FI64952B - FREQUENCY REQUIREMENT FOR THE GENETIC STABILIZATION OF CELLINERS - Google Patents

Description

1 --- »- -ί Μ NOTICE sac \ rr \ M c ') UTLAe (SNI ^ GSSKRIFT 64952' '—'—' (51) K * A.Wa.3 C 12 Μ 5/00, A 61 K 39/42 FINLAND —FINLAND (71) Patent application —Patantanaftkning 7Ö1Ö18 (22) HikemtapiW * —AroÄknlnjadaj 07.06.78 '(23) Alkuplly »—GUtlghetadag 07.06.78 (41) * «,

Patents and registrants '' Amman uttagd odi utUkdftwi publicerad. xu. oj (32) (33) (31) utuoikeui —Begird prtoritet 13 * 06.77 USA (US) 806857 (71) The Wistar Institute, Thirty-Sixth Street at Spruce, Philadelphia, Pennsylvania 19101+, USA (US) (72) Hilary Koprovski, Wynnewood, Pennsylvania, Walter U. Gerhard,

Philadelphia, Pennsylvania, Carlo M.Croce, Philadelphia,

The present invention relates to a method for the production of genetically stable cell lines which are capable of continuously producing "in vivo" - Pennsylvania, USA (US) (7 * 0 Leitzinger Oy (5 * 0 Method for the production of genetically stable cell lines) or in culture medium with large amounts of a viral monoclonal antibody, especially an influenza or rabies virus antibody.

The production of antibodies specific for antigenic determinants of viruses is important for both immunotherapy and medical research. Monoclonal antibodies to viral antigenic determinants have been produced in vitro in cultures of virus-infected spleen cells. However, the production of antibody to spleen foc in culture has so far decreased after 30 to 40 days and completely stopped after 90 days.

We have developed a new method of producing antibodies by growing a new cell line that is genetically stable and can be cultured and sub-cultured continuously and that produces large amounts of 2,6452 antibodies against viruses and their antigenic determinants.

The method according to the invention is characterized in that viral antibody-producing cells and myeloma cells compatible therewith, preferably mouse cells, are fused to hybrid cells as is known per se, the preferred influenza virus antibody-producing cell being stored under the symbol ATCC CL 189.

These new cell lines are (a) myeloma cells, i. fusion cell hybrids of malignant cells of primary tumors of the bone marrow, and (b) viral antibody-producing cells, preferably spleen or lymph node cells of virus-treated animals. A particularly preferred cell line is a fusion cell hybrid between virus-treated mouse spleen cells and mouse myeloma cells. These cell lines can be maintained substantially infinitely in a culture medium such as selective hypoxanthine-amino-pterin-thymidine medium, and the cell lines consistently produce antibodies specific for the antigenic determinants of the virus.

Cells can also be grown in vivo in a histologically acceptable animal, accumulating large amounts of antibodies in the animal's serum and ascites fluid.

Myeloma cells themselves are unique in that such cells are capable of producing antibodies, although the specificity of these antibodies is not yet known. The animal species from which the myeloma and spleen cells are derived are not critical as long as it is possible to fuse cells of another species with cells of another species, i. mouse and rat, rat and human. However, it is recommended to use the same animal species as a source of cells producing both myeloma and viral antibodies. Excellent results have been obtained with somatic cell hybrids between BALB / c mouse previously immunized with virus, virus antibody-producing spleen cells and BALB / c mouse myeloma cells. Particularly preferred myeloma cells are the myeloma cells of clone P3 x 63 Aq8, called the MOPC-21 generation, presented by Köhler et al., Nature, Vo. 256, 395-397 (1975).

3 64952 Köhler et al. Have previously described in the literature, Nature, Vol. 256, 495-497 (1975) and Eur. J. Immunol., Vol. 6, 511-519 (1976), fusion cell hybrids of BALB / c spleen cells and BALB / c myeloma cells.

However, these hybrids were not derived from mice immunized with viruses but from mice immunized with sheep red blood cells. Hybrids readily form between sheep antibody-producing red blood cells and myeloma cells, but the antibodies produced by the hybrids of Köhler et al. Are specific for sheep red blood cells and have no effect on viruses. Prior to this invention, it was not known whether hybrids could be formed between viral antibody-producing cells and myeloma cells.

The following is a procedure for preparing a cell generation of hybrid cells by fusing spleen cells from a BALB / c mouse previously immunized with influenza virus with BALB / c mouse myeloma cells. Although a particular influenza virus strain is used in the method, other viruses such as rabies, mumps, vaccinia virus, influenza strain 6-94, simian virus 40, poliovirus, etc. may also be used.

(a) Preparation of spleen cells for fusion

To prepare spleen cells for fusion, BALB / c mice injected intraperitoneally 2-3 months with 1250 hemagglutination units of purified influenza virus (A.PR/8/34 (HONI)) were given an intravenous homologous virus of 100 haemagglutination units. Mice were sacrificed after 7 days and Gerhard et al., Eur. J. Immunol., Vol. 5, 720-725 (1975), a spleen cell suspension was prepared. Red blood cells were dissolved by incubation for 15 minutes at 4 ° C in ammonium chloride (0.83%). The resulting cell suspension was treated by centrifugation (800 x g) once with heat-inactivated calf serum and once in protein-free buffered (pH 7.2) medium, RPMI 1640.

64952 (b) Preparation of myeloor cells for fusion

Dr. Cesar Milstein, Nature, Vol. 256, 495-497 (1975), BALB / c (P3 x 63 Ag8) myeloma cells derived from the MOPC-21 generation and deficient in HPRT (EC2.4.2.8) were maintained in Eagle's medium (minimal essential medium (MEM)) containing 10% fetal calf and 10 * horse serum. Growth of P3 x 63 Ag8 myeloma cells was selectively inhibited by hypoxanthine-aminopterin-thymidine medium.

(c) Preparation of hybrids

Hybrids were prepared by mixing ten million BALB / c p (P3 x 63 Ag8) myeloma cells and 10 spleen cells obtained from virus-infected BALB / c mice. The cell mixture was centrifuged at 800 x g and the cells were resuspended in a 50% solution (w / v) of polyethylene glycol-1000 (PEG) diluted in serum-free MEM medium for fusion. Davidson et al., Somat. Cell Genet.

2, 1977, 175-176, the polyethylene glycol was first diluted with serum-free MEM medlum and then with serum, after which the cells were placed in five 75 cm Falcon flasks in hypoxanthine-aminopterin-thymidine (HAT) selective medium. Cultures were incubated at 37 ° C in a 95% air / 5 * CO 2 atmosphere and every 7-10 days the culture medium was partially replaced with fresh HAT medlum (1/2 to 1/3).

After incubating cultures prepared by fusing spleen cells from PR8-immunized mice with P3 x 63 Ag8 cells for 10 to 15 days, cell growth was observed in one flask. All growth in HAT medium is indicative of successful hvbri dissociation of mouse spleen cells and mouse myeloma cells. These cells, called HK-PEG-1 cells, were grown continuously in HAT medium and cloned into microplates (Linbro) as a limiting dilution. The other four flasks, which showed no cell growth, were discarded three weeks after fusion of li 5 64952. A cell generation culture, designated HK-PEG-1, has been deposited with the American Type Culture Collection under accession number ATCC CL 189.

(d) Caryological analysis

As shown in Table 1, P3 x 63 Ag8 base cells contained an average of 63 chromosomes and BALB / c spleen cells 40 chromosomes. Thus, the 92 chromosomes of HK-PEG-1 cells are approximately the sum of the chromosomes of the two base cells. The karyology of the hybrid clone HK-PEG-1 and subclones was monitored for 2 months without significant karyotope change.

table 1

Number of chromosomes in stem and hybrid cells

Cells Average number of chromosomes per cell P3 x 63 Ag8 63 * BALB / c spleen cells 40 HK-PEG-1 92 * ♦ contains two metacentric labeling chromosomes.

(e) Analysis of immunoglobulin prepared with hybrid cells The culture medium of HK-PEG-1 and P3 x 63 Ag8 cells was analyzed in a radioimmunoassay (RIA) for the presence of immunoglobulins (Ig) reactive with PR8. P3 x 63 Ag8 is known to secrete IgG1, k. As shown in Table 2, p3 x 63 Ag8 culture solutions did not contain immunoglobulin with anti-PR8 reactivity. In contrast, HK-PEG-1 produced large amounts of IgG anti-PR8 antibodies.

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Table 2 Class of anti-PR8 antibody produced by HK-PEG-1 cultures in the RIA method CMP detected in the RIA method, antiserum examined culture-labeled with 125j

solution * Anti-F (ab ') 2 Anti-IgM Anti-IgA Anti-IgG

P3 x 53 Ag8 44 + 24 ** ND *** ND 185 HK-PEG-1 4255 ± 158 *** 204 26 8335 * Mean of cultures with the same cell concentration.

Analysis was performed on 15 μl (P3 x 63 Aq8) or 7.5 μl (HK-PEG-1) replicates of culture medium.

** Mean of 6 determinations + SE. Other figures: Average of two RIA determinations performed in parallel.

*** ND = Not determined.

The values in Table 3 show that the viral antibodies (anti-PR) produced by the hybrid cells are specific for the PR8 antigen (hemagglutinin). This is evident from functional assays in which antibodies (roughly about 40 μm / ml) inhibit hemagglutinin (HI) but not detectably neuraminidase (NI).

Table 3 Antibody activity of HK-PEG-1 cultures in functional assays against PR8 virus In vitro HI titer of HK-PEG-1 cultures * NI titer * secreted antibody, concentrated (log 10) (log 10) by doping with ammonium sulfate - 2.98 <1.00 accounts ** n 64952 ♦ Hemagglutination inhibition (HI) and neuraminidase inhibition (NI) against PR8 virus.

** 50 ml of HK-PEG-1 culture medium was precipitated with ammonium sulfate at 4 ° C at 42% (v / v) saturation. The precipitate was dialyzed against PBS and concentrated by ultrafiltration to 0.6 ml. Based on the RIA assay, this final sample contained 47% of the antiviral antibody in the initial volume of culture medium, so that the antiviral antibodies were roughly 40-fold concentrated.

(f) Clone Origin of Hybrid Cells The clone origin of HK-PEG-1 culture was tested using three independent criteria: (i) Concentrated Ig from mass cultures of HK-PEG-1 (see explanation in Table 3) was focused isoelectrically as shown in Figure 1, wherein anti-PR8 antibodies with IgG1 heavy chain determinant (s) accumulated in a limited pH range.

(ii) In a radioimmunoassay, the antibody was tested for cross-reactivity against various viruses known to antigenically modify PR8 and was found to be specific for the PR8 hemagglutinin (HA) determinant. However, 20-25% splenic PR8 treatments on precursor B cells have this strain-specific anti-HA (PR8) reactivity. Thus, antibody reactivity alone is a somewhat weak criterion for determining the monoclonal nature of HK-PEG-1 cultures.

(iii) To rule out the possibility that antiviral antibody-producing cells were only a small fraction of the hybrid disin cell population, the anti-PR8 antibody and its specificity were determined from culture solutions derived from twelve generations of the HK-PEG-1 hybrid generation. clone. As shown in Table 4, eleven of the twelve clones produced anti-PR8 antibody. In addition, these antibodies were specific for the HA determinant of PR8 virus.

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Table 4 Anti-PR8 antibody produced by clones of HK-PEG-1 cultures Culture of HK-PEG-1 culture - Concentration of anti-PR8 HA antibody clone No. traathion * (pg / ml) 2 2 3 1.2 5 2.4 6 3.4 7 4.7 9 6.9 10 4.7 11 5.8 12 <0.02 12 2.8 14a 2.7 14b 2.6 * Quantitative RIA determination was performed with 125 I-anti-P (ab ') 2 (g) Tumorigenicity of hybrid cells

To determine the tumorigenicity of the hybrid cells, either HK-PEG-1 (1 x 107) or P3 x 63 Ag8 (1 x 107) cells were injected subcutaneously into the abdominal wall of adult BALB / c mice.

As shown in Table 5, 5/5 mice injected with HK-PEG-1 cells and 3/5 mice injected with P3 x 63 Ag8 base cells developed tumors at the inoculation site 10 to 12 days after implantation. In the second experiment, 4/5 mice developed tumors after inoculation with hybrid cells and 3/5 mice after inoculation with P3 x 63 Ag8 cells. The i.p. after injection (Experiment 3, Table 5), tumors were found to grow in mass in the peritoneal cavity. To form ascites fluid, mice had to be injected with tumor cells resuspended in Freund's adjuvant. In the fourth experiment, 9 64952 mice, pristane-treated mice developed an aqueous humor after cells of HK-PEG-1 clones were inoculated i.p.

Table 5

Experiment No. Inoculation Inoculation The ratio of mice in which the habit developed

Subcutaneous tumor ascites 1 subcutaneous HK-PEG-1 5/5 --- * P3 x 63 Ag8 3/5 --- 2 subcutaneous HK-PEG-1 4/5 --- P3 x 63 Ag8 3/5 --- 3 intraperiton- HK-PEG-1 --- 3/3 neural in CFAr HK-PEG-1 --- 0/3 4 intreperito- HK-PEG-1 cl 6 --- 4/4 neal ** HK-PEG- 1 cl 7 --- 4/4 HK-PEG-1 cl 12 --- 4/4 * No tumors or abdominal tumors had developed within 30 days after inoculation.

** Mice treated with pristane isooctadecane.

The results of these studies show that hybrid cells between mouse myeloma and normal spleen cells behave like malignant stem cells without a reduction in malignancy.

The concentration of anti-PR8 antibody was determined by serum and ascites fluid obtained from tumor-bearing mice at different time points after injection.

The results are shown in Table 6.

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Table 6

Anti-PR8 antibodies in sera and ascites fluids obtained from mice injected with HK-PEG-1 cells

Experiment Material Yield Material Antiviral Response Time in Days Injection Quality Estimated Concentration After Concentration mg / ml 1 28 Serum 1.35

Serum 1.1

Serum 2.3 2 19 Serum 0.9

Serum 1.6

Serum ** 1.2 ascites ** 0.5 3 15 ascites 0.5 cl 6 ascites 0.45 cl 7 ascites <0.002 cl 12 * Quantitative RIA was performed with ^ 5l-anti-F (ab *) 2.

** From the same mouse.

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

Serum electrophoresis from mice suffering from HK-PEG-1 tumors showed the presence of three predominantly Iq-64952 populations: One corresponded to parenteral (P3 x 63 Aq8) IgG1, one was characteristic of IgG, and the third was between the two. To determine which class of immunoglobulins the anti-viral antibody was associated with, ascites fluid collected from twenty pristane-treated mice was pooled and immunoglobulins were separated. The separation was performed using the following technique.

Ascites fluid collected from mice was pooled and the pool was neutralized with phosphate-buffered saline (PBS). At 4 ° C, an equal volume of saturated ammonium sulfate was added and the precipitated protein was dissolved in PBS and dialyzed against 0.01 M Tris buffer at pH 8.0. The precipitate formed after overnight dialysis was redissolved in PBS and reprecipitated by dialysis against 0.01 M Tris buffer at pH 8.0. The original dialysate supernatant was absorbed onto a DE-52 (Whatman) column equilibrated with 0.01 M Tris buffer, pH 8.0, and eluted using a linear NaCl gradient (0.05 to 0.15 M NaCl). .01 M in Tris buffer, pH 8.0).

After IgG3 was doped by dialysis against 0.01 M Tris buffer, it was found by electrophoresis that the remaining supernatant did not contain one component observed in the ascites pool before fractionation. Chromatography of this material on a DE-52 (Figure 2) column did not completely separate the remaining components, but electrophoresis of the individual batches showed the presence of two main components: One corresponded to parenteral (P3 x 63 Ag8) IgG1 and the other indicated hybrid Ig: en, which is between IgG1 and IgG3 and was also found in the serum of mice suffering from HK-PEG-1 tumors (see above).

The anti-PR8 concentration was determined by R1A using? 5'-anti-F (ab ') 2. The specific anti-PR8 activity was calculated as the ratio of anti-PR8 (mg / ml) to 2lg (mg / ml). wherein the latter estimate was based on an OD 280 measurement assuming a mouse Ig absorption capacity (1%, 1 cm) of 14.0. The specific anti-PR8 activity determined in the low salt precipitate was roughly two 12 64952 times higher than the peak of anti-PR8 activity in DE-52 chromatography (0.06 in fraction 21). Analysis of the samples in the RTA with? 5I anti-IgG1 showed that about 15% of the anti-PR8 antibodies in the low salt precipitate expressed G1 determinants. In contrast, 1 I-anti-P (ab ') 2 quantified anti-PR8 antibodies equally efficiently, in different fractions of DE-52 chromatography.

Additional sets of experiments were performed with the rabies virus.

BALB / c mice were treated with a vaccine containing inactivated rabies virus (ERA strain) and a month later were given the same vaccine as an intravenous booster. Mice were sacrificed 3 days after booster administration, and hybrid cells were formed by fusing spleen cells and P3 x 63 Ag8 myeloma cells as previously described. A large number of hybrid cells produced anti-rabies virus antibodies. In contrast, when mice were sacrificed 10 days after booster inoculation, the number of hybrid cells producing anti-rabies virus antibodies was quite low. The hybridomas used in the tests shown in Tables 7, 8 and 9 were prepared by this method.

The rabies virus strains used in the following tests are known in the art. For example, the ERA strain is described, inter alia, in DSA patents 3,423,505 and 3,585,266. HEP-Flury is described, inter alia, in U.S. Patent 2,768,114 and the SAD strain is described in Can. J, of Microbiology, (5, 606 (1960)) The analytical tests used are also well known in the art.

52 hybridomas producing anti-rabies virus antibodies by radioimmunoassay (RIA) were tested against three different rabies virus strains (BATCH, CVS and HEP-Flury strains). In addition, four different assays were used, i. viral neutralization (VN), cytotoxicity test (CT), membrane fluorescence (M), and nucleocapsid fluorescence (NC). The test results are shown in Table 7.

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Table 7

Antiviral activity in different assays

Of hybrid Domien

amount of ERA CVS HEP

_VN CT M NC VN CT M NC VN CT M NC

7_ + + + - + + + - + + + - 2_ + + + - + + + - + + + - 2_ + + + - + + + - - - - - 11_ + + + - - -4- 4 - + _ 2_ + + + + - - + + + + + 2_ + + + + - - + - - + 1 + + + - 2_-ί + + - - + + + + + 1 - ------- + + + - 22 _--- + - - + - - +

As Table 7 shows, antibodies that react with one strain may not react with another strain of rabies virus. As shown in Table 7, 9 of 52 hybridoma cells secreting rabies antibodies reacted with all three rabies virus strains, BATCH, CVS, and HEP, in virus neutralization, cytotoxicity, and membrane immunofluorescence assays; fifteen react with ERA and HEP strains; two with ERA and CVS strains; three with ERA virus only and one with HEP virus only. Twenty-eight hybrid cultures produced antibodies that reacted with the nucleocapsids of all three virus strains; and 23 of these react only with nucleocapsids and not with any other viral component.

The antigenic ratio between street and fixed strains of rabies virus was analyzed using ten other strains of different geographical origins. Hybridomas were prepared as described above using the ERA strain. The results in Table 8 show that the antibodies secreted by only one hybridoma (No. 120) neutralize the fixed strain Kelev virus and the antibodies secreted by only two hybridomas neutralize the recently discovered South African street virus (Duvenhage). In contrast, all street virus strains appear to be cross-reactive in the VN assay except for the AP strain, which does not appear to react with antibody # 193. Hybridoma antibodies were found to be quite heterogeneous in VN assays using fixed strains of the virus. For example, PM and CVS strains, both derived from the Pasteur strain, reacted differently from the Pasteur strain and reacted differently from each other. Instead, SAD virus and its ERA derivative react in an identical manner with antibodies secreted by the same hybridomas. Finally, the non-virulent HEP strain was found to be cross-reactive with the Kelev strain in the VN assay using No. 120 antibodies. In addition, however, antibodies from the other three hybridomas neutralize the HEP strain.

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16 64952

Cells infected with all viruses, regardless of origin, showed intracytoplasmic fluorescence after being fixed and exposed to antibodies produced by hybridoma 104 (NC assay), although none of the viruses reacted with the same hybridoma in the VN assay. This confirms that although hybridoma antibodies readily distinguish fixed rabies virus strains in the VN, CT, and MF assays, the antibodies reacted with all rabies virus strains in the NC assay.

The results shown in Tables 7 and 9 show that hybrid cells producing anti-water scavenger virus antibodies are very useful for research and analytical purposes.

Hybrid cultures that secrete antibodies that neutralize rabies viruses protect mice from the lethal effects of rabies virus. BALB / c mice were subcutaneously inoculated with C-1 and B-1 cells from hybridoma cultures and clone 110-5 (see Table 9). The serum obtained from these mice two weeks after inoculation with C-1 and 110-5 hybridomas had a 100-fold higher concentration of rabies antibodies than the medium obtained from tissue culture of the same hybridomas. Five days after hybridoma inoculation, mice were challenged intracerebrally with a lethal dose of PM rabies virus. As shown in Table 9, mice inoculated with hybridoma cultures secreting VN antibody survived viral exposure, whereas mice receiving hybridomas that did not secrete VN antibody did not show a protective effect.

I! 17 64952

Table 9

Protective effect of hybridoma antibodies on mice against rabies virus exposure

Hybridoma Number of cells inoculated VN Number of protected mice / number of exposed Cl 5 x 106 + 6/6 110-5 5 x 105 + 9/10 5 x 104 + 6/6 Bl 5 x 106 - 0/6 - none - 0 / 6 The cell generation of the present invention refers to a hybrid culture that has the properties of both normal spleen cells and myeloma stem cells and appears to be derived from a single fusion event. The cells are hybrid in nature because: (a) The cell generation has been grown for several months in a selective HAT medlum that inhibits the growth of parenteral P3 x 63 Ag8 myeloma cells but not normal spleen cells, which in turn are unlikely to survive more than 4-5 weeks in vitro; (b) The number of chromosomes in the hybrid cells is close to the sum of the chromosomes in 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 Ag8, but also IgG3 and probably hybrid molecules; and (d) the hybrid produces antivirally active antibodies, which P3 x 63 Ag8 does not.

Although antibodies are best produced by growing a hybrid cell generation in vitro, antibodies can also be generated by injecting a histologically compatible animal with a hybrid cell generation and producing antibodies in vivo. For example, mice, which are 18 64952 relatively inexpensive animals, can be injected with mouse-producing antibody-producing cells to generate recoverable antibodies. Table 6 previously discussed shows the amount of antibody obtained under such conditions.

Antibodies produced by hybrids can be recovered by standard techniques and used in analytical medical research to identify viral antigens in blood samples and culture media. Antibodies produced in vitro are homogeneous and highly specific for the viral antigen. When a variety of homogeneous antibodies are available, each of which is highly specific for a particular antigen, researchers can quickly determine the specificity of the antigen and / or characterize the viral antigens. In addition, antibodies can be administered to diseased animals and help control the disease.

Claims (6)

19 64952 1. A method for producing genetically stable cell lines capable of continuously producing large amounts of a monoclonal viral antibody, in particular an influenza or rabies virus antibody, in vivo or in a medium, characterized in that the virus antibody-producing cells and myeloma cells compatible therewith , preferably mouse cells, are fused to hybrid cells as is known per se, the preferred influenza virus antibody-producing cell line being deposited under the symbol ATCC CL 189. The method of claim 1, characterized in that the cell producing the viral antibody is a spleen cell. A method according to claim 2, characterized in that the spleen is a mouse spleen and the myeloma is mouse myeloma. Method according to claim 3, characterized in that the mouse is a BALB / c mouse. Method according to claim 3, characterized in that the myeloma is P3 x 63 Ag8 derived from the MOPC-21 line. The method of claim 1, characterized in that the BALB / c mouse is injected with a virus to induce mouse spleen cells to generate an antibody, a virus-producing spleen cells and a BALB / c myeloma cell clone from the MPO-21 line are generated. (P3 x 63 Ag8) fusion cell hybrid.