KR900007950B1 - Production of monoclonal antibodies - Google Patents

Abstract

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Description

Method for producing monoclonal antibody

The present invention relates to non-human primate monoclonal antibodies, hybridoma cell lines and methods of making them, and the use of such cell lines to produce such antibodies.

Hybridomas are cells formed for the fusion of immortalizing cells, such as myeloma cells, mainly non-transformed paired cells whose ability to produce certain predetermined substances is usually selected (partner cell). (E.g., lymphocyte cells producing antibodies). The resulting hybrid cells can be selected and cloned to obtain cell lines that produce substances with monostructure and / or properties. In particular, hybridomas formed with lymphocytes can be used to produce monoclonal antibodies.

Recent advances in hybridoma technology have led to the obtaining of cell lines with high and specific productivity of certain substances which are stable and preferably obtained. Because of their historical origins, there has been a diversified approach to this problem with a high concentration in the field of mission noglobulins / antibodies.

The study of prior activity in this field provides insight into the types of bland problems and the various solutions attempted. The original desire for research on hybrid cell lines that produce monoclonal products originated in the field of immunology where the product is a monoclonal antibody. Conventional anti-serum usually contains a very large number of antibodies that structurally differ in antigen binding sites, but which bind to the same antigen with large or small specificity and / or specificity, respectively. In addition, conventional anti-serum also contains a number of antibodies which are induced in addition to other antigens and exhibit a prior protective response for each host from which the antiserum was obtained. Although such "broad" antiserum is bulky for most purposes, the provision of additional specificity and reproducibility will make significant advances and provide a scientific tool in the field of quite powerful, especially diagnostic and therapeutic.

Early and present classical solutions pre-immunized mice

Kohler and Milstein, NATURE, (256), 495-497 (1975), who have successfully fused to myeloma cells. Such immortal fusion cells can be cloned ex vivo and cloned from the single cell level to form the same cell population that produces the same antibody population (monoclonal antibodies). The choice between many unique cells and selective cloning makes it possible to obtain and grow cells that produce antibodies with the desired antigen specificity.

Mouse antibodies produced in this way have proved useful in research and diagnostics and some have been used therapeutically in humans. However, if primate antibodies other than humans or humans with similar specificity and reproducibility are to be obtained in this way, great advances should be made in the treatment of human mission noglobulin. This should also reduce the risk of sensibilisation. Several approaches to the problem of producing human antibodies in vitro have been attempted but have not been very successful. These are as follows. (V) Transformation of Epstein-Barr virus (EBV) and normal human lymphocytes, this method requires a long and tedious process on which these types of cells are based and is extremely difficult to clone and select But it succeeded. (Ii) Fusion of human myeloma cells with normal (human) lymphocytes This method shows a clear similarity to mouse-mouse hybridomas, but only one myeloma cell line in human-systems is widely available and this leads to successful fusion. Faced with the problem of being infected with interfering mycoplasma. (Iii) Fusion of normal human lymphocytes to the EBV-transformed human lymphocyte B-cell line. While this is perhaps the most robust and reproducible method described so far, there is a fundamental drawback to impinging lymphocytes in vitro: antibodies are extremely difficult to clone and because they represent an early stage in B-cell differentiation titration Production and secretion capacity is about 10 times lower than that of real myeloma. (Iii) Fusion of human lymphocytes to mouse myeloma. This method produces cells that have the same and superior biological properties as mouse-mouse hybridomas, but with the major disadvantage that such hybrids have inherent genetic instability. A particularly difficult result of this is that the human chromosome is pushed out where the genome is located to form the kappa light chain of mission noglobulin.

Eventually, by using xenogenic hybridomas as parents in fusion to other cells, a highly enhanced stability of hybridomas can be obtained (Ostberg, et al., HYBRIDOMA (2), No. 4,361). , 1983), these heterologous hybridomas have been reported to exist in a milder environment for enhanced stability in terms of chromosomal loss.

The present invention relates to a hybridoma cell line wherein the parental immortalized cells are fused with heterologous partner cells and the resulting immortalized cells are subsequently fused to cells capable of producing non-human primate monoclonal antibodies. This latter cell must be genetically compatible with the partner cell in the heterologous hybridoma. Genetic matching means that the cells involved are similar to the species and genus of origin, so as to avoid large loss of chromosomes and thus maintain desirable stability after fusion.

The production and use of primate monoclonal antibodies other than humans produced with such heterologous hybridoma fusion technology is presently expected to be a useful approach in the therapeutic and diagnostic applications of humans using monoclonal antibodies. There is no prior record concerning the preparation and isolation of primate monoclonal antibodies other than humans.

It is therefore an object of the present invention to provide a method for producing stable cell lines that produce non-human primate monoclonal antibodies useful for human treatment and diagnostics. It is another object of the present invention to provide a mission noglobulin derived from primate lymphocytes other than humans that can be effectively used in humans. Primates other than humans included in this aspect of the present invention are basically monkeys such as orangutans, gorillas, or chimpanzees, and New World monkeys such as cebus monkeys and rhesus monkeys. They are tailed monkeys like the Old World monkeys on their backs. Between apes and monkeys, the use of animals small enough to be easily manipulated in laboratory conditions for practical purposes is desirable, but it is clear that this view encompasses non-human primates.

In particular terms, heterologous hybridomas, selected as immortalized cells, are myeloma hybrids that have lost their ability to produce mission noglobulin. An example of such a dual hybridoma cell is between myeloma cells and lymphocyte cells. Examples of suitable acid myeloma cells are obtained from mice and rats and preferably do not produce mission noglobulin. These myeloma can be hybrids (eg, mouse myeloma x mouse lymphocytes) that are themselves virtually myeloma. Such cells are, for example, mouse sp-2 myeloma cell lines. This may then be fused to, for example, monkey lymphocytes to produce monkey monoclonal antibodies.

As described in the aforementioned Hybridoma 2 (as well as the corresponding patent application, for example British Patent Application No. 2,113,715A, published August 10, 1983), heterologous hybridomas used in immortalization are also fused. It is then made drug resistant, which lymphocytes to be fused to are preferably obtained after pre-sensitization for enhanced production of the desired material. Drug resistance and pre-sensitization methods are conventional as well as methods for fusing these cells. The selection and cloning of the desired hybrid is also already described and is carried out by conventional methods, for example as disclosed in the above cited material.

In certain embodiments according to the invention sp-2 cell line (Korea Depositary: Korea Advanced Institute of Science and Technology, date of deposit 14 May 1986, Accession No .: KCTC 8193p) was used. This is originally a mouse spleen cell producing antibodies against positive red blood cells and hybridomas themselves at p3-XG3-Ag8 weeks and lost their ability to produce antibodies. (CFM Shulmann et al., NATURE (276) ), 269, 1978).

This can be obtained, for example, from the NIGMS Human Genetic Mutant Cell Repository Ref. GM35669 A (see US DHHS 1982 Cell Line List). This cell line becomes drug resistant and is then fused with normal human peripheral lymphocytes by conventional techniques [C.F.G. Galfre et al., NATURE 266, 550 (1977) and R Nowinski et al., SCIENCE (210), 537 (1980)].

A large number of hybrids are obtained, and five clones are selected that show rapid growth after about five weeks, but no antibodies. These cells are screened for resistance to 8-azaguanine, and variants can be obtained that are resistant to 20 μg / ml of 8-azaguanine in three of these weeks. These cells are susceptible to Hypoxanthine-Aminopterin-Thymidine (HAT) medium, which simultaneously shows that they have lost their ability to produce hypoxanthinphosphoribosyl tronsferase. One of these strains is SPAZ 4, which can also be used to fuse to yield antibody producing hybridomas.

Simple hybridoma cell lines of the present invention that produce primate monoclonal antibodies other than humans may also be fused with monkey lymphocytes and the like, if desired, to obtain cells that are still highly stable or with many different desired properties. Antibodies according to the invention can be used to treat infectious diseases, malignant medications, allergies in humans, and for other purposes known in the art, such as in toxicity by drugs such as digoxin upon exclusion of non-epidermal grafts. Can be used. They can also be used for diagnostic applications such as kits. Examples of cell line types to which the present invention pertains include primate cell lines other than (mouse x human) x human, eg, (mouse x human) x monkey hybridoma cell lines, or (other than mouse x human) produced from mouse-human xenogenes. Primate cell) other than humans, for example, (mouse x monkey) x monkey cell line, such as mouse myeloma cells and cell lines containing human and non-human primate cells. Cell lines provided according to the present invention also include primate cell lines other than [(mouse × human) × human other than primate] × human, for example [(mouse × human) × sebus monkey] × sebus monkey cell line. , [(Mouse x person) x Rhesus monkey x Rhesus monkey cell line, [(mouse X person) X chimpanzee] X chimpanzee cell line, and [(mouse X person) x Rhesus monkey] x chimp cell line.

Monoclonal antibodies produced according to the present invention can be prepared and administered in a conventional manner for therapeutic applications, or can be made into conventional kits for diagnostic use.

The following examples illustrate the invention.

Example 1

Digoxin is dissolved in 2 ml of absolute ethanol, 2 ml of 0.1 m sodium periodate is added dropwise and the mixture is incubated for 45 minutes. Add 60 μl of ethylene glycol. After 5 minutes of incubation, 50 mg of keyhol limpet hemocyanin (KLH) in 10 ml of water at pH 9.5 is added. When stabilized to pH 9.5, 0.3 g of NaBH _{4 in} 2 ml of water is added. The resulting product is dialyzed overnight for brine.

Approximately 4 mg of KLH-digoxin in 0.5 ml volume is emulsified with 0.5 ml of Freund's complete adjuvant. The emulsion is injected intramuscularly in both femurs of Cebu's monkey. Following this first injection, the animal is incubated four times with a similar amount of KLH-digoxin or this time emulsified in 0.5 ml of Freund's incomplete supplement. Test immunization after this immunization has shown that animals can produce antibodies that react with digoxin, which can inactivate the toxicity of digoxin in biomass studies. It can be seen that the dilution of monkey serum neutralizes the toxic (lethal) effect of digoxin (or its analog oubain) on susceptible human lymphocytes in cell culture. The animals are allowed to rest for 2 months and then another injection of 0.5 ml of KLH-digoxin in saline is administered intravenously.

Three days after the last intravenous injection, cells are harvested from the animals for fusion.

; Pharmacia, Inc., Piscataway, N. J. )의 쿠션(cushion)상에서 구배 원심분리에 의해 정제한다. 원심분리후 세포를 행크 평형 염용액(Hank's balanced salt solution; Gibco Laboratories, Grand Island, N.Y. , catalog no. 310-4020, 상업적으로 널리 구매 가능함)에서 세척한다.a. Blood is transferred from the peripheral vein to a syringe containing 50 μl heparin solution (10,000 usp units / ml). PERCOLL BLOOD LAMPS

; Purification by gradient centrifugation on a cushion of Pharmacia, Inc., Piscataway, N. J.). After centrifugation the cells are washed in Hank's balanced salt solution (Gibco Laboratories, Grand Island, N.Y., catalog no. 310-4020, commercially available).

b. Using sterile surgical techniques, lymph nodes are obtained from the inguinal area. Lymphocytes are passed through a fine sieve of steel sieve to produce a single cell suspension. The isolated cells are washed in Hank's balanced salt solution.

c. Using sterile surgical techniques, partial nasal resection is performed in the animal. Single cell suspensions are prepared from spleen fragments by passing the organs through a fine sieve of steel sieves. This isolated single cell suspension is washed in Hank's balanced salt solution.

Nearly 2 × 10 ⁷ isolated lymphocytes are mixed with a similar amount of SPA-4 myeloma cells (obtained according to methods described in the art, such as the hybridoma 2 data described above) in serum-free medium, and centrifuge Pellets together at low speed. The cell pellet is treated with 1.0 ml of 50% PEG-1500 in Hank's balanced salt solution at pH 8.0 for 1 minute. After incubation at 37 ° C. for 1 minute, 1 ml of Hank's balanced salt solution is slowly added. After 1 minute, 10 ml of Hank's solution is added. Cells were harvested by centrifugation, Dulbecco's Modified Eagle Medium (No. 3) containing serum of 20% thiasso and using HAT (Hyphosanthine-Aminopterin-Thymidine) as the selection system. 430-2100, Gibco Laboratories), VIROLOGY (8), 396 (1959); VIROLOGY (12), 185 (1960); Tissue Culture Standards Commitee, IN VITRO, Vol. 6, No. 2, P 93] and resuspended in 20 ml.

After about 3 weeks, if good growth is observed in the culture wells, the supernatant is collected from the culture to test. Tests are performed by ELISA technique using digoxin bound to bovine serum albumin (BSA) as the antigen (BSA-digoxin is prepared in a similar manner to KLH-digoxin). Positive clones are detected using a rabbit anti-human or anti-monkey immunoglobulin-peroxidase system. Positive clones are scaled up and cloned by limiting dilution in microtiter wells using mouse thymocytes as dilution cells at the same time. Upon obtaining a large number of cells, the clones are frozen at −196 ° C. in liquid nitrogen.

Antibodies are produced by growing hybridoma cell lines in stationary or rotational cultures using Dulbecco's MEM containing a serum maximum of 10% fetal bovine. Antibodies, typically 20-50 μg per ml, are prepared by affinity chromatography on a Sepharose Protein A column (Pharmacia Inc.), placing the material to be chromatographed on the column, saturating the column binding capacity, and the column phosphate buffered saline. Purify by washing with. After unbound material is removed from the column, the column is eluted with 0.05 M sodium citrate containing 0.5 M sodium chloride (pH 3.0). Neutralize rapidly by adding 1M Tris-HCl (pH 8.0) to the eluate.

Example 2

Treatment of cells according to the cell production and selection of Example 1, which has already been read for the ability to generate mission noglobulin, in the presence of 8-azaguanine 20 μg / ml, makes the drug resistant, so that the HAT is treated in a conventional selection system. Make it available. The resulting cells are fused to immunized Cebu's monkey lymphocytes, again treated with HAT-medium, and positive cell cultures are selected using the same method as described in Example 1 above.

According to this method, three kinds of antibodies were obtained from fusion using the cells of Cebu's Monkey. These hybridomas are designated 7-1, 11-1 and 11-3, respectively. All three antibodies were tested for their ability to inhibit digoxin toxicity on the human lymphocyte cell line, IM-9. The following results were obtained when the antibody was used at a final concentration of 100 μg / ml:

The 7-1 antibody was also tested for its specificity for other types of digitalalis alkaloids. The results of this specificity test are summarized in the table below.

Taken together, these data show that the antibodies produced by this method are protective against the toxic effects of drugs, and in the case of digitalis alkaloids, two drugs (digoxin and digitoxin) in which one antibody is used as a drug for outpatient clinical patients and parenterally It is shown that it can cover for one drug used (deslanoside). There is no activity against other parenteral drugs (Ovine).

Example 3

In a manner similar to that described in Example 1, KLH-digoxin is used as the immunogen of mature male chimps. After five injections of this antigen, basically two weeks apart, finally, 0.5 ml of KLH-digoxin (about 4 mg of protein) is administered intravenously. Blood samples are taken from the animals six days after the last intravenous injection and used for cell fusion with the SPAZ-4 myeloma cell line, using the method that has already been prepared after one day of bleeding. From the samples obtained on day 6, IgG antibody hybridomas labeled CH 4-14 and CH 4-25 with lambda and cappa light chains are obtained, respectively. Light chain characterization is carried out using reagents for human mission noglobulin in conventional manner. Such reagents produce obvious results and the reactivity is stronger than the anti-monkey reagents prepared for cynomolgus monkeys. Both of these (mouse × human) × chimpanzee antibodies have significantly higher affinity for digoxin than Cebu's lower body 11-1 mentioned in Example 2 above.

Claims (12)

Xenogeneic hybridoma cells are made to be drug resistant, and these cells are fused to antibody-producing cells genetically matched with partner cells in the heterologous hybridoma, thereby selecting a desired hybrid. Immortalizing cells fused to primate monoclonal antibody producing cells other than human, wherein the immortalizing cells are double hybridoma cells fused from parental immortalized cells and partner cells, and the antibody producing cells are the paired cells. And genetically matched thereto). The method of claim 1, wherein the selection of the desired hybrid is performed based on a lack of susceptibility to HAT and an assay for the ability to produce primate monoclonal antibodies other than humans. The method of claim 1, wherein said antibody producing cells are of the same genera as paired cells in a heterologous hybridoma cell parent. The method of claim 1, wherein said antibody producing cells are of the same species as the partner cells in the heterologous hybridoma cell parent. The method of claim 3, wherein the primate monoclonal antibody producing cells other than human are lymphocytes. The method of claim 1, wherein the non-human primate monoclonal antibody is a monkey monoclonal antibody. The method according to claim 1, wherein the parental immortalized cells are SP-2 cell lines (Korea Depositary: Korea Advanced Institute of Science and Technology, Deposit Date: May 14, 1986, Accession No .: KCTC 8193P). The method of claim 1, wherein the antibody produced is a monkey monoclonal antibody. The method according to claim 1, wherein the produced hybridoma cell line is a primate hybridoma cell line other than (mouse x human) x human. The method according to claim 1, wherein the manufactured hybridoma cell line is a primate hybridoma cell line other than (primates other than mouse x human) x human. The method according to claim 9, wherein the generated cell line is (mouse x human) x monkey hybridoma cell line. The method according to claim 10, wherein the generated cell line is (mouse x monkey) x monkey hybridoma cell line.