JPH02501160A - Method of treating mammals using antibodies against early pregnancy factor (EPF) - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Title of the invention: Treatment of mammals using antibodies against early pregnancy factor (EPF) treatment method Technical field The present invention provides antibodies against early pregnancy factor (EPF) or active fragments thereof. The present invention relates to a method of treating a mammal, including: These antibodies help detect and destroy tumors. Particularly useful for destruction. These are a means of detecting pregnancy and, if necessary, artificially delivering premature birth. It can be used as a means of

Background of the invention Early pregnancy factor (EPF) regulates the first and second trimester of pregnancy in mammals. It is a protein detected in serum and urine during pregnancy, and is a protein detected in serum and urine during pregnancy. Initial and continued production is dependent on the presence of viable fetuses. early pregnancy factor Used to detect the presence of (E　P　F) and to act as a pregnancy diagnostic agent. A method for producing monoclonal antibodies that can be used was developed by the present inventor, and It is disclosed in Laria Patent Application No. 55897/86.

Modified rosette biological assay for detection of early pregnancy factor (EPF) in cells Although it is well known as a suppression test, it is an indirect test and does not contain early pregnancy factors (E It is not a calibration for the detection of PF). Moreover, the suppression tests are redundant and consume considerable time. It is a long process and is recommended for bulk assays as a routine laboratory test to identify tumor cells. is not appropriate.

Because transformed cells and neoplastic cells in the active growth phase also produce early pregnancy factors. , such tumor cells can be easily detected or clearly for destruction. This is a clear advantage if you are a potential target.

Disclosure of invention The present inventor has developed monoclonal and polyclonal antibodies against early pregnancy factor (EPF). The body or its active fragments are injected to identify and destroy tumor cells. I found it easy to use.

The antibodies and active fragments thereof can be produced by any suitable technique known in the art. , a synthetic peptide that matches the appropriate portion of the amino acid sequence of early pregnancy factor (EPF). as well as derived from sources or by methods involving recombinant DNA techniques. It can be manufactured for early pregnancy factor (EPF).

Therefore, according to the first embodiment of the present invention, a method for identifying tumor cells is provided. The method includes (1) injecting antibodies against early pregnancy factor (EPF) or fragments thereof into patients; adding to serum; (21 Monitoring the response due to the presence of early pregnancy factor (EPF), It consists of

Both monoclonal and polyclonal antibodies against early pregnancy factor (EPF), or Fragments thereof can be used, and monoclonal antibodies are particularly preferred.

A second embodiment of the invention provides a method for destroying tumor cells in a patient, The law is Antibodies against early pregnancy factor (EPF) or active fragments thereof are administered to patients to infect tumor cells. Administer in sufficient quantity to destroy It consists of

Experiments were conducted by subcutaneously injecting anti-EPF antibodies into mice at the same time as tumor cells. these Tumors failed to grow in mice. Tumor cells were injected, but anti-EPF antibody There was tumor growth in a control group of mice that were not injected.

Similarly, tumor cells were unable to grow in culture in the presence of anti-EPF antibodies.

If the patient is a female of reproductive age, what reaction occurs to EPF antibodies? Further tests are needed to confirm that the symptoms are not due to pregnancy.

However, another advantage of the present invention is that polyclonal antibodies against EPF can be used as monoclonal antibodies against EPF. Used in pregnancy tests in mammals, including humans, either in isolation or in combination with cloned antibodies can.

Therefore, according to a third embodiment of the present invention, there is provided a method of pregnancy diagnosis, which The method is (1) Monoclonal antibody or active fragment thereof against early pregnancy factor (EPF) Mixing with serum, blood or urine from a mammal believed to be pregnant (Whether prisoner early pregnancy factor (EPF) is present in said serum, blood or urine; It consists of monitoring the reaction to measure the

Mammal urine is collected from the floor of the premises, for example, so human resources are required to handle the mammals. Not. Like China's giant pandas, these animals are sensitive to human contact. This is important when testing dangerous animals that are made sensitive.

A fourth embodiment of the present invention provides a method for artificial premature birth of a mammal, The method is Antibodies against early pregnancy factor (EPF) or active fragments thereof administered to mammals. to remove EPF from mammalian serum. It consists of

When EPF levels decrease, mammals abort their fetuses and undergo artificial preterm birth.

Since EPFO deficiency impedes the development of fertilized eggs, EPF antibodies can be incorporated into "post" treatments. It can be used in the middle of the day or later in pregnancy.

In experiments with Amas, acid mice were given EPF antibodies after mating (early pre-implantation stage). injections over a period of 32 to 56 hours. In a significant number of cases, pregnancy also does not occur. There was no sign of miscarriage.

In all suitable embodiments of the invention, a second polyclonal or monoclonal antibody or active fragments thereof are used to monitor the reaction.

Example A monoclonal antibody against early pregnancy factor (EPF) was injected into the chorionic carcinoma cell line BeWo. (ATCC Deposit No. CCL22) Australian Patent Application No. 55897/86 from mice immunized with Prepared as described. The planned experiment included in vivo neutralization of mouse EPF. Therefore, we selected mice immunized against mouse pregnant EPF with a high titer antibody. . The fused cells were incubated in a medium containing hypoxanthine, aminopterin and thymidine (HAT). Selected by growing hybridomas in ineffective medium (vehicle) was subjected to separation to obtain anti-EPF immunoglobulin (Ig). However, the parent bone marrow As observed with tumor cells (observed with all cancer cells), hybridoma cells in culture cells produce EPF, and therefore specific antibody-secreting hybridomas produce EPF and anti-EPF. Both PFs are manufactured. Therefore, before testing anti-EPF antibodies, culture supernatants should be treated. It is necessary to remove both EPF without antibody binding and EPF with antibody binding. .

1uΩ of 0.2M Glycine-HcQ mixed with medium (20uQ) for 30 minutes at room temperature. , pH 2.0 to pH 2.5, Waters C1, Seppupa (Waters Association, Massachusetts, USA) I let it pass. Waters C1I Sep Pack (Waters C, Sep pak) was pre-filled with 5uΩ acetonitrile (Model, Kentucky, USA). Activate with link rot), wash with distilled water, and level with glycine-HcQ just before use. Balanced. The fluid fragments were collected and the pH was adjusted to 8.0 with solid Tris (Sigma). Raise it to 0, Dialysis against PB5 was followed by testing the specificity of the anti-EPF enzyme as follows. process A sample of 0.1 uΩ of the supernatant and diluted 100 times with PBS was diluted to 500 pgm. Incubate at 37°C for 30 minutes using Ω-1 O, 1uΩ purified mouse EPF. did. After incubation, the mixture was tested for EPF activity in the rosette inhibition test. did.

Ig thought to be EPF specific was bound to the tube by solid-phase immunoassay. Sheep anti-mouse IgM, IgG, IgG2a, IgG, b and IgGs (Nordic, Tilburg, Netherlands; 4000x with binding buffer) Tested on classes and subclasses. Two actively proliferating spiders Loans 7/342 and 5/341 were selected and male BALB/C mice were It was maintained by hybridoma cells grown as ascites. Both clones are Ig M was produced. IgM control preparations were separated from those that produced IgM without anti-EPF specificity. It was prepared from a clone of Hybridoma (10 cells per mouse) Bristane (2,6,10,14-tetra) was administered to 12-week-old BALB/C mice. Methylpentadecane, Sigma) was injected starting 78 days before cell injection. ascites fluid Cut, coagulate, centrifuge 3000g for 20 minutes at 4°C, then The fat was removed by suction. The pH of the ascites fluid was adjusted to glycine-HaQp82. using 0 to adjust the pH to 2.5, and then pass it through a C1l seppak cartridge. F was combined.

The IgM in the flowable fragments was dialyzed against 5mM Tris-HcQ, pH 7.5. Precipitate and wash the precipitate twice in dialysis buffer, then PBS 10.0 It was reconstituted with 5% sodium azide and stored at a temperature of 4°C.

5DS-PAGE horizontally with an exponential acryl acid gradient from 4% to 22.5% Run on a 250X110X0.5mm gel, 0.375M Tris-HaQ, pH 8°8 was prepared in 0.1% SDS, 0.375 mol Tris-glycine, A sample developed in 0.1% SDS at pH 8.3 was added to 2% 5DS10. During IMDTT diluted with an equal volume of 0.075 mol Tris-HaQ, pH 8.8, then for 3 min. After boiling and cooling to room temperature, 2 uΩ was applied to the gel. For 1 hour and 45 minutes, Electrophoresis was performed by applying a voltage of 600 V, and then the gel was incubated at 20% for 30 minutes. Coagulate in dichloroacetic acid (TCA) and incubate at 0.05% Coomassie Brill overnight. 15 including Ant Blue R250 (LKB located in Bromma, Sweden) After staining in %TCA, stains were removed with 12.5% TCA.

About Mr750 for monoclonal antibodies 7/342.5/341 and 7/331, respectively. The results showed that it was IgM with heavy and light chains indicated by 00 and 25000. , the specimen was virtually free of contaminants.

Host animals (e.g. rabbits, donkeys or other suitable hosts) can be grown from a variety of sources [e.g. Chorionic carcinoma cell line BeWo (ATCC Deposit No. CCL98), Mazin Darby (Madin Darby) Bovine kidney cell line MDBK (ATCC deposit number C Polyclonal antibodies were produced using EPF purified from a medium modified by CL22). was prepared by immunizing. Purified EPF (5 μg to 30 μg) was added to Freund Emulsify in complete or incomplete adjuvant and inject into animals after 1 month. Ta. For the first time after the fourth injection, serum from the immunized animal was added to the solid-phase immunosorbent. The binding ability of the antibodies to EPF was tested by assay. Sulfate appropriate antibody Purified from serum by sodium precipitation method and then purified using DEAE Affigel Blu - (Bio-Rad Richmond, California, USA) It was subjected to chromatography.

Samples from rabbits 7 and 8 were used in the experiment. This is where 5DS-PAGE was described earlier. The results showed that the antibodies were approximately Mr55,000 and 25000, respectively. It was found to be substantially free of contaminants with heavy and light chains designated oO. single Similar to clone preparations 7/342 and 5/341, both of these preparations also contain purified EP Neutralize the activity of F, e.g. 500 pg (0.04 pmoQ) EPF by 5 ng. clone (0,03pmo Q) and 50ng monoclonal antibody (0,05pmo Depending on the L, lng (0, OO6pmo Q) multiple clones or 5ng (0 , 005 pmo Q) Neutralization was achieved independent of any single clone. single claw Antibodies 7/342 and 5/341 bound to different epitopes of the EPF molecule. So it is shown. Control monoclonal antibody 7/331 without anti-EPF specificity Prepared for use in the experiments described below. At any concentration it neutralizes EPF There wasn't.

In experiments, transformed neoplastic cells in the active growth phase produce EPF; It was shown that the production ceases after growth arrest or cell differentiation. In the experiment, only low Anti-EPF antibodies showed these effects both in culture and after transplantation into mice as solid tumors. It was found to have a negative effect on the growth of tumor cells. Fibrosarcoma cell line (MCA) 2) Mouse methylcholanthrene was used in both in vivo and in vitro experiments. The mouse melanoma cell line B16 was used in test tube experiments.

The results of these experiments are shown in wiring diagrams in FIGS. 1 and 2.

Two single lone anti-EPF antibodies, 7/342 and 5/341, were used in test tube experiments. It was done. Tumor cells (10') were incubated in 2 mΩ medium (Darbetzkos' modification of Eagle medium). (form) and fetal bovine serum and added various amounts of 7/342 antibody to each Petri dish. Additionally, final concentrations ranging from 1 M to 0.05 uM were applied (6 concentrations in triplicate). (tested).

Cells were examined microscopically for 48 hours after addition of antibodies; anti-EPF7/342, control anti- There were no differences between cells incubated with 7/331 or without antibody. At low concentrations, cells were separated and aggregated. Cell viability 24 hours after antibody addition , 3H-thymidine incorporation and trypan blue exclusion. Antibodies are thin As shown in the figure, the effect on cell viability was dose-dependent at antibody concentrations of 0° and 16 μM. There were 50% non-viable cells. 7 at the same concentration used in 7/342. /331 There was no phase difference in 3H-thymidine incorporation of cells incubated with control antibody. There was no difference. Viability of fresh pancreas incubated in parallel was not affected by either antibody. It didn't resonate. As a result, an EPF molecule different from that binding 7/342 Obtained with anti-EPF monoclonal antibody 5/341, which is well known for its binding to epitopes. It was the same.

In vivo studies have also been conducted. In a preliminary test, 10" MCA-2 cells were placed in CBA mice. It was found that it was palpable 7 days after subcutaneous injection into the body (3.0 mm 5 D 0 ．． 8゜n=4). On the 10th day, the tumor size was 1 to 5.7 mm (SD ± 1°2), on the 13th day. 13. O[[1[[1 (SD±2.8)]. anti-EPF7/3 In experiments at 42, 10'' of MCA-2 cells were injected subcutaneously and treated with various doses of anti-inflammatory agents. The drug was administered to the body several times at different times. 1 m injected during inoculation with MCA-2 cells. 7/342 g had no effect on tumor growth. However, 500 ALg per day Mice treated daily for 4 days had no tumor growth on the 13th day.

These results indicate that the proliferation of tumor cells could be significantly inhibited by anti-EPF antibodies. It was shown that

Outbred Acid Quackenpush mice were caged overnight with males. Pregnancy is vaginal contents It started on the morning of object detection (first day), and the time of copulation was estimated at 2 a.m. that day. . Serum for EPF detection by rosette inhibition test (EPF bioassay) The first dose of antibody was taken from post-copulated females. EPF in serum is 24 Twenty-three mice were tested at the same time to confirm that they were definitely pregnant. We conducted an EPF test 24 hours after the first test, and examined the embryos 10 days later, and found that this group 15 mice became pregnant within 10 days, and 14 mice showed positive EPF after 24 hours of testing. , one fish came out negative. All 8 cats that were not pregnant at 10 days were pregnant at 24 hours. The test came out negative.

Therefore, only mice with positive EPF in the bioassay were included in the study. .

In the first experiment, mice were given a single loan of anti-E at 32 and 56 hours after mating. i, p of PF antibody 7/342.

Patients received two injections, with doses ranging from 64 μg to 500 μg per injection. Ta. The control group consisted of 1) a mouse monoclonal antibody with a specificity other than anti-EPF, and 2) the same antibody. They received 0.9% NaCΩ according to the experimental design (Table 1).

In the second experiment, polyclonal anti-E Received 4 injections of PF antibodies 7 and 8 (500 μg 1st injection dose). The control group was 1) normal rabbit IgM with a similar experimental design, or 2) o.

Received 9% NaaQ. The mice died 7 days after mating, and the number of embryos was as shown in Table 2. there were.

Table 1 Passive immunization of mating-confirmed mice administered with anti-EPF alone (Mab) improves fetal viability. Impact on.

2, i, p, 250 115" 133, i, p, 125 215" 7 ．． 134, i, p, 64 1/3" 127/331 5. i, p, 2 50　515　13.2±1.6(5) (Control) 6. i, p, 125 4/4° 12.3±0.8 (4) 0.9% NaC17,i,p, ---17/18 14.5±0.4 (17) The number of embryos/mouse is listed in the test group column.

Results in the control group are expressed as mean ± s, e, m (the number of animals is in parentheses). It's out. Mammals who became pregnant at 10 days in each group from 1st to 6th administered Mab The numbers in the group 7 were compared with those in group 7, which received 0.9% NaCQ (X" test).

#p<0.001. ” p<o, 01.’ p anti-EPF rich loan antibody #7 and Effect of passive immunization of mating-confirmed mice administered #8 on fetal viability.

Anti-EPF IgG 1, #7 500μg 4 2/6" 11,152, #8 500μg 4 1 76° 8 contrasts 3. Rabbit IgG 500 μg 4 4/4 15,1216.17 4. Salt solution 4 979 14.5 Various modifications may be made without departing from the scope of the invention as detailed in the claims below. and modifications may be made to the embodiments described above.

XO too international search report WO8605498AU55897/86　■　2192634

Claims (1)

[Claims] 1) (a) Antibodies against early pregnancy factor (EPF) or active fragments thereof in the patient's serum (b) monitoring the response due to the presence of early pregnancy factor (EPF); and, How to identify tumor cells consisting of. 2) using either a second polyclonal or monoclonal antibody or an active fragment thereof; The method according to claim 1, characterized in that the reaction is monitored by 3) Make the antibody polyclonal and monitor the reaction using monoclonal antibodies. A method according to claim 2, characterized in that: 4) Whether antibodies or active fragments thereof are produced against EPF induced by recombination techniques. or synthetic peptides that match appropriate portions of the amino acid sequence of EPF. The method according to any one of claims 1 to 3, characterized in that Method. 5) Claims 1 to 3, characterized in that the antibody is antibody 7/342. The method according to any one of Item 4. 6) The antibody is selected from polyclonal antibody 7 or polyclonal antibody 8. A method according to any one of claims 1 to 4. 7) Using antibodies against early pregnancy factor (EPF) or active fragments thereof to destroy tumor cells A method for destroying tumor cells, the method comprising administering to a patient only an amount sufficient to destroy tumor cells. 8) (a) Polyclonal antibodies against early pregnancy factor (EPF) or active fragments thereof , mixing with serum, blood or urine from an animal considered to be pregnant, ( b) Determining whether early pregnancy factor (EPF) is present in serum, blood or urine monitoring reactions to ensure A pregnancy diagnosis method consisting of: 9) Perform the reaction with either a second polyclonal or monoclonal antibody or an active fragment thereof. Pregnancy diagnosis method according to claim 8, characterized in that the pregnancy diagnosis method is carried out using a Law. 10) Early pregnancy factor (EPF) to remove early pregnancy factor (EPF) from mammalian serum comprising administering to a mammal an antibody against EPF or an active fragment thereof. Artificial preterm birth method for animals.