NO782081L - PROCEDURE FOR PREPARING ANTIBODIES - Google Patents

Abstract

Procedure for the production of antibodies

Description

The present invention relates to a method for producing antibodies from living cells in vitro.

The production of antibodies specific for the antigenic determinants of viruses is of importance both for immunotherapy

pi and for medical research.

Monoclonal antibodies to antigenic determinants of viruses have been produced in vitro in cultures of spleen cells infected with a virus. Until now, however, the production of the antibody by splenic foci in culture has decreased after 30-40 days and ceased completely after 90 days.

New techniques have been developed for the production of antibodies, more particularly the propagation of new cell types which are genetically stable, can be cultivated and further cultivated indefinitely and produce large quantities of antibodies against viruses and their antigenic determinants. The new cell types are fused cell hybrids of. (a) myeloma cells, i.e. malignant cells from primary

re tumors in the bone marrow, and (b) antibody-producing cells, preferably those from the spleen or lymph nodes of virus-infected animals. A particularly preferred cell type is a fused cell hybrid between virus-infected mouse spleen cells and mouse myeloma cells. These cells can be maintained essentially indefinitely in a culture medium such as hypoxanthine-aminopterin-thymidine-selected medium and continue to produce antibodies specific for the antigenic determinants of the virus. The cells can too

is grown in vivo in a tissue compatible animal to accumulate large amounts of antibodies in the animal's serum and tissue fluid.

Myeloma cells themselves are unique in what such cells are

able to produce antibodies though the specificity of

these antibodies are still unknown. The particular species of animal

■ from which the myeloma and spleen cells are obtained is not critical as it is possible to fuse the cells of one species with another, i.e. mouse to rat or rat to human. However, it is preferred to use the same animal species as the source of both myeloma and viral antibody-producing cells. Excellent results have been obtained with somatic cell hybrids between the viral antibody-producing spleen cells from a BALB/c mouse previously immunized with a virus and myeloma cells from a BALB/c mouse. Particularly preferred myeloma cells are those of the M0PC-21 type called clone (P3 x 63. Ag8) and discussed by Kohler et al in Nature, Vol. 256, 495 - 497

(1975).

Fused hybrids of BALB/c spleen cells and BALB/c myeloma cells have previously been described in the literature by Kohler et al in Nature, Vol. 256, 495-497 (1975) and Eur. J. Immunol., Vol. 6, 5H-519 (1976). However, these hybrids were not obtained from. mice immunized with viruses, but instead from mice immunized with red blood cells from sheep. Hybrids between sheep or red blood antibody-producing cells and myeloma cells are easily formed, but antibodies produced by the hybrids according to Kohler et al are specific for sheep red blood cells and have no . effect on viruses. Before the present invention, it was not known whether hybrids could be formed between viral antibody-producing cells and myeloma cells.

The following is a method for producing a cell type of hybrid cells by fusing the spleen cells

a BALB/c mouse pre-immunized with influenza virus with myeloma cells of a BALB/c mouse. Although the method utilizes a particular strain of influenza virus, other viruses such as rabies, mumps, vaccines, influenza strain 6-94, simian virus 40, polio, etc. can be used.

(a) Preparation of spleen cells for fusion

To prepare the spleen cells for fusion, BALB/c mice, which had previously been given an intraperitoneal injection of 1250 hemagglutination units of purified influenza virus (A.PR/8/34) 2-3 months before, received intravenously a booster dose of 100 hemagglutination units of the homologous virus. The mice were euthanized 7 days later and one spleen cell suspension was prepared as described by Gerhard et al, Eur. J. Immunol., 5, 720-725 (1975). Red blood cells lyse.t by incubation for 15 minutes at 4° C in NH^Cl (0.83%). The resulting cell suspension was washed by one centrifugation (800 x g) through heat-inactivated calf serum and one centrifugation in protein-free medium (RPMI 1640, buffered with 7.5 mM HEPES, pH 7.2).

(b) Preparation of myeloma cells for fusion

BALB/c (P3 x.63 Ag8) myeloma cells obtained from the MOPC-21 type and deficient in HPRT (E.C.2.4.2.8) as described by

Dr. Cesar Milstein in Nature, Vol. 256, 495-497 (1975) was maintained in Engle's minimal essential medium (MEM) containing 10% fetal calf and 10% horse serum. The growth of P3 x . 63 Ag8 myeloma cells are inhibited by selective hypoxanthine-aminopterin-thymidine medium.

(c) P roduction of hybrids

Productions of hybrids were performed by mixing ten million BALB/c (P3 x 63 Ag8) myeloma cells with 10 8 spleen cells obtained from the virus-infected BALB/c mice. The cell mixture was centrifuged at 800 x g and the cells were resuspended for fusion in a 500 g/l solution of polyethylene glycol-1000 (PEG) diluted in minimum essential medium (MEM) without serum. By following the fusion methods set forth by Davidson et al, Somat, Cell Genet. 2, 175 - 176 , polyethylene glycol was first diluted with MEM without serum and then with serum before the cells were seeded into five 75-ml Falcon flasks in hypoxanthine-aminopterin-thymidine (HAT) selective medium. The cultures were incubated at 37° C. in an atmosphere of 95% air/5% CO 2 , and every 7 or 10 days the culture medium was partially replaced with fresh HAT medium (1/2 - 1/3).

10 to 15 days after incubation of the cultures formed by fusion of spleen cells of PR8 immunized mice with P3 x 63 Ag8 cells, cell growth was observed in a flask. Any growth in HAT medium is indicative of successful hybridization between mouse spleen and mouse myeloma cells. These cells, designated as HK-PEG-1, were propagated continuously in HAT medium and were cloned in microplates

(Linbro) by limiting dilution. The other 4 flasks, none of which showed cell growth, were discarded 3 weeks after fusion. A sample of the cell type culture designated as HK-PEG-1 has been deposited with the American Type Culture Collection and has been assigned the collection number ATCC CL 189.

(d) K aryological analysis

As shown in Table 1, the P3 x 63 Ag8 stem cells contained an average of 63 chromosomes and the BALB/c spleen cells 40 chromosomes. The 92 chromosomes in the HK-PEG-1 cells thus approximately represented the sum of chromosomes of 2 stem cells. The physiology of the hybrid clone HK-PEG-1dg subclones was monitored for 4 months without any significant change in the karyotype.

(e) Analysis of immunoglobulin produced by the hybrid cells Culture fluids of HK-PEG-1 and P3 x 63 Ag8 cells were analyzed for the presence of immunoglobulins (ig) that react in the radioimmunoassay (RIA) with PR8. P3 x 63 Ag8 is known to secrete IgCl, k. As shown in Table 2, P3 x 63 Ag8 culture fluids did not contain Ig with anti-PR8 reactivity. In contrast, large amounts of IgG anti-PR8 antibodies were produced by HK-PEG-1. Data in Table 3 show that the viral antibodies (anti- ' PR), formed by the hybrid cells are specific for the antigen (hemagglutinin) of PR8. This is evident from the functional determinations in which the antibodies (approximately 40 ug/ml) showed a homeostasis lutein inhibition (HI), but no detectable neuraminidase inhibition (NI).'

(f) C lonal origin of the hybrid cells

The clonal origin of HK-PEG-1 was investigated by 3 independent criteria: (i) Concentrated Ig obtained from HK-PEG-1 mass culture (see legend to Table 3) was subjected to isoelectric focusing, anti-PR8 antibodies with IgCl heavy chain determinants accumulated in a limited pH range. (ii) The antibody was tested in RIA for its cross-reactivity against various viruses known to be antigenically related to PR8 and was found to be specific for a determinant of hemagglutinin (HA) of PR8. However, 20 to 25% of splenic PR8-primed precursor B cells exhibited this species-specific anti-HA (PR8) reactivity. Taken alone is therefore anti- the drug reactivity a rather weak criterion for determining the monoclonality of HK-PEG-1. (iii) To exclude the possibility that cells producing antiviral antibody constitute only a small part of the hybrid cell population, culture fluids from 12 clones of HK-PEG-1 hybrid types were analyzed for the presence of anti-PR8 antibody and its specificity. As shown in Table 4, 11 of the 12 clones produced anti-PR8 antibody. Moreover, these antibodies were specific for a determinant of the HA of the PR8 virus.

(g) Tumor induction of the hybrid cells

To determine the tumorigenicity of the hybrid cells, adult BALB/c mice were injected subcutaneously in the abdominal wall with either HK-PEG-1 (1 x 10<7>) or P3 x-63 Ag8 (1 x 10<7>) cells.

As shown in fig. 5, ten to twelve days after implantation, tumors developed at the inoculation site in 5/5 mice injected with HK-PEG-1 cells and in 3/5 mice injected with the P3 x 63 Ag8 stem cells. In another experiment, 4/5 mice developed tumors after inoculation with hybrid cells and 3/5 after inoculation with P3 x 63 Ag8 cells. After intraperitoneal injection of cells (Experiment 3, Table 5), tumors were found to grow as masses in the abdominal cavity. To produce ascitic fluid, it was necessary to inject the mice with tumor cells resuspended in complete Freund's adjuvant. In a fourth experiment, pristane-pretreated mice developed ascites after i.p. inoculation of cells by

HK-PEG-1 clones.

The results of these investigations indicate that hybrid cells between mouse myeloma and normal spleen cells behave like the malignant origin without suppressing the malignancy.

Sera and tissue fluids obtained from tumor-bearing mice at various intervals after injection were examined to determine the concentration of anti-PR8 antibody by Ria. The results are shown in table 6.

Sera from mice injected subcutaneously with HK-PEG-1 cells (experiments 1 and 2) contained anti-PR8 antibodies at a concentration of 1-3 mg/ml. Anti-PR8 antibodies were also found in the ascitic fluid of mice injected i.p. (experiment 4) with hybrid cells, although the concentration of antibodies was 3 to 4 times lower than in the serum.

Electrophoresis of serum obtained from mice bearing HK-PEG-1 tumors showed the presence of 3 major Ig populations: One corresponding to stock (P3 x 63 Ag8) IgG1; one characteristic of IgG3; and one third lying between the other two. For. to determine with which group of immunoglobulins the antiviral antibody was associated, ascitic fluid was collected from 20 pristane-pretreated mice, pooled and the immunoglobulins separated therefrom. Separation was performed using a method as follows: Ascitic fluid collected from mice was pooled and neutralized with phosphate buffered saline (PBS). An equal volume of saturated ammonium sulfate was added at 4°C, and the precipitated protein was dissolved in PBS and dialyzed against 0.01 M tris-

buffer at pH 8.0. The precipitate, formed after dialysis overnight, was redissolved in PBS and precipitated again by dialysis against 0.01 M tris buffer at pH 8.0. The supernatant from the original dialysate was absorbed on a DE-52 (Whatman) column equilibrated with 0.01 M Tris buffer, pH 8.0, and eluted using a linear sodium chloride gradient (0.05 - 0.15 M

• NaCl in 0.01 M tris, pH 8.0).

After purification of IgG3 by dialysis against 0.01 M tris buffer, the remaining supernatant was found by electrophoresis to lack one of the components of the ascites fluid prior to fractionation. Chromatography of this material on DE-52 did not lead to complete separation of the remaining components, but electrophoresis of the individual samples showed the presence of two main components: one corresponding to the original (Ps x 63 Ag8) IgGl, and the other indicating a hybrid Ig, lying between IgGl and IgG3, which was also repeated in the serum of HK-PEG-1 tumor-bearing mice (see above).

The concentration of anti-PR8 antibody was determined 125

in RIA using I-anti-(F(ab')2 and the specific anti-PR8 activity was calculated as the ratio of anti-PR8 (mg/ml) 2Ig (mg/ml), the latter calculation being based on OD .. the 280 measurement assuming an absorbency (1%, 1 cm) for mouse Ig of 14.0 The specific anti-PR8 activity determined in the low-salt sweep was approximately twofold higher than at the peak of the anti-PR8 activity of DE-52 chromatography (0.06 in fraction 21).Analysis of the samples in RIA with <123>I-anti-IgGl indicated that approximately 15% of the anti-PR8 antibodies in the low-salt precipitate expressed Gl determinants In contrast, 12 5 I-anti-F(ab') 2 was equally effective in quantitating the anti-PR8 antibodies in the different fractions of the DE-52 chromatography.

Another series of experiments was carried out with the rabies virus.

BALB/c mice were pretreated with vaccine containing inactivated rabies virus (ERA strain) and one month later they received an intravenous re-inoculation of the same vaccine. The mice were killed 3 days after the booster inoculation and the hybrid cells were formed by fusion of the spleen cells with P3 x 63

Ag8 myeloma cells as described previously. A large number

of the hybrid cells produced anti-rabies virus antibodies.

However, when the mice were sacrificed 10 days after the booster inoculation, the number of hybrid cells that produced anti-rabies antibodies was quite low. This method was used to obtain the hybridomas used in the experiments shown in Tables 7, 8

.and 9.

The strains of rabies virus used in the following samples are known species. For example, strain ERA is described, among other things, in US patent 3,423,505 and 3,585,266. HEP-Flury is described, among other things, in US patent 2,768,114 and SAD is described in Can. J. of Microbiology, 6, 606 (1960). Likewise, the analytical samples used are well known in the art.

52 hybridomas that produced anti-rabies virus antibodies as determined by radioimmunoassay (RIA) were tested against three different strains of rabies virus (ERA, CVS and HEP - lury strains). In addition, four different determination methods were used, "Virus Neutralization" (VN), "Cytotoxic Test" (CT), "Membrane Fluorescence" (M), and "Nucleocapsid Fluorescence" (NC). The results of the experiments are shown in table 7.

As Table 7 shows, antibodies that react with one strain need not react with another strain of rabies virus. As shown in Table 7, 9 out of 52 hybrid cells secreting rabies antibodies reacted in virus neutralization, cytotoxicity and membrane immunofluorescence assays with all three strains of rabies, ERA, CVS and HEP flury; 15 reacted with the ERA and HEP strains; 2 with ERA and CVS; 3 with only and with only HEP virus. 28 hybrid cultures produced antibodies that reacted with nucleocapsids of all 3 virus strains; and of those, 23 reacted only with nucleocapsids and not with other viral components.

Analysis of the antigenic relationship between street strains and specific strains of rabies virus was studied using a further ten strains of different geographical origin. The hybridomas were prepared as described above using ERA. Results given in Table 8 indicate that Kelev virus of a particular strain is only neutralized by antibodies secreted by a hybridoma (No. 120) and the recently discovered southern,

.African street viruses (Deuvenhage) only by antibodies secreted by two hybridomas. In contrast, all gate viruses appear to be cross-reactive in VN except the AF strain which does not appear to react with antibody of No. 193. Hybridoma antibodies were found to be quite heterogeneous in VN determinations with particular strains of virus. For example, the PM and CVS strains, both of which were obtained from the Pasteur strain, react differently from the Pasteur strain and from each other. On the other hand, SAD virus and its derivative ERA reacted identically with antibodies secreted by the same hybridomas. Finally, the non-virulent HEP was found to be cross-reactive with the Kelev strain in VN with No. 120 antibodies. In addition, however, HEP was neutralized by antibodies secreted by three other hybridomas. Cells infected with all viruses of any origin showed intracytoplasmic fluorescence after fixation and exposure to antibodies raised by hybridoma 104 (NC determination),

although neither virus reacted with the same hybridoma in VN. This confirmed that although the hybridoma's antibodies easily distinguish strains of certain rabies viruses. in VN, CT and MF samples, the antibodies react with all strains of rabies in the NC determination.

The results shown in Tables 7 and 8 demonstrate that anti-rabies virus antibody-producing hybrid cells are very useful for research and analytical purposes.

Hybrid cultures secreting rabies virus-neutralizing antibodies protect mice against the lethal effects of rabies virus. Cells of hybridoma mass cultures C-l and B-l and

. of clone 110-5 (see Table 9) was implanted subcutaneously in BALB/c mice. Serum obtained from these mice within 2 weeks after implantation of the C-1 and 110-5 hybridomas showed a hundredfold

higher concentration of rabies antibodies than medium from tissue cultures of the same hybridoma. 5 days after implantation of hybridomas, the mice were intracerebrally given a lethal dose of PM rabies virus. As shown in Table 9, mice implanted with hybridoma cultures secreting VN antibody survived the viral challenge, whereas mice bearing hybridomas not secreting VN antibody were not protected.

The cell type according to the present invention represents a hybrid culture that exhibits characteristics of both the normal spleen and the myeloma stem cells and appears to be derived from

a single merger case. The cell is hybrid in nature, because:

(a) The cell type has been cultured for several months in selective HAT medium which inhibits the growth of the P3 x 63 Ag8 myeloma stem cells, but not the normal spleen cells which, in turn, presumably would not survive more than 4 to 5 weeks in vitro; (b) The number of chromosomes in the hybrid cells is close to the sum of that of the normal mouse and•myeloma stem cells; (c) the HK-PEG-1 hybrid produces not only IgG1, which is also secreted by P3 x 63 Ag9, but also IgG3, and presumably hybrid molecules; and (d) The hybrid produces antibodies with antiviral activity, whereas P3 x 63 Ag8 does not.

Although the antibodies are best produced by propagating the hybrid cell type in vitro, antibodies can also be produced by injecting a histocompatible animal with the hybrid cell type and producing the antibodies in vivo. For example, mice, relatively cheap animals, can be injected with the mouse-derived antibody producing cells according to the invention, to produce recoverable antibodies in sera and body fluids. Table 6, discussed earlier, shows the amount of antibody obtained under such conditions.

The antibodies produced by the hybrids can be harvested using standard methods and can be used in analytical medical research to identify viral antigens in blood samples and culture media. The antibodies produced in vitro are homogeneous and highly specific for the viral antigen. A supply of different homogeneous antibodies, each highly specific for a particular antigen, allows researchers to rapidly determine the specificity of an antigen and/or characterize viral antigens. In addition, the antibodies can be administered to diseased animals to help them fight the disease.

Claims (22)

1. Method for the production of viral antibodies, characterized in that a fused cell hybrid is produced between a viral antibody-producing cell and a myeloma cell, that the hybrid is cultivated and the viral antibodies are collected. 2. Method according to claim 1, characterized in that the hybrid is cultivated in vitro. 3. Method according to claim 2, characterized in that the hybrid is cloned and grown in a medium containing hypoxanthine-aminopterin-thymidine. 4. Method according to claim 1, characterized in that the hybrid is injected into a histo-compatible animal and -cultivated in vivo. 5. Method according to claim 4, characterized in that the hybrid is a clone. 6. Method according to claim 1, characterized in that the viral antibody-producing cell is a cell selected from the group consisting of spleen cells and lymph node cells. 7. Method according to claim 5, characterized in that the viral antibody-producing cell is a spleen cell. 8. Method according to claim 6, characterized in that the spleen is mouse spleen and the myeloma is mouse myeloma. 9. Method according to claim 7, characterized in that the mouse is a BALB/c mouse. 10. Method according to claim 9, characterized in that the myeloma is (P3 x 63 Ag8) derived from the MOPC-21 line. 11. Method according to claim 1, characterized in that the viral antibody-producing cell is obtained from an animal immunized with a virus. 12. Method according to claim 2, characterized in that the viral antibody-producing cell is obtained from one animal immunized with a virus. 13. Method according to claim 7, characterized in that the viral antibody-producing cell is kept from a mouse immunized with a virus. 14. Method according to claim 12, characterized in that the virus is an influenza virus. 15. Method according to claim 12, characterized in that the virus is a rabies virus. 16. Method for the production of antibodies, characterized in that a BALB/c mouse is injected with a virus to cause antibody formation by spleen cells in the mouse, that a fused cell hybrid is formed of the viral antibody-producing spleen cells with a BALB/c myeloma cell clone (P3 x 63 Ag8) obtained from the M0PC-21 line, the hybrid is cultivated in vitro in selective HÅT medium and the antibodies produced are harvested. 17.. Method according to claim 16, characterized in that the virus is an influenza virus. 18. Method according to claim 16, characterized in that the virus is influenza virus strain 6-94. 19. Method according to claim 16, characterized in that the virus is simian virus 40. 20. Method according to claim 16, characterized in that the virus is a rabies virus. 21• Preparation comprising a continuous cell line producing influenza antibodies in vitro in hypoxanthine-aminopterin-thymidine medium comprising a fused cell hybrid of influenza-affected BALB/c mouse spleen and MOPC-21 mouse myeloma and a culture medium therefore. 22. Preparation according to claim 21, characterized in that the continuous cell consists essentially of clone HK-PEG-1.