FI93469B - Method for producing a hybridoma cell line and monoclonal antibody produced by it - Google Patents

Description

93469

A method for producing a hybridoma cell line and an antibody produced by it

The present invention relates to methods for producing primate (non-human) monoclonal antibodies and hybridoma cell lines.

Hybridomas are cells obtained by combining an immortalizing cell, such as a myeloma cell, most often with an untransformed partner cell, usually selected for its ability to produce a particular, predetermined compound (e.g., a lymphocyte that produces antibodies). to clone and thus obtain cell lines that produce compounds with a particular structure and / or property. In particular, those hybridoma cells in the production of which lymphocytes have been used can be used in the production of monoclonal antibodies.

In recent years, advances in hybridoma technology have focused on the production of cell lines that are stable and efficient and specifically produce the desired compound. This problem has been approached in a variety of ways, largely due to their historical origins and widely focused on immunoglobulin / antibody research.

25 A review of previous work in this area provides an overview of the problems encountered in the past and the solutions sought.

The initial impetus to study monoclonal-producing hybrid cells came from the field of immunobiology, where these products were monoclonal antibodies. Conventional antisera usually contain a large number of antibodies that differ in the structure of the antigen binding site, but each bind to the same antigen with greater or lesser avidity and / or specificity. In addition, 93469 2 __ conventional antisera also contain large amounts of antibodies against other antigens, and thus describe the previous defense reactions of the host individual from which the antiserum was obtained. Although such "broad-spectrum 5" antisera were sufficient for most uses, it was felt that greater specificity and reproducibility would represent significant progress and would be a scientifically potential tool, especially in diagnostics and therapy.

10 The first and now already classic solution was described

Kohler and Milstein (NATURE, 256, 495-497 (1975)) who succeeded in fusing preimmunized mouse spleen isolated cells with drug-sensitive mouse myeloma cells. These fused cells thus immortalized could be grown in vitro and cloned from a single cell stage to produce homogeneous cell populations that produced a homogeneous antibody population (monoclonal antibodies). By selection from many unique cells and selective recloning of selected cells, it was possible to create and grow cells that produced antibodies with the desired antigen specificity.

Mouse antibodies produced in this manner have been shown to be useful in research and diagnostics and some have even been used for therapeutic purposes in humans. However, it should be noted that human immunoglobulin therapy must make significant progress in order to achieve equally specific and reproducible antibody production in humans or other primates. This would also reduce the risk of sensitization. There have been several attempts to address the in vitro production of human antibodies, but they have so far largely failed. These methods include the following: (i) Transformation of normal human lymphocytes with 35 Epstein-Barr virus (EBV). This has not been a very successful method, as these types of cells require a long and laborious process before they can be properly grown and these cells are very difficult to clone and select, (ii) Fusion of normal human lymphocytes with human mye-5 animal cells. This method represents an obvious analogy for the production of mouse-mouse hybridoma cells, but the problem is that only one myeloma cell line has been widely available to humans that has been found to be contaminated with mycoplasma, and this prevents successful fusions.

(Iii) Fusion of normal human lymphocytes with a human lymphoblastoid B cell line transformed with EBV. Although this is probably the most reliable and reproducible method of all tried so far, its basic problem is in vitro culture, which is always encountered in lymphoblastoid cell culture: they are very difficult to clone and because they represent an early stage in B cell differentiation. and the secretion of antibodies is about 10 times lower than in actual myelomas.

(Iv) Fusion of human lymphocytes with mouse myeloma cells. This method can be used to produce cells with the same excellent in vitro properties as mouse-mouse hybridomas, but their major drawback is that such hybrids are genetically unstable in nature. One very detrimental indication of this is that they push out of the cell the human chromosome that houses the genome encoding the immunoglobulin kappa light chain production.

It has since been reported that by using a xenogeneic 30 hybridoma cell as a partner in another fusion with another cell, it is possible to develop a much more stable hybridoma (Ostberg et al., HYBRI-DOMA 2, No. 4,361, 1983) and that these xenogeneic hybridomas represent a more favorable habitat for improving stability with respect to chromosome loss. This invention relates to a method for producing a hybridoma cell line comprising an immortalizing cell fused to a primate (non-human) monoclonal antibody-producing cell, wherein the immortalizing cell comprises a xenogeneic hybridoma cell which is an immortalizing cell , and said antibody-producing cell is genetically compatible with said partner cell. By genetic compatibility is meant that these cells are related at the species and genealogical level in order to avoid significant chromosome loss and to achieve the desired degree of post-fusion stability. The method is characterized by what is set forth in claim 1. The method of the invention for producing a primate (non-human) monoclonal antibody is characterized by what is claimed in claim 5.

It now appears that the production and use of primate (non-human) monoclonal antibodies produced by xenogeneic hybrid technology may prove to be a useful way to treat humans with monoclonal antibodies as well as in diagnostic applications. The production or isolation of primate (non-human) monoclonal antibodies has not been reported in the past.

Accordingly, it is an object of the present invention to provide a method for producing stable primate (non-human) monoclonal antibody-producing cell lines useful in the treatment and diagnosis of humans. Another object of the present invention is to provide immunoglobulins derived from primate (non-human) lymphocytes which can also be utilized in humans.

The primates included in this part of the invention included substantially large tailless monkeys, e.g., orange-gutangs, gorillas, or chimpanzees, and actual monkeys, such as New World monkeys, e.g., cebus monkeys, and Old World monkeys, e.g., rhesus monkeys. With respect to different monkey species, it is clear that, for practical reasons, the use of sufficiently small species that are easy to handle under laboratory conditions is preferred, but this part of the experiment sought to cover all primates (other than 10 non-human).

In one aspect, the xenogeneic hybridoma selected as the immortalizing cell is a myeloma hybrid that has lost its ability to produce immunoglobulin. An example of such a xenogeneic hybridoma cell 15 could be a hybrid of a myeloma cell and a lymphocyte. Examples of suitable myeloma cells are mouse and rat myeloma cells, which at best do not produce immunoglobulin. These myelomas may themselves be hybrids (e.g., mouse myeloma x mouse lymphocyte) that are actually myelomas. Such a cell is, for example, a murine SP-2 myeloma cell line. This can then be fused to a monkey lymphocyte, e.g., to produce monkey monoclonal antibodies.

As described in HYBRIDOMA 2, previously cited (as well as in corresponding patent applications, e.g. UK Patent Application 2, 113, 715A, published August 10, 1983), the xenogeneic hybridoma used to immortalize is rendered drug resistant prior to fusion, and the lymphocyte is fused to a mie-30 reading pre-sensitized because it enhances the production of the desired compound. Conventional methods previously described and published, e.g., in the reference cited above, have been used for drug resistance and presensitivity treatments.

6 93469

The SP-2 cell line is used in the practice of the present invention. It is originally a hybridoma of the P3-X63-Ag8 line itself and of mouse spleen cells that produce antibodies to sheep red blood cells but has since lost its ability to produce antibodies (C.F.M. Shulmann et al., NATURE 276, 269, 1978).

It is available, for example, from the NIGMS Human Genetic Mutant Cell Repository under reference number GM 35669 A (see U.S. DHHS 1982 list of cell lines). This cell line is first rendered drug resistant and then fused with normal human peripheral lymphocytes by conventional methods (C.E. Galfre et al., NATURE 266, 550 (1977) and R. Nowinski et al.

SCIENCE 210, 537 (1980).

The result is a large number of hybrids, from which, after 5 weeks, 5 clones with a good growth rate and which do not produce antibodies are selected. These cells were selected for their 8-azaguanine resistance, and mutants 20 resistant to 20 pg / ml 8-azaguanine can be obtained from three of these lines. These cells are concomitantly sensitive to hypoxanthine-aminopterin-thymidine (HAT) medium, indicating that they have lost their ability to produce hypoxanthine phosphoribosyltransferase. One of these lines is SPAZ4, which can be further used in fusions to create an antibody-producing hybridoma.

The simple hybridoma cell lines described above that produce primate (non-human) monoclonal antibodies, if necessary or desired, can be further fused, e.g., with a monkey lymphocyte, to provide an even more stable cell or cell with a slight various unwanted features. The antibodies of this invention can then be used in human medicine to combat infectious diseases, cancer, and allergies, as well as to alleviate transplant responses and possible other uses in the art, such as intoxication conditions of drugs such as digoxin. They can also be used in diagnostics, for example by including them in test kits.

Examples of cell types to which this invention pertains include cell lines consisting of a mouse myeloma cell and a primate cell, such as a mouse-human xenogeneic cell that produces a (mouse x human) x non-human primate cell line, e.g. mouse x human) x monkey hybridoma cell line, or (mouse x non-human primate cell line) x non-human primate cell line, e.g., (mouse x monkey) x monkey cell line. According to the invention, cell lines such as 15 [(mouse x human) x non-human primate] x non-human primate cell line can also be produced, e.g. [(mouse x human) x cebus monkey] x cebus monkey cell line [(mouse x human) x rhesus monkey] x rhesus monkey cell line, [(mouse x human) x chimpanzee] x chimpanzee cell line and [(mouse 20 x human) x rhesus monkey] x chimpanzee cell line.

The antibodies produced in accordance with this invention can be prepared and administered in a conventional manner for therapeutic use and can also be included in a variety of test kits in diagnostics. The following examples illustrate this invention.

Example 1

The digoxin is dissolved in 2 ml of absolute ethanol, to which 2 ml of 0.1 molar sodium periodate are added dropwise and this solution is incubated for 45 minutes. 60 microliters of ethylene glycol are then added. After incubation for 5 minutes, 50 mg of volcanic shell hemocyanin (KLH) dissolved in 10 ml of water, pH 9.5, are added. When the pH has stabilized at 9.5, 0.3 g of NaBH4 dissolved in 2 ml of water are added to the solution. This preparation is dialyzed against saline overnight.

β 93469

Approximately 4 mg of KLH digoxin in 0.5 ml is emulsified with Freund's complete adjuvant (0.5 ml). This emulsion is injected intramuscularly into both thighs of the cebus monkey. After this first injection, the animal is given four more booster injections with the same amount of KLH digoxin, but this time emulsified in 0.5 ml of Freund's incomplete adjuvant. A blood sample taken after this immunization indicates that the test animal had produced antibodies that reacted with digoxin-10 so that its toxicity was inactivated in in vitro experiments. This was manifested by the fact that dilutions of monkey serum were found to neutralize the toxicity (lethality) of digoxin (or its analogue oubain) to sensitive human lymphoblastoid cells in cell culture. After two months of rest, the test animal is given a new intravenous injection of Q, 5 ml of KLH digoxin (dissolved in saline). On the third day from the last injection, animal cells are isolated for fusion.

(a) A blood sample is taken from a peripheral vein into a syringe 20 containing 50 μΐ heparin solution (10,000 USP units / ml).

Blood lymphocytes are purified by gradient centrifugation with a PERCLLL layer (Pharmacia, Inc., Piscataway, N.J.). After centrifugation, the cells are washed in Hank's balanced salt solution (Gibco Laboratories, Grand Island, 25 N.Y., Cat. No. 310-4020, commercially available).

(b) The lymph nodes are removed aseptically from the area of the groin folds. The lymph nodes are pressed through a dense steel sieve to obtain unicellular suspensions. The isolated cells are washed in Hank's balanced salt solution.

(C) Part of the spleen is removed from the test animal by aseptic surgical means. To obtain a single cell suspension, the spleen tissue is pressed through a dense steel sieve. This isolated single-cell suspension is washed with Hank's balanced salt solution.

9 93469

Approximately 2 x 10 7 isolated lymphocytes are mixed with the same amount of SPAZ-4 myeloma cells (obtained using the methods described, e.g., in the aforementioned reference HYBRIDOMA 2) in serum-free medium and pelleted by slow centrifugation. The cell pellet is treated for one minute with 1.0 ml of 50% PEG-1500 in Hank's balanced salt solution, pH

8.0. After incubation for one minute at 37 ° C, 1 ml of Hank's balanced salt solution is added slowly. Over the next 10 minutes, add an additional 10 mL of Hank's balanced salt solution. Cells are harvested by centrifugation and mixed with 20 ml of Dulbecco's modified Eagle's medium (Dulbecco's Modified-Eagle Medium, No. 430-2100, Gibco Laboratories) VIROLOGY 8, 396 (1959); Virology 12, 185 15 (1960); Tissue Culture Standards Committee, IN VITRO, Vol.

6, NO. 2, p. 93, containing 20% fetal bovine serum, and using HAT (hypoxanthine-aminopterin-thymidine) as the selection system. After about three weeks, when the cultures show good growth, 20 supernatants are removed for testing. Testing is performed by ELI-SA using digoxin bound to bovine serum albumin as antigen (BSA digoxin is produced in the same way as KLH digoxin). Positive clones are detected using a rabbit anti-human or an-25 monkey immunoglobulin peroxidase system. Positive clones are harvested for propagation culture, and at the same time cloned by limiting dilution in the wells of microtiter plates and using mouse thymocytes as filler cells. As soon as the number of cells is large enough, 30 clones are frozen in liquid nitrogen at -196 ° C

Antibodies are produced by growing hybridoma cell lines in either stationary or rotating culture medium using Dulbecco's MEM medium supplemented with 10% fetal bovine serum. Antibodies, usually at a concentration level of 20 to 50 pg / ml, are purified by affinity chromatography on a Sepharose * Protein A column (Pharmacia, Inc.) by loading the material to be chromatographed on the column, saturating the column binding capacity, and washing the column with phosphate buffered saline. After removal of unbound material, the column is eluted with 0.05 M sodium citrate with 0.5 M sodium chloride (pH 3.0). The eluate is rapidly neutralized by the addition of 1 M Tris-HCl, pH 8.0). Example 2 Cells produced according to the production and selection process described in Example 1, which over time have lost their ability to produce immunoglobulin, are rendered drug resistant by treatment with a solution of 20 pg / ml 8-azaguanine, thus enabling the HAT system. use as a selection system in a conventional manner. The cells thus obtained are fused to immunized cebus monkey lymphocytes, and after a new HAT treatment, positive cell cultures are selected as described in Example 1.

According to this method, three antibodies were obtained from fusions using cebus monkey cells. For these hybridomas, codes 7-1, 11-1, and 11-3 are used. All three antibodies have been tested for their ability to reduce digoxin toxicity in the human lymphoblast-25 discellular line IM-9. At a final antibody concentration of 100 pg / ml, the following results were obtained:

Addition Amount of digoxin / which does not cause harm 30

Phosphate buffered

saline 2.9 x 10'8 M

7-1 4.7 x 107 M

11-1 9.4 x 10’7 M

35 11-3 2.3 x 10-7 M

II

11 93469

Antibody 7-1 has also been used in other specificity tests against other types of digitalis alkaloids. The results of these specificity tests are summarized in the following table: 5 Drug Amount of drug with no effect

Phosphate buffered saline

Oubain 2 x 10'8 M 2 x 10'8 M

Digitoxin 5.9 x 10'9 M 2.3 x 1CT8 M

Deslanoside 2.3 x 10 * 8 M 1.9 x 10 "7 M

Taken together, these results indicate that the antibodies produced by this method can protect against drug toxicity, and for digitalis alkaloids, one of the antibodies is effective in reducing the toxicity of two outpatients (digoxin and digitoxin) and one intravascular agent (delanoside). It has no effect on another medicine used intravenously (oubain).

20 Example 3

KLH-dioxin is used as an immunogen in adult male chimpanzees as described in Example 1. After a total of five injections given at two-week intervals, one more intravenous injection of 0.5 ml of KLH digoxin (approximately 4 mg protein) is given. On the sixth day, a blood sample is taken from the test animal from the last i.v. injection, and the day after sampling, the cells are used in fusion with the SPAZ-4 myeloma cell line as described above. A sample taken on the sixth day yields IgG antibody-producing hybridomas, designated CH 4-14 and CH 4-25, with a lamda and kappa light chains, respectively. Light chains are characterized using reagents against human immunoglobulins in a conventional manner. These reagents give a very reliable result and their reactivity to anti-human reagents is stronger than that to anti-monkey reagents prepared against cynomolgus monkey. Both of these (mouse x human) x chimpanzee antibodies have an affinity for digoxin that is clearly higher than that of cebus antibody 11-1 mentioned in Example 2.

Claims (6)

A method of producing a hybridoma cell line comprising an inunortalizing cell fused with a cell producing monoclonal antibodies of a primate other than human, wherein the inunortalizing cell comprises a xenogenic hybridoma cell constituting the fusion product of an inunortalizing stem cell. and a partner cell, and said antibody producing cell is genetically compatible with said partner cell, characterized in that it fuses the inunortalizing stem cell line SP2 with a partner cell, which is a lymphocyte, which has lost its ability to produce antibodies. thus obtained xenogenic hybridoma cell line drug resistant, after which it is fused with said antibody producing cell and selected the desired hybrid. 2. A method according to claim 1, characterized in that one produces (mouse x human) x primate other than human hybridoma cell line. 20 3. The method of claim 1, characterized in that one produces a [(mouse x human) x primate other than human] x primate other than human hybridoma cell line. Method according to any of claims 1-3, characterized in that the primate other than human is monkey or chimpanzee. Method for producing monoclonal antibodies of a primate other than human, characterized in that a hybridoma cell line comprising an inunortalizing cell fused to a cell producing monoclonal antibodies of a primate other than human is produced, the inunortalizing the cell comprises a xenogenic hybridoma cell which is the fusion product of an inunortalizing stem cell and a partner cell, and said cell producing antibodies is genetically compatible with said partner cell, by fusing the immortalizing stem cell line SP2 with a partner cell, which is a lymphocyte, which has lost its ability to produce antibody pairs, and render the well-obtained xenogeneic hybridoma cell line drug resistant, fuse it with said antibody producing cell and select the desired hybrid and isolate the antibodies it produces. 6. A process according to claim 5, characterized in that monoclonal antibodies of monkey or chimpanzee are produced.