LU86437A1 - MONOCLONAL ANTIBODIES - Google Patents

Description

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The present invention relates to monoclonal antibodies to non-human primates of hybridoma cell lines and methods for their production, as well as the use of these v 5 cell lines to produce said antibodies.

Hybridomas are cells formed by the fusion of an immortalizing cell, such as a myeloma cell, with most often an untransformed cell chosen for its ability to produce a particular predetermined substance, for example a lymphocyte, to produce antibody, The resulting hybrid cell can be selected and cloned to obtain cell lines producing substances having a unique structure and / or property. In particular, these hybridomas formed with lymphocytes can be used to produce monoclonal antibodies.

The development of hybridoma technology in recent years has been directed towards the production of cell lines which are both stable and produce in high yield and specifically a particular substance which is desired. There are various approaches to this problem which, due to their historical origin, have been largely concentrated in the field of immunoglobulins / antibodies.

A study of previous activities in this area gives an overview of the types of problems encountered and the various solutions attempted so far.

the initial impetus for research in hybrid cell lines manufacturing monoclonal products came from the field of immunobiology, 05-22-86 13:22 T-SRNBOZ PAT 61/577532 “291 -06 the products being antibodies monoclonals.

Conventional antisera usually contain a very large number of antibodies which differ structurally by the site of binding of the antigen, but each bind to the same antigen with a greater or lesser degree of avidity and / or specificity.

In addition, conventional antisera also contain a large number of antibodies to other antigens and reflecting the pre-labiated defense reactions of the receptor individual S from which the antiserum was obtained. Although these "large" antisera are sufficient for most applications, it has been felt that greater specificity and increased reproducibility would mark significant progress and provide a scientific instrument with considerable potential, particularly in the area of diagnosis and of therapy.

The first solution now conventional was that described by Köhler and Milstein [Nature, 20 256, pages 495-497 (1975)], who succeeded in fusing spleen cells of mice pre-immunized with myeloma cells of mice sensitive to "drug". The thus immortalized fused cells could be cultured in vitro and cloned from the single cell stage to produce a homogeneous cell population producing a homogeneous antibody population (monoclonal antibodies). By selection among the many single cells and selective recloning, Π has been possible to obtain and cultivate cells which produce antibodies with the desired specificity vls-3-vis the antigen.

Antibodies from mice produced in this way have been found useful for research and diagnostic 05-22-66 13:23 T-SAHDDZ PW 61/577532 tt291 -07 * t · * 3 and some of them have even been used in therapy in humans.

However, considerable progress in human therapy with immunoglobulin would be achieved if human or non-human primate antibodies of similar specificity and reproducibility could be obtained in this way. This would also reduce the risk of awareness. Several approaches to the problem of in vitro production of these human antibodies have been attempted, but have so far largely failed.

They include: (i) Transformation of normal human lymphocytes by Epste1n-Barr virus (EBV), This method has met with little success, since cells of this type require a long and tedious process for their establishment and are extremely difficult to clone and select.

(Ii) Fusion of normal (human) lymphocytes with human myeloma cells. This method represents an obvious analogy with the mouse-mouse hybridoma approach *, however, the problem is that * in the human system, only one myeloma cell line has been able to be produced widely and only 'it was found to be contaminated with mycoplasma which prevents successful mergers.

(iii) Fusion of normal human lymphocytes with an EBV transformed human lymphoblastotic B cell line. Although this method is probably the most reliable and the most reproducible which is described until 05-22-86 13:23 T-SANDOZ PAT 61/577532 “291 -08 4 present, it has the basic disadvantage in vitro encountered with lympho-blastold cells: they are extremely difficult to clone and, as they represent an earlier stage 5 in the line B of differentiation of B cells, their capacity to produce and secrete antibodies is approximately ten times weaker than that of true myelomas.

(iv) Fusion of human lymphocytes with 1 q mouse myeloma cells. This method·; produces cells with the same excellent characteristics in vitro as mouse-mouse hybridomas, but with the major drawback that these hybrids have intrinsic genetic instability. A particularly troublesome result of this is that they expel the human chromosome on which the genome is located for the formation of the light kappa chain of immunoglobulin.

It has been mentioned below that it was possible, thanks to the use of a xenogenic hybridoma cell serving as a mother cell for a subsequent fusion with another cell, to obtain a hybridoma having markedly improved stability. - (Ostberg et al. / HYBRXDOm 2, N * 4,361 25 1983), and that this "xenogenic" hybridoma present an environment more suitable for improving stability, expressed by chromosomal loss.

The present invention relates to a hybridoma cell line, comprising a mother immortalizing cell, fused with an associated xenogenic cell, the resulting immortalizing cell then being fused with a cell capable of producing a non-human primate monoclonal antibody. This last cell must be genetically compatible with said associated cell in the xenogenic dome. 05-22-86 13:24 T-SANDÜZ PAT 61/577532 # 291 -09 5 "Genetically compatible" means the characteristic of resembling the related cell, in terms of species and genus of origin, while avoiding significant loss of chromosomes, and therefore retaining a desirable degree stability after fusion.

It now turns out that the preparation and use of monoclonal antibodies to non-human primates prepared by this technique of fusion of xenogenic hybrids may prove to be a useful approach to the treatment of human beings by the use of antibodies. monoclonals, as well as in diagnostic applications. There is no prior record of preparation or isolation of monoclonal antibodies from non-human primates.

The present invention therefore relates to a method for preparing stable cell lines producing monoclonal antibodies to non-human primates which can be used in the treatment of human beings and in diagnostics. The subject of the invention is also immunoglobulins derived from non-human primate lymphocytes and which can have an effective use in humans. The non-human primates considered in the context of the invention essentially include the great apes, for example the orangutans, the 25 or chimpanzees, and the little apes like the New World monkeys, for example the cébus monkeys, and Old World monkeys, for example rhesus monkeys. With regard to a distinction between great apes and little apes, it is clear that, for practical reasons, it is preferred to use sufficiently small animals which can easily be handled under laboratory conditions, but in the context of the invention is meant all of the non-human primates.

05-22-86 13:25 T-SANDOZ PAT 61/577532 14291 -10 r 6

In a particular embodiment of the invention, the xenogenic hybridoma chosen as the immortalizing cell is a myeloma hybrid which has lost its own ability to produce immunoglobulin. An example of such a xenogenic hybridoma cell would be that obtained from a myeloma cell and a lymphocyte cell, Examples of suitable myeloma cells are those obtained from mice and rats, and , preferably, they do not produce immunoglobulins.

These myelomas can themselves be hybrids (eg mouse myeloma x mouse lymphocyte), which are I * *, * '.

actually myelomas. Such a cell will, for example, be the mouse SP-2 myeloma cell line. The latter can then be fused, for example, with a monkey lymphocyte, to give a monkey monoclonal antibody.

As described in the HYBRIDOMA 2 article mentioned above (as well as in the corresponding patent applications, for example UK patent application No. 2 113 715A, published on August 10, 1983), the xenogenetic hybridoma 20 used for immortalization is made resistant to drugs before a new fusion, and the lymphocyte with which it is fused is preferably obtained after presensitization, to improve the production of the desired substance. The methods used in drug resistance and presensitization treatments are conventional, as is the process of fusing these cells. Likewise, the selection of the hybrids desired for cloning are carried out in a conventional manner, as described previously and published for example in the referenced references mentioned above.

In a particular embodiment of the invention, the SP-2 cell line is used. It was initially a hybridoma of the line P3 ~ X63 ~ Ag8 and spleen cells of mice which produce antibodies * 05-22-86 13:25 T-SAND02 PAT 61/577532 # 291 -11 7 against sheep erythrocytes, and which has lost its ability to produce antibodies (cf. M. Shulmann et al ,, NATURE 276, 269, 1978).

It can be obtained, for example, from NXGMS 5 Human Genetic Mutant Cell Repository, ref. GM 35669 A (see US DHHS 1982 Catalog of Cell Lines). This cell line is made resistant to drugs, then it is fused with normal human peripheral lymphocytes by conventional techniques (cf. G. Galfre et al., NATURE 266, 550 (1977) and R. Novdnski et al.,. SCIENCE 210, 537 (1980)).

10 - Oh obtains a large number of hybrids and, after approximately five weeks, five clones are selected which exhibit rapid growth and no production of antibodies. These cells are selected for their resistance to 6-azaguanine and, with three of these lines, it is possible to obtain mutants which resist 20 ug / ml of 8-azaguanine. These cells are at the same time sensitive to the hypoxanthine-aminopterin-thymidine (HAT) medium, which has shown that they have lost their ability to produce hypo-xanthine-phorphoribosyl-transferase. One of these lines is 2q, the SPAZ 4 line, which can be used for a subsequent fusion with the aim of obtaining an antibody-producing hybridoma.

The simple hybridoma cell lines according to the invention, which produce monoclonal antibodies to non-human primates, can also, if desired or if necessary, undergo a new fusion, for example with another cell of monkey lymphocyte, to give cells which either have even greater stability or have somewhat different desired characteristics.

The antibodies according to the invention can be used in humans to fight against infectious diseases, malignant tumors, allergies, and they can be used to slow down rejection after transplantation 05-22-86 13:26 T-SANDOZ PAT 61/577532 8291 -12 8 and in other applications known in the art, such as in the case of toxicity caused by pharmaceutical substances, for example digoxin. They can also be used in diagnostic applications, for example in kits and the like.

The types of cell lines which the present invention relates to are, for example, cell lines comprising mouse myeloma cells and human and non-human primate cells *, originating for example from a mouse-human xenogenic cell to give a cell line (mouse x man) x non-human primate, for example a hybridoma cell line (mouse sï man) x monkey, or a urie cell line (mouse x non-human primate) x non-human primate, such as a cell line (mouse x monkey) * monkey · Other cell lines obtained according to the invention comprise, for example, a cell line ((mouse x man) x non-human primate) x non-human primate, for example a line cell ((mouse.x man) x cebu monkey) x cebus monkey, a cell line ((mouse x man) x rhesus monkey) x rhesus monkey, a cell line ((mouse x man) x chimpanzee) and a chimpanzee cell line ((mouse x human) x rhesus monkey) x chimpanzee.

The monoclonal antibodies prepared according to the invention can be formulated and administered in a conventional manner in therapeutic applications, and also can be presented in conventional kits for diagnosis.

The following examples illustrate the invention.

30 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. Then added 60 µl of ethylene glycol. After 5 minutes of incubation, 50 mg of lepas hemocyanin is added (keyhole limpet hemo-. 05-22-86 13:27 T-SAWDOZ PAT 61/577532 14231 -13 i 4! V. '9 cyanin, KLH ) in 10 ml of water at pH 9.5. When the pH has stabilized at 9.5, 0.3 g of NaBH 4 is added in 2 ml of water,

The preparation obtained is dialyzed overnight against a saline solution.

. g Qn emulsifies approximately 4 mg of KLH-digoxin in a volume of 0.5 ml at the same time as 0.5 ml of the complete Freund's adjuvant. The emulsion is injected intramuscularly into the two thighs of a cébus monkey. After this first injection, four booster injections are made, with an analogous amount of KL'H-digoxin but, this time, emulsified in 0.5 ml of incomplete Freund's adjuvant. After this immunization, a test bleed shows that the animal has produced antibodies which react with digoxin and which can inactivate the toxicity of digoxin in the context of in vitro studies. It is shown by observing that dilutions of monkey serum neutralize the toxic (lethal) effect of digoxin (or of sbn analogous lcubain) on sensitive human lypiphoblastold cells in cell culture. After allowing the animal to rest for two months, another intravenous injection of. 0.5 ml KLH-digoxin in a salified solution.

On the third day after the last intravenous injection, the cells of the animal are extracted for fusion.

25 a. Blood is taken from a peripheral vein in a syringe containing 50 μl of a heparin solution (10,000 USP units / ml). The blood lymphocytes are purified by centrifugation in a gradient centrifuge on a needle & sin of PERCQLL (Pharmacia # Inc., Piscataway, N, J.). After centrifugation, the cells are washed in a de-Hank balanced saline solution (Gibco Laboratories, Grand Island, N.Y., catalog No. 310-4020, which is readily available on the market).

b. Using aseptic surgical techniques, $ 3 is extracted from the inguinal area the lymph nodes.

05-22-86 13:27 T-9GNDGZ PAT 61/577532 14291 -14 V 1 t * 10

The lymph nodes are crushed through a steel mesh with a fine mesh opening, to prepare suspensions of individual cells. The isolated cells are washed in Hank's balanced saline solutions.

5 c. Using aseptic surgical techniques, a partial splenectomy is performed on the animal. A suspension of individual cells is prepared from the fragment of the spleen, by grinding the organ through a wire, metallic steel with a small mesh opening. This 1 q suspension of isolated individual cells is washed in Hank's balanced saline solution.

About 2.T0 isolated lymphocytes are mixed with a similar amount of spaz-4 myeloma cells (which can be prepared by methods described in the art, as in the reference HYBRIDOMA 2 mentioned above), in a medium free of serum, and pelletized in a low speed centrifuge. The cell tablet is treated for one minute with 1.0 ml of 50% PEG-1500 in a balanced Hank saline solution at pH 8.0. After 2Q one minute of incubation at 37 eC, 1 ml of Hank's balanced saline is slowly added. Over the next minute, an additional 10 ml of Hank's solution is added. The cells are recovered by centrifugation and resuspended in 20 ml of modified Eagle's medium from Dulbecco (N® 430-2100, Gibco Laboratories), VIROLOGY 8, 396 (1959); VIROLOGY U, 185 (I960)? Tissue Culture Standards Committee, IN VITRO, vol. 6, N * 2, p. 93, containing 20% of fetal beef serum and using the HAT system (hypoxanthine-aminopterin-30 thymidine) as a selection system,

After about three weeks, when good growth is observed in the culture tanks, the supernatant is extracted from the cultures for testing. The test is carried out by an ELISA technique, using digoxin coupled to beef serum albumin (BSA) as an antigen 05-22-86 13; 28 T-S6ND02 PAT 61-577532 # 291 -15 * "11 (BSA-digoxin is prepared in a manner analogous to the KLH-digoxin system). Positive clones are detected using an anti-human or anti-rabbit monkey immunoglobulin peroxidase system. Positive clones are taken for multiplication and, simultaneously, cloned by limited dilution in microtiter tanks using mouse thymocytes as loading cells. As soon as a large number of cells is available, the clones are frozen. in liquid nitrogen at -196eC.

ΙΟ Antibodies are obtained by growing hybridoma cell lines either in stationary culture or in mobile culture, using MEM medium from Dulbecco, with a maximum of 10 * fetal bovine serum. The antibodies, in a representative manner at a concentration] of 20 to 50 μg / ml, are purified by affinity chromatography on columns of Sepharose Protein A (Pharmacia, Inc,), placing the product to be chromatographed on the column , saturating the column binding capacity and washing the column with saline buffered with phosphate buffer. After removal of the unbound products from the column, the column is eluted with 0.05 M sodium citrate containing 0.5 M sodium chloride (pH 3.0). The eluate is quickly neutralized by addition of 1 M Tris-HCl, pH 8.0.

9K Example 2

Cells prepared by the methods of producing and selecting cells of Example 1, which cells have, over time, lost their ability to produce immunoglobulin, are made resistant to drugs by treatment in the presence of 20 ug / ml of 8-azaguanine, which allows a HAT system to be used in a conventional manner as a selection system. The cells obtained are then fused with immunized cebu monkey lymphocytes and, after a new treatment with HAT medium, positive cell cultures are chosen using • v 05-22-86 13:29 T — SANDOZ PAT 61/577532 “291 -16 12 m the same procedures as those described in Example 1 above.

According to this procedure, three antibodies were obtained from fusions using cells from the cebu monkey. Hybridomas are designated respectively by 7-1, 11-1 and 11-3. The ability of the three antibodies to inhibit the toxicity of digoxin to a human lymphoblastoid / IM-9 cell line was studied. The following results were obtained when the λ 1Q antibodies were used a final concentration of 100 ag / ml:

Addition Dose of digoxin not leading to A

_no lesions ____

Buffered saline solution

2.9.10 M phosphate buffer

15 7-1 4,7.10-7: M

9.4.10-7 "11-3 2.3.10-7> 4

The specificity of the antibody 7-1 with respect to other types of digital alkaloids was also studied. The results of this specificity test are summarized in the table below.

Drug. Application of drug causing no injury '

Saline solution buffered 25 with phosphate buffer 7-1

- Cuban 2.10 ® M 2.10 M

Digitoxin 5.9.10-9 M 2.3.10-8 M

DdsXanoside 2,3.1Q8M 1.9.10 M

These results, taken together, show that the antibodies produced by this procedure can be 'protective against the toxic effect of drugs and that, in the case of digitalis alkaloids, an antibody can cover two of the medicines used in outpatients (digoxin and digitoxin) and against 35 medicines used parenterally (deslanoside), 05-22-86 13:29 T-SANDOZ PAT 61/577532 8291 -17 13

It has no activity against another drug used parenterally (Cuban).

Example 3 In a manner analogous to that described in Example 1, the KLH-digoxin system is used as an immunogen in an adult male chimpanzee. After a total of five injections of this antigen, administered essentially every two weeks, provocation is carried out by intravenous administration of 0.5 ml of KLH-digoxin (approximately 10 mg of protein). The blood sample is taken from the animal on the sixth day after the last injection i, v. and "one day after bleeding" it is used for cell fusion with the myeloma cell line SPAZ-4, using the procedure described above. From the sample obtained on day 6, hybridomas producing IgG antibodies are obtained, called CH 4-14 and CH 4-25, having respectively a lambda light chain and a kappa light chain. Characterization of the light chains is carried out using the reagents against human immunoglobulins in a conventional manner. These reagents give unambiguous results, and the reactivity is, vis-à-vis anti-human reagents, stronger than vis-à-vis the anti-monkey reagents prepared against the cynomolgus monkey.

Two of these antibodies (mouse x man) x chimpanzee have an affinity for digoxin, markedly higher affinity than in the case of the cebus 11-1 antibody mentioned in Example 2 ei-dessuô.

Claims (14)

05-22-86 13:30 T-SANDOZ PAT 61/577532 Ö291 -18 * * * 14 " 1. A hybridoma cell line comprising an immortalizing cell fused with a cell capable of producing a monoclonal non-human primate antibody, the immortalizing cell comprising a xenogenic hybridoma cell originating from a cell fusion immortalizing mother with an associated cell, said cell capable of producing the antibody being genetically compatible with said associated cell. 2. A hybridoma cell line according to claim 1, characterized in that said cell capable of producing the antibody is of the same genus as the associated cell in the parent xenogenic hybridoma cell. 3. “A hybridoma cell line according to claim 1, characterized in that said cell capable of producing the antibody is of the same species as the associated cell in the mother xenogenic hybridoma cell, 4. A hybridoma cell line according to claim 2, characterized in that the cell producing the non-human primate monoclonal antibody is a lymphocyte. 5. A hybridoma cell line according to claim 1, characterized in that the non-human primate monoclonal antibody is a monkey monoclonal antibody. 6.- A hybridoma cell line. 30 according to claim 1, characterized in that the mother immortalizing cell is an SP-2 cell line, (JD- {Lé. — DD I -DMINUUC ΓΗ I bl- ^ D f (ZSCld. Hei Dl —1D. I C 15 7. - A process for the preparation of hybridoma cell lines according to claim 1, characterized in that an xenogenic hybridoma cell is made resistant to drugs, this cell is fused with a cell which is capable of produce an antibody which is genetically compatible with the associated cell in the xenogenic hybridoma "and the desired hybrid is selected. 8. A method according to claim 7, 10 characterized in that the selection of a desired hybrid is based on the absence of sensitivity to HAT and on its ability to produce a non-human primate monoclonal antibody. 9. A cell fusion system which comprises a cell producing a non-human primate monoclonal antibody and a xenogenic hybridoma cell originating from the fusion of an immortalizing cell with an associated cell genetically compatible with said antibody producing cell. ,. In a nutritive culture medium together with an agent promoting the fusion of said cells. 10. Non-human primate monoclonal antibody. 11. An antibody according to claim 10, - characterized in that the antibody is a monoclonal monkey antibody. 12. A hybridoma (mouse x man) x non-human primate cell line, 13. A cell line according to claim 12, characterized in that the cell line is a hybridoma (mouse x man) x monkey cell line. , 05-22-86 13:31 T-SGNDOZ PAT 61/577532 # 291 -20 16 14.- A cell line according to claim 13, characterized in that the cell line is a hybridoma cell line (mouse x monkey) x monkey, 5 15., - A hybridoma cell line comprising a triome cell fused with a cell producing a non-human primate monoclonal antibody, the triome cell comprising an immortalizing cell fused with a non-human primate lymphocyte, the immortalizing cell comprising a xenogenic hybridoma cell originating from the fusion of a mother immortalizing cell with an associated cell, said lymphocyte being genetically compatible with said associated cell and with said antibody producing cell. 17, - A hybridoma cell line according to claim 16, characterized in that said antibody producing cell is of the same kind as the lymphocyte * 20 18, - A hybridoma cell line according to claim 17, characterized in that the lymphocyte is of the same genus as the associated cell and that said antibody producing cell. 19, - A cell line according to reven-25 dicàtlon 16, characterized in that the cell line is a cell line [(mouse x man) x non-human primate] x non-human primate, 20, - A cell line according to claim 19, characterized in that the cell line is a cell line [(mouse x man) x chimpanzee] x chimpanzee, 21, - A cell line according to claim 14, characterized in that the cell line, 05-22-85 13:32 T-SANDOZ PAT 61/577532 “291 -21 17 is a cell line (mouse x man) x chimpanzee. 22, - An antibody according to claim 10, characterized in that the antibody is a chimpanzee monoclonal antibody.