are concentrated to about 5 mg/ml by ultrafiltration. This results, for example, in an antibody of the IgGl kappa subtype which, on isoelectric focusing, appears in several bands in the pH range 6.2 - 6.5 and to which PP4 binds in the presence of calcium ions.

Suitable immunoaffinity gels can be prepared in the following way: one of the monoclonal antibodies whose preparation is described above is immobilized on a suitable insoluble support material (for example agarose, cellulose, polyacrylamide and its derivatives) by processes familiar to the person skilled in the art (for example activation by cyanogen bromide, epoxides or carbodiimides). In a preferred procedure, it is possible for such monoclonal antibodies to be coupled to RSepharose 4B (Pharmacia) which has been activated with cyanogen bromide in accordance with the manufacturer's instructions. For example, 5 mg of antibody can be bound per 1 ml of gel. These gels can then be used for the immuno15 affinity chromatography of PP4.

The examples which follow illustrate the invention: Example 1 Purification of PP4 from human placenta and determination of the biological activity 36 kg of human placenta were comminuted, washed several times with 0.15 M NaCl and subsequently lyophilized. The lyophilisate (3 kg) was extracted with 50 1 of 0.02 M Tris/HCl, pH 7.5, 0.15 M NaCl, 0.1 M trisodium citrate, mixed with ammonium sulfate (33% saturation) and incu25 bated with 4 1 of phenyl-RSepharose in a batch process.

After the resin had been washed, PP4 was eluted all at once with water, precipitated by adding ammonium sulfate to 80% saturation, pelleted by centrifugation, taken up in 0.02 M Tris/HCl, pH 8.0 (buffer A) and dialyzed against the same buffer. After addition of CaCl2 to a final concentration of 0.005 M, the dialysate was mixed in a batch process with 1 1 of a heparin-RSepharose and stirred at room temperature for 60 min, and the supernatant solution was separated off. The adsorbent was then - 7 washed with buffer A with the addition of 0.005 M CaCl2. PP4 was eluted by a linear gradient from 0 to 0.5 M NaCl in calcium-containing buffer A. After dialysis of the PP4-containing fractions, EDTA was added to a final concentration of 0.001 M, and the dialysate was contacted with 200 ml of a heparin-RSepharose equilibrated in buffer A containing 0.001 M EDTA and stirred at room temperature for 60 min, and the supernatant PP4-containing solution was separated off. If necessary, the PP4-containing solution was contacted with a DEAE-RSepharose, and the protein was eluted with a linear NaCl gradient. The PP4 purified in this way appeared as a band with a molecular weight of 33 kDa in SDS polyac aryl amide gel electrophoresis (PAGE). PP4 showed no cross-reactions with polyclonal antibodies against five other annexins.

The anticoagulant activity of PP4 was examined in a modified prothrombin time tests 50 μΐ of citrated plasma (standard human plasma) were mixed with 150 μΐ of a solution of 0.05 M Tris/HCL, pH 7.5, 0.15 M NaCl, and μΐ of buffer or sample and 25 μΐ of calcium-free thromboplastin. This mixture was incubated at 37“C for 3 min. Coagulation was initiated by adding 25 μΐ of a solution of 0.02 M CaCl2. The coagulation time was determined using a Schnitger and Gross coagulometer. A calib25 ration curve was constructed using the PP4 purified from placenta as described above.

Example 2 Purification of PP4 from human placenta by immunoaffinity chromatography Mature human placenta was washed, lyophilized and extracted with citrate buffer as described in Example 1. This extract was dialyzed against 0.02 M Tris/HCl, pH 7.5. A sample of the dialysate which contained 4.5 mg of PP4 (determined by the enzyme immunoassay described in Example 4) was mixed with CaCl2 (final concentration 0.02 M) and pumped through an affinity gel (20 mg of monoclonal antibodies coupled to 4 ml of CNBr-activated RSepharose 4B). Unbound protein was subsequently washed off the gel with 100 ml of TBS/calcium (0.02 M Tris, 0.15 M NaCl, 0.02 M CaCl2, pH 7.4). The gel was then washed with 30 ml of TBS without calcium, whereupon PP4 was eluted from the antibody. The yield of antigen and activity from the immunoaffinity chromatography were 92 and 90% respectively. PP4 appeared as a band with a molecular weight of 33 kDa in SDS-PAGE.

Example 3 Purification of recombinant PP4 from E. coli by immunoaffinity chromatography The cDNA coding for PP4, isolated and characterized from 15 a human placental gene bank, was cloned and used for the expression in E. coli by means of the vector pTrc99A.

Recombinant PP4 (r-PP4) was expressed by fermentation of the E. coli strain W31101acIQ. The E. coli was fermented by procedures known to the person skilled in the art.

After completion of the fermentation, the cells were harvested by centrifugation. The pellet was resuspended in 0.02 M Tris/HCl, pH 7.5, and 0.01 M EDTA, and the cells were lysed using a French press. Cell fragments were removed by centrifugation, and the supernatant was then purified by filtration through a 0.45 μτα. filter. The r-PP4 -containing solution was mixed with ’’Triton X-100 and dialyzed against a buffer of 0.02 M Tris/HCl, pH 7.5. A sample of the dialysate which contained 3.8 mg of PP4 (determined by the enzyme immunoassay described in Example 4) was mixed with CaCl2 (final concentration 0.02 M), the resulting precipitate was spun down, and the supernatant was purified by immunoaf finity chromatography as described in Example 2. The yield of PP4 antigen and its activity (determined by the enzyme immunoassay described in Example 4 and by the activity assay described in Example 1 respectively) were 94 and 93% respectively. The protein appeared homogeneous with a molecular weight of 33 kDain an SDS gel.

Example 4 Sandwich ELISA for quantification of PP4 antigen A sandwich ELISA was developed for the quantification of PP4 in biological fluids: Microtiter plates were coated with polyclonal antibodies against PP4 (1-20 Mg/ml). The PP4-containing sample (for example placental tissue extract, E. coli lysates or plasma) was incubated on the antibody-coated plates.

Incubation was subsequently carried out with a biotinylated monoclonal antibody (2 /ig/ml) followed by a complex of avidin and biotinylated peroxidase. The procedures for the enzyme immunoassay and the detection of the peroxi15 dase activity were as described under Assay for PP4 antibodies. A calibration line was constructed using the PP4 purified from placenta as described in Example 1. It was possible to determine PP4 in a concentration range between 125 ng/ml and 2 ng/ml (Table 1).

Table 1 PP4 concentration (ng/ml) | Absorption at 405 nm 1 125 | 1.601 62.5 | 1.393 31.25 | 1.067 15.63 | 0.812 7.82 | 0.597 3.91 | 0.298 1.96 | 0.076

Claims (5)

HOE 90/B 019 Patent claims:

1. A monoclonal antibody against PP4.

2. A monoclonal antibody as claimed in claim 1, which 5 binds to PP4 only in the presence of divalent metal ions.

3. A monoclonal antibody as claimed in claim 1, which binds to PP4 only in the presence of calcium ions.

4. A process for preparing a monoclonal antibody as 10 claimed in claim 1, which comprises injecting PP4 into a mammal, obtaining lymphocytes from a lymphatic organ of this animal, fusing them with cells of a myeloma cell line, cultivating the hybridomas formed, testing the cell supernatants for antibodies 15 reacting with PP4, cloning hybridomas, cultivating resulting clones, and obtaining monoclonal antibodies against PP4 from the cultures. 5. The process as claimed in claim 4, wherein a monoclonal antibody which binds to PP4 only in the 20 presence of divalent metal ions is obtained. 6. The use of a monoclonal antibody against PP4 for purifying PP4. 7. The use as claimed in claim 6, wherein the antibody is bound to an insoluble material. 25 8. The use of a monoclonal antibody against PP4 as diagnostic aid or in a diagnostic method. 9. A process for purifying PP4, which comprises binding a monoclonal antibody against PP4 to an insoluble material suitable for a chromatographic purification 30 process, adsorbing PP4 from a solution onto the 11. 11. 12. 12. 13. 13. 14 . 14 . 15. 15. 16. 16. bound antibody, and eluting PP4. The process as claimed in claim 9, wherein the monoclonal antibody is one to which PP4 binds only in the presence of divalent metal ions, and wherein PP4 is eluted with a solution which contains such metal ions in complexed form or not at all. A monoclonal antibody according to claim 1, substantially as hereinbefore described and exemplified. A process for preparing a monoclonal antibody according to claim 1, substantially as hereinbefore described and exemplified. A monoclonal antibody according to claim 1, whenever prepared by a process claimed in any one of claims 4,

5. Or 12. Use according to claim 6 or 8, substantially as hereinbefore described. A process according to claim 9 for purifying PP4, substantially as hereinbefore described and exemplified . PP4 whenever obtained by a process claimed in any one of claims 9, 10 or 15. Dated this the 17th day of April, 1991