JPS61271982A - Monoclonal antibody - 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 The present invention provides non-human primate monoclonal antibodies,
The present invention relates to hybridoma cell lines and methods of producing them, and to the use of said cell lines to produce said antibodies.

Hybridomas are immortalized cells (immortalizin).
g cell), for example, myeloma cells.

are often fused to what is an untransformed partner cell, said partner cell being specifically fused with a predetermined substance (
For example, antibodies are usually selected for their ability to produce antibodies (lymphocytes) that produce antibodies. The resulting hybrid cells can be selected and cloned to obtain cell lines that produce substances with the same structure and/or properties. In particular, such hybridomas formed using lymphocytes can be used to produce monoclonal antibodies.

Recent developments in hybridoma technology have been directed toward obtaining cell lines that are stable and at the same time highly and specifically produce the specific substance of interest. There have been various approaches to this problem. This is because their historical origins have largely focused on the field of immunoglobulins/antibodies.

A study of prior activity in this field provides an overview of the types of problems encountered and the various solutions that have been attempted.

The original inspiration for the study of cell lines that produce monoclonal antibodies came from the field of immunobiology, and the products are monoclonal antibodies. Conventional antisera usually contain a large number of antibodies with structurally different antigen-binding sites, each binding to the same antigen with greater or lesser degree of avidity and/or specificity. Furthermore, conventional antisera also contain a large number of antibodies directed against other antigens and reflecting the previous protective response of the individual from whom the antiserum was obtained. For most purposes, such "broad" antisera are
However, we feel that gaining greater specificity and reproducibility represents a significant advance and offers a scientific tool with much potential, especially in the fields of diagnosis and therapy. It was done.

The first and now classic solution was the cola (K.

h l e r) and Milste
in) [Nature, 256°4
95-497 (1975)],
They were able to fuse tile cells from previously immunized mice to 'drug'-sensitive mouse myeloma cells. The thus immunized and fused cells could be grown in vitro and cloned from live cell lines to produce a population of homologous cells that produced a population of homologous antibodies (monoclonal antibodies). By selecting on a large number of unique cells and selectively cloning one cell, it was possible to obtain and grow cells that produced antibodies with the desired antigen specificity.

Mouse antibodies produced in this manner have proven useful for research and diagnostic purposes, and some have even been used therapeutically in humans. However, if human or non-human long animal antibodies with similar specificity and reproducibility could be obtained in this way, it would represent a considerable advance in human immunoglobulin therapy. This would also reduce the risk of sensitization. Several approaches to the problem of producing human antibodies in vitro have been attempted, but hitherto largely without success.

These include: (i) Nibstein-Barr (E p s t
Transformation of normal human lymphocytes using the Sin-Barr virus (EBY), as this type of cell requires a long and tedious process to establish and is extremely difficult to clone and select. This method had little success.

CL i) Fusion of normal (human) lymphocytes with human myeloma cells, this method is strikingly similar to the mouse-mouse hybridoma approach, but only one cell line is widely accessible in the human system. , and this runs into problems when it is found to be contaminated with mycoplasma, which prevents effective fusion.

(iii) Fusion of +1 normal human lymphocytes into the EBV-transformed human lymphoblastoid B-cell lineage, which is probably the most reliable and reproducible method ever described. However, it suffers from the fundamental in vitro drawbacks faced using hydrinhoblastoid cell types: they are extremely difficult to clone, and they are difficult to clone in the origin of B-cell differentiation. Since they represent stage 19 stages, their ability to produce and secrete antibodies is about 10 times lower than that of true myeloma.

(iv) fusion of human lymphocytes to mouse myeloma;
Although this method produces cells with the same outstanding in vitro properties as mouse-mouse hybridomas, this one is particularly troublesome, with the drawback that such hybrids have inherent genetic instability. The result is that they shed human chromosomes, on which are located the genomes that form the kappa light chains of immunoglobulins.

By using the xenohybridoma cell as a parent for further fusion into other cells, it is possible to obtain hybridomas with greatly improved stability [Ostberg, Hybridoma].
v (HYBRIDOMA) Winter, No, 4.361.1
[983] and these interspecies hybridomas were subsequently reported to provide a more benign environment for improved stability upon chromosome loss.

Undeveloped II refers to a hybridoma cell line consisting of a parental immortalized cell fused to a xenogeneic four partner cell; 1. The resulting immortalized cell is then fused to a non-human primate cell capable of producing monoclonal antibodies. . This latter cell should be genetically compatible with its partner cell in a cross-species hybridoma. Genetically compatible means properties similar to related cells with respect to species and genus of origin, such as avoiding significant loss of chromosomes and therefore retaining a desired degree of stability after fusion. do.

Here, the preparation of non-human primate monoclonal antibodies by such cross-species hybridoma fusion techniques and the use of the prepared antibodies is a useful approach in human therapeutic and diagnostic applications using monoclonal antibodies. I think I can prove something. There are no known prior reports of the preparation or isolation of non-human primate monoclonal antibodies.

Accordingly, another aspect of the invention is to generate non-human anti-animal monoclonal antibodies that are useful in human therapy and diagnosis. Another object of the present invention is to provide a method for producing stable cell lines.Another object of the present invention is to provide immunoglobulins derived from non-human primate lymphocytes that can be used effectively in humans. 0 Non-human primates included in this aspect of the invention include:
In terms of wood, AIDS, for example, organutans (O
rgangut anS), gorillas, or chimpanzees, and monkeys, such as new world monkeys, such as cebus monkeys.
s), and Old World monkeys (old
world monkeys), such as rhesus monkeys. For practical purposes, the use of animals as small as 1 minute is preferred for ease of handling in laboratory conditions, but this aspect does not apply to all non-human primates. It is clear that 14 is intended to encompass.

In certain aspects, the xenohybridoma selected as the immortalized cell is a myeloma hybrid that has lost its own ability to produce immunoglobulins. One example of such a cross-species hybridoma cell would be between a myeloma cell and a lymphocyte; examples of suitable myeloma cells are obtained from mice and rats and preferably produce immunoglobulins. It's something you don't do. These bone marrow insects can themselves be hybrids that are actually myeloma (e.g., mouse myeloma x mouse lymphocytes); such cells are
For example, the mouse SP-2 myeloma cell line 0 can then be fused to, for example, monkey lymphocytes to produce monkey monoclonal antibodies.

The aforementioned HYBRIDOMA article (as well as the corresponding patent application 1, e.g.

British patent application 2,1 published in August 1983
The cross-species hybridoma used for immortalization is then rendered drug resistant prior to fusion, and the lymphocytes with which it is fused are preferably purified of the desired substance, as described in 13,715A). Obtained after being presensitized for production. The methods used in the treatment of drug resistance and presensitization are conventional, as are the methods of fusing these cells. Selection and cloning of the desired hybrids is also carried out in a conventional manner as described above and published, for example, in the references cited in 1.

In a particular embodiment according to the invention, the SP-2 cell line is used. It is originally P3-X63-Ag
A hybridoma between a strain of mouse tile cells that produces antibodies against sheep red blood cells and has lost its ability to produce antibodies [C, F, M, Sur? 7 (
Shu 1mann) et al., Nature
, 2ヱ1.269 (1978)].

It is, for example, the NIGMS Human Genetic Mutant Φ Cell Repository (Huma
n Mutant Genetic Cell
Repository)RefGM 35669
Human PNT ability from A (US D of cell line)
HH31982 catalog), this cell line is made drug resistant and then fused with normal human peripheral lymphocytes by conventional techniques [C, F, G, Galfre (Ga)
Nature (l fre) et al.
), 266.550 (1977)] and R. Nowinski et al., Science (SCIE
NCE) Mujosuke, 537 (1980)].

A large number of hybrids are obtained and after about 5 weeks, 5 clones are selected that show rapid growth and do not produce antibodies. These cells were selected for resistance to 8-azaguanine, and three of these lines were used to obtain mutants that were resistant to 20 gg/ml of 8-7 zaguanine. It is possible. These cells were simultaneously sensitive to hypoxanthine-aminopterin-thymidine ()IAT), indicating that they had lost the ability to produce hypoxanthine phosphoribosyltransferase. One of these cell lines is SPAZ 4, which can be used for further fusions to obtain antibody-producing hybridomas.

Hybridoma cell lines of the present invention that produce non-human primate monoclonal antibodies can also be used, if desired or necessary.

For example, further fusions can be made with further lymphocytes from monkeys to provide cells with even more stable or somewhat different desired properties.

Then, one antibody according to the present invention can be used to treat infectious diseases in humans,
It can be used to control malignant tumors, allergies, and to suppress rejection of transplants and other IJ agents known in the art, such as preparations such as those caused by digoxin 11i. Can be used for sex. They are also 1, the use of rare questions,
For example, it can be used in a kit.

Examples of types of cell lines related to the present invention are cell lines consisting of mouse myeloma cells and human and non-human primate cells, such as cell lines from mouse-human xenogeneic cells (mouse x human). ×Non-human primate cell 1 For example, (mouse × human) × monkey hybridoma cell cell line, or (mouse × non-human primate) ×
A non-human primate cell line 1, for example, producing a (mouse x monkey) x monkey cell line and another cell line provided according to the present invention may be, for example, [(mouse x human) x human. other than 'j: 1 anti-animal] x non-human primate cell line 1 e.g.

[(mouse x human) )X rhesus macaque]×chimpanzee cell lines.

Monoclonal antibodies produced according to the invention can be formulated and administered in therapeutic applications in a conventional manner, and can also be used in the construction of conventional kits for use in diagnosis.

The following examples illustrate the invention.

Dissolve digoxin in 2 ml of absolute ethanol and add dropwise 2 ml of 0.1 molar sodium periodate and incubate this mixture for 45 minutes.6 Then 60 microliters of ethylene glycol. Add. After incubation for 5 minutes, 1
Add 50 mg of keyhole limpet hemocyanin (KLH) in 0 ml of water at pH 9.5. p
0 in 2 ml of water when H stabilized at 9.5
．． Add 3g NaBH4. The resulting preparation is dialyzed against saline overnight.

Volume 0, 5 m 1 (7) Emulsify approximately 4 mg of KLH-digoxin with 0.5 ml of 70 India complete adjuvant. This emulsion was applied to CebuS Monkey (cebuS).
Inject intramuscularly into both thighs of the monkey. After this first fl injection, the animals are stimulated four times with the same acid, but this time with KLH-digoxin emulsified in 0.5 ml of Freund's incomplete adjuvant. After this immunization,
The bleeding test shows that the animals produce antibodies reactive with digoxin and that this antibody is capable of inactivating the toxicity of digoxin in in vitro studies.

This suggests that dilutions of monkey serum can induce IXi of digoxin [or its cousin, oubain] to susceptible hydrinphoblastoid cells in cell culture.
This is demonstrated by focusing on the fact that it neutralizes the lethal effects. After subjecting the animals to body IF for 2 months,
0.5 ml of KLH-digoxin in saline was injected intravenously.

After the last intravenous injection: at tS311, cells are taken from the animals for fusion.

a. 50g1 heparin solution (10,000US
Blood is taken from peripheral vessels into a syringe containing P units/ml). The lymphocytes of this blood were treated with PERCOLL [7 Pharmacia, Inc.].
Piscataway, N.

J, )] cushion 1. Purify by gradient centrifugation.

After centrifugation, cells were soaked in Hank's balanced salt solution (Hank's Balanced Salt Solution).
balanced 5alt 5solution
) [Gibco Laboratories (Gibc.

Laboratories, Grand l5land, N, Y, Catalog No. 310-
4020. It is widely used commercially and washed with r-Iif].

b. By using aseptic surgical technique, the lymph nodes are excised from the area of the root of the thigh. The lymph nodes are ground through a fine-mesh steel mesh to prepare a medium-fine suspension. ? Wash the detached cells with the bank's balanced salt solution.

A partial stagectomy is performed on the animals by using C1 sterile surgical technique.

The organ is ground through a fine steel mesh to prepare a medium-fine suspension from the tablet. Wash this neutralized cell suspension with Hang's salt solution.

I4 SPAZ-4 myeloma cells [prior art, e.g., previously described hybridoma 2
(L1+) capacity] and precipitated together in a centrifuge at low speed. Cell pellets were collected at pH 8.

50% PEG-1500 in O hang's balanced salt solution
c7) Treat with 1.0 ml for 1 minute. After incubation for 1 minute at 37°C, slowly add 1 ml of Hang's balanced salt solution. After another minute, another 10m1
Add the hang solution. Collect cells by centrifugation,
and 20 ml of Dulbecco's modified Eagle medium (Du
lbecco's Modified Eagle
Medfum) (No. 430-2100, Gibco Laboratories) [VIROLOGY
Dan, 396 (1959), VIROLO
GY)↓ヱ, 185 (1960), Tissue Culture Standards Committee 0
(Tissue Culture Standard
s Comm 1ttee), in vitro (IN VIT)
RO), Vol. 6, No. 12, Page 93, containing 20% bovine Jffi baby serum and using HAT (hypoxanthine-aminopterin-thymidine) as the selection system].

About 3il! After 1 hour, good 11f growth was observed in the culture well (we
1- Clear fluid is taken from the culture for testing when observed during tl). The test is carried out using digoxin conjugated to bovine blood blue albumin (BSA) as antigen (BSA-digoxin is produced in a similar way to KLH-digoxin) by the ELISA technique. Positive clones are detected using rabbit anti-human or anti-monkey immunoglobulin-peroxidase systems. Positive clones were subjected to JIF, l (scaling
up) for taking 1. At the same time, mouse thymocytes were used as filler cells in a microtiter.
Cloning by limited dilution in ter).

As soon as a large number of cells are available, clone one by one.
Freeze in liquid nitrogen at 96°C.

Static culture or roller culture using Dakubetsuko's MEM containing up to 10% calf serum.
Antibodies are produced by growing hybridoma cell lines in the r culture. Antibodies are typically 20-50 micrograms/mI of 1HJ
ff, SepharoSe protein A
By affinity chromatography (Pharmacia Inc.), the substance to be chromatographed is applied to this column and the binding volume of the column t
β is saturated and purified by washing the lamb with phosphate buffered saline. After removing unbound material from the column, the column is eluted with 0.05 M sodium citric acid (pH 3.0) containing 0.5 M sodium chloride. The eluate is rapidly neutralized by the addition of 1 molar Tris-HCl, pH 8.0.

Example 2 Cells produced according to the cell production and selection method of Example 1 (the cells have lost the ability to generate immunoglobulins over time) were subjected to drug resistance in the presence of 201 Lg/ml of 8-azaguanine. 0 then 1! which allows the use of HAT as a selection system in the conventional manner. IIT cells were fused to lymphocytes of an immunized Seb's monkey, and IIT
After treatment with HAT-medium and HAT-medium, positive cell cultures are selected according to the L order of .

Following this ruled order, three antibodies will be produced from the fusion using cells from Cebus Monkey! I was beaten. These hybridomas are designated as 7-1, 11-1 and 11-3, respectively. All three antibodies were tested for their ability to block the sexing of digoxin to the hydrinphoblastoid cell line, IM-9. Using the antibody at a final concentration of 1100 JL/ml, the following results were obtained:
9.4X10-7 mol 11-3 2.3X10
The -7mol7-1 antibody was also tested for its specificity against other types of digitalis alkaloids. The results of this specificity test are summarized in the following table:
10-” Digitoxy 5, 9X 2
, 3Xn 10-9
10-8 Deslanoside 2, 3X
l, 9X(desla 10-”
10-'nos i d) Considering these data together, it is clear that the antibodies produced by this method are protective against the action of the drug and, in the case of digitalis alkaloids, 1 One antibody is out-clinic (out-cl).
inic) that can cover two types of drugs (digoxin and digitoxin) used in patient criteria and one
It depends on the type of drug used for parenteral purposes (deslanoside). It has no activity against other parenteral drugs (wooapain).

In a method similar to that described in Example 1, KLH
- Using digoxin as an immunogen in adult male chimpanzees. After a total of 5 injections of this antigen, after an interval of essentially 2 weeks, a final counter-dose of 0.5 ml was administered.
Administer intravenously using KLH-digoxin (4 mg of protein), take a sample of white fluid orally 6 days after the last intravenous injection, and 1 day after bleeding. [Use for cell fusion with the SPAZ-4 myeloma cell line according to the L order outlined in 1. From the sample obtained on the 611th, CH4
IgG-producing hybridomas designated as -14 and CH4-25 were obtained, which have lambda and kappa light chains, respectively. Light chain characterization is carried out in a conventional manner using reagents for human immunoglobulins; such reagents give unambiguous results, and the reactivity is similar to that of macaque monkeys (C.
ynomolgus monkey) than against anti-human reagents prepared against
Both of these (mouse x human) x chimpanzee antibodies
s) WJ'A has a higher affinity for digoxin than antibody 11-1.

Claims (1)

[Scope of Claims] 1. Consists of an immortalized cell fused to a monoclonal antibody-producing cell of a non-human primate, said immortalized cell being a fused cross-species hybridoma cell from a parent immortalized cell and a partner cell. A hybridoma cell line comprising: said antibody-producing cell being genetically compatible with said partner cell. 2. The hybridoma cell line according to claim 1, wherein the antibody-producing cell is of the same genus as the partner cell in the parent of the xenogeneic hybridoma cell. 3. The hybridoma cell line according to claim 1, wherein the antibody-producing cell is of the same species as the partner cell in the parent of the xenogeneic hybridoma cell. 4. The hybridoma cell line according to claim 2, wherein the non-human primate monoclonal antibody-producing cells are lymphocytes. 5. The hybridoma cell line according to claim 1, wherein the non-human primate monoclonal antibody is a monkey monoclonal antibody. 6. The hybridoma cell line according to claim 1, wherein the parental immortalized cell is the SP-2 cell line. 7. Making the xenohybridoma cells drug resistant, fusing the latter cells with antibody-producing cells that are genetically compatible with the partner cells in the xenohybridoma, and selecting the desired hybrid. A method for producing a hybridoma cell line according to claim 1. 8. The method of claim 7, wherein the selection of the desired hybrid is carried out on the basis of assays for lack of sensitivity to HAT and the ability of non-human primates to produce monoclonal antibodies. 9. Promoting the fusion of non-human primate monoclonal antibody-producing cells with immortalized cells and fused cross-species hybridoma cells from partner cells that are genetically compatible with the antibody-producing cells; A cell fusion system characterized in that it is contained in a nutrient medium together with a factor. 10. Non-human primate monoclonal antibodies. 11. The antibody according to claim 10, which is a monkey monoclonal antibody. 12. (mouse x human) x non-human primate hybridoma cell line. 13. (Mouse x non-human primate) x non-human primate hybridoma cell line. 14. The cell line according to claim 12, which is a (mouse x human) x monkey hybridoma cell line. 15. The cell line according to claim 13, which is a (mouse x monkey) x monkey hybridoma cell line. 16. Consisting of trioma cells fused to monoclonal antibody producing cells of a non-human primate, said trioma cells consisting of immortalized cells fused to lymphocytes of a non-human primate, said immortalized cells a hybridoma cell consisting of a fused xenohybridoma cell from a parental immortalized cell and a partner cell, wherein said lymphocyte is genetically compatible with both said partner cell and said antibody-producing cell. system. 17. The hybridoma cell line according to claim 17, wherein the antibody-producing cells belong to the same genus as lymphocytes. 18. The hybridoma cell line according to claim 17, wherein the lymphocytes are of the same genus as both the partner cell and the antibody-producing cell. 19. [(mouse x human) x non-human primate] x non-human primate cell line Claim 1
Hybridoma cell line according to item 6. 20. The hybridoma cell line according to claim 19, which is a [(mouse x human) x chimpanzee] x chimpanzee cell line. 21. The cell line according to claim 14, which is a (mouse x human) x chimpanzee cell line. 22. The antibody according to claim 10, which is a chimpanzee monoclonal antibody.