United States Patent 2 [19]
`Koprowski et al.
`
`[11]
`[45]
`
`4,196,265
`Apr. 1, 1980
`
`[54] METHOD OF PRODUCING ANTIBODIES
`[75] Inventors: Hilary Koprowski, Wynnewood;
`Walter U. Gerhard; Carlo M. Croce,
`both of Philadelphia, all of Pa.
`[73] Assignee: The Wistar Institute, Philadelphia,
`Pa.
`[21] Appl. No.: 933,092
`[22] Filed:
`Aug. 11, 1978
`
`Related US. Application Data
`Continuation-in-part of Ser. No. 806,857, Jun. 15, 1977.
`
`[63]
`
`[51] Int. 01.2 . . . .
`. . . .. A01N 1/02; A61K 39/42
`[52] US. Cl. ........................................ .. 435/2; 424/85;
`424/86
`[58] Field of Search .................... .. 424/85, 86; 195/ 1.8
`
`[56]
`
`References Cited
`'
`U.S. PATENT DOCUMENTS
`2,768,114 10/1956 Koprowski et al. ................. .. 424/89
`
`FOREIGN PATENT DOCUMENTS
`
`1300391 12/ 1971 United Kingdom .
`
`OTHER PUBLICATIONS
`Gerhard & Koprowski, Nature, vol. 266, Mar. 4, 1977,
`pp. 360-361.
`Milstein & Herzenberg, Immune System, “Genetics &
`
`Regulation,” edited by Sercarz et al., (Academic Press),
`Dec. 1977, pp. 273-275.
`Koprowski, Gerhard & Croce, Proc. Nat. Acad. of Sci.
`(USA), vol. 74, Jul. 1977, pp. 2985-2988.
`Milstein & Kohler, “Antibodies in Human Diagnosis &
`Therapy”, edited by Haber & Krause (Raven), Feb. 17,
`1977, pp- 271-284.
`The Lancet, editorial, Jun. 11, 1977, pp. 1242-1243.
`Kohler, Pearson & Milstein, Somatic Cell Genetics, vol.
`3, Feb. 7, 1977, pp- 303-312.
`’
`Kohler et al., Nature, vol. 256, (Aug. 7, 1975), pp. 495 to
`497.
`Kohler et al., Eur. J. Immunol., vol. 16, (1976), pp.
`511-519.
`Galfre et al., Nature, vol. 266, (Apr. 1977), pp. 550-552.
`Welsh, Nature, vol. 266, (Apr. 1977), p. 495.
`Primary Examiner-Sam Rosen
`[57]
`ABSTRACT
`Continuous cell lines of genetically stable fused cell
`hybrids capable of producing large [amounts of mono
`clonal antibodies against speci?c viruses and their anti
`genic determinants have been developed. The cell lines
`are fused cell hybrids between viral antibody producing
`cells and myeloma cells. Fused cell hybrids between
`in?uenza virus-primed mouse spleen cells and mouse
`myeloma cells can be maintained inde?nitely in culture
`and continue to produce large amounts of anti-in?uenza
`antibody.
`
`18 Claims, No Drawings
`
`Merck Ex. 1073, pg 1588
`
`

`
`1
`
`METHOD OF PRODUCING ANTIBODIES
`
`RELATED APPLICATIONS
`This is a continuation-in-part of application Ser. No.
`806,857, ?led June 15, 1977.
`BACKGROUND OF THE INVENTION
`This invention relates to antibody culture and in par
`ticular the production of antibodies by living cells in
`vitro.
`The production of antibodies speci?c for the anti
`genic determinants of viruses is of importance both for
`immunotherapy and for medical research.
`Monoclonal antibodies for antigenic determinants of
`viruses have been produced in vitro in cultures of spleen
`cells infected with a virus. Up to now, however, pro
`duction of antibody by spleen foci in culture declined
`after 30-40 days and ceased altogether after 90 days.
`We have developed new techniques for producing
`antibodies, more particularly, the propogation of new
`cell lines which are genetically stable, can be cultivated
`and subcultivated inde?nitely and produce large
`amounts of antibodies against viruses and their antigenic
`determinants. The new cell lines are fused cell hybrids
`of (a) myeloma cells, i.e., malignant cells from primary
`tumors of bone marrow, and (b) viral antibody produc
`ing cells preferably those of the spleen or lymph nodes
`of virus primed animals. A particularly preferred cell
`line is a fused cell hybrid between virus-primed mouse
`spleen cells and mouse myeloma cells. These cell lines
`can be maintained substantially inde?nitely in a culture
`medium such as hypoxanthine-aminopterin-thymidine
`selected medium and continue to produce antibodies
`speci?c for the antigenic determinants of the virus. The
`cells can also be grown in vivo in a histocompatible
`animal to accumulate large amounts of antibodies in the
`serum'and ascitic ?uid of the animal.
`Myeloma cells are themselves unique in that such
`cells are capable of producing antibodies albeit the spec
`i?city of these antibodies is as yet unknown. The partic
`ular species of animal from which the myeloma and
`spleen cells are derived is not critical insofar as it is
`possible to fuse the cells of one species with another,
`i.e., mouse to rat, rat to human. We prefer, however, to
`use the same species of animal as a source of both my
`eloma and viral antibody producing cells. Excellent
`results have been obtained with somatic cell hybrids
`. between the viral antibody producing spleen cells of a
`BALB/c mouse previously immunized with a virus and
`myeloma cells of a BALB/c mouse. Particularly pre
`ferred myeloma cells are those of the MOPC-2l line
`called clone (P3 X 63 Ag8) and disclosed by Kohler et a!
`in Nature, Vol. 256, 495-497 (1975).
`’
`Fused cell hybrids of BALB/c spleen cells and
`BALB/c myeloma cells have been previously described
`in the literature by Kohler et al in Nature, Vol. 256,
`495-497 (1975) and Eur. J. ImmunoL, Vol. 6, 511-519
`(1976). These hybrids, however, were not derived from
`mice immunized with viruses, but rather from mice
`immunized with sheep red blood cells. Hybrids between
`sheep red blood antibody producing cells and myeloma
`'_ cells are easily formed but the antibodies produced by
`65
`the hybrids of Kohler et al are speci?c to sheep red
`blood cells and have no effect upon viruses. Prior to this
`invention, it was not known whether hybrids could be
`
`4,196,265
`2
`formed between viral antibody producing cells and
`myeloma cells.
`Following is a procedure for preparing a cell line of
`hybrid cells by fusing the spleen cells of a BALB/c
`5 mouse previously immunized with in?uenza virus with
`myeloma cells of a BALB/c mouse. Although the pro
`cedure utilizes a particular strain of in?uenza virus,
`other viruses such as rabies, mumps, vaccinia, in?uenza
`strain 6-94, simian virus 40, polia, etc., can be used.
`(A) PREPAATION OF SPLEEN CELLS FOR
`FUSION
`In order to prepare the spleen cells for fusion,
`BALB/c mice, primed by an intraperitoneal injection of
`1250 hemagglutinating units of puri?ed in?uenza virus
`(A.PR/8/34 (HONI)) 2 to 3 months previously, re
`ceived intravenously a booster dose of 100 hemag
`glutinating units of the homologous virus. The mice
`were sacri?ced 7 days later and a spleen cell suspension
`was prepared in the manner taught by Gerhard et al,
`Eur. J. ImmunoL, 5, 720-725 (1975). Red blood cells
`were lysed by incubation of 15 minutes at 4° C. in
`NH4Cl (0.83%). The resulting cell suspension was
`washed by one centrifugation (800Xg) through heat
`inactivated calf ser and one centrifugation in protein
`free medium (RPMl 1640, buffered with 7.5 mM
`HEPES, pH 7.2).
`(B) PREPARATION OF MYELOMA CELLS FOR
`FUSION
`BALB/c (P3X63 Ag8) myeloma cells derived from
`the MOPC-2l line and de?cient in HPRT (E.C.2.4.2.8)
`as described by Dr. Cesar Milstein in Nature, Vol. 256,
`495-497 (1975), were maintained in Englc’s minimal
`essential medium (MEM) containing 10% fetal calf and
`10% horse serum. The growth of P3 X63 Ag8 myeloma
`cells is inhibited by selective hypoxanthine-aminopter
`in-thymidine medium.
`(0) PRODUCTION OF HYBRIDS
`Production of hybrids was accomplished by mixing
`ten million BALB/c (P3 X 63 Ag8) myeloma cells with
`108 spleen cells obtained from the virus infected
`BALB/c mice. The cell mixture was centrifuged at
`800Xg and the cells were resuspended for fusion in a
`50% solution (w/v) of polyethylene glycol-1000 (PEG)
`diluted in minimum essential medium (MEM) without
`serum. Following fusion procedures taught by David
`son et a1, Somat, Cell Genet. 2, 175-176, the polyethyl
`ene glycol was diluted ?rst with MEM without serum
`and then with serum before the cells were seeded in ?ve
`75-cm Falcon ?asks in hypoxanthine-aminopterin
`thymidine (HAT) selective medium. The cultures were
`incubated at 37° C. in an atmosphere of 95% air/5% .
`CO2, and every 7 to 10 days the culture medium was
`partially replaced by fresh HAT medium (5 to 5).
`Ten to ?fteen days after incubation of cultures pro
`duced by fusion of spleen cells of PR8 immunized mice
`with P3 X 63 Ag8 cells, cell growth was observed in one
`?ask. All growth in HAT medium is indicative of suc
`cessful hybridization between mouse spleen and mouse
`myeloma cells. These cells, referred to as HK-PEG-l,
`were propagated continuously in HAT medium and
`were cloned in microplates (Linbro) at limiting dilution.
`The other 4 ?asks, none of which showed cell growth,
`were discarded 3 weeks after fusion. A deposit of the
`cell line culture identified as HK-PEG-l is on deposit
`
`20
`
`25
`
`60
`
`Merck Ex. 1073, pg 1589
`
`

`
`3
`with the American Type Culture Collection and is as
`signed the accession number ATCC CL 189.
`(D) KARYOLOGICAL ANALYSIS
`As shown in Table 1, the P3X63 Ag8 parental cells
`contained an average of 63 chromosomes and BALB/c
`spleen cells of 40 chromosomes. Thus, the 92 chromo
`somes in the HK-PEG-l cells represented approxi
`mately the sum of chromosomes of 2 parental cells. The
`kuryology of the hybrid clone I-IK-PEG-l and of sub
`clones was monitored for 4 months with no signi?cant
`change in the karyotype.
`TABLE 1
`Number of Chromosomes in Parental and Hybrid Cells
`Average Number of Chromosomes per Cell
`
`Cells
`
`P3 X 63 Ag8
`BALB/c spleen
`cells
`
`63'
`40
`
`'
`
`‘Including 2 metacentric marker chromosomes.
`
`20
`
`4,196,265
`4
`tric focusing, anti-PR8 antibodies with IgGl heavy
`chain determinant(s) accumulated in a restricted pH
`range.
`(ii) The antibody was tested in the RIA for its cross
`reactivity against various viruses known to be antigeni
`cally related to PR8 and was found to be speci?c for a
`determinant of the hemagglutinin (HA) of PR8. How
`ever, 20% to 25% of splenci PR8-primed precursor B
`cells exhibit this strain-speci?c anti-HA (PR8) reactiv
`ity. Taken alone, the antibody reactivity is, therefore, a
`rather weak criterion for determining monoclonality of
`HK-PEG-l.
`(iii) In order to exclude the possibility that cells pro
`ductin antiviral antibody constituted only a small frac
`tion of the hybrid cell population, culture ?uids derived
`from 12 clones of I-IK-PEG-l hybrid line were assayed
`for the presence of anti-PR8 antibody and its speci?city.
`As shown in Table 4, ll of the 12 clones produced
`anti-PR8 antibody. Furthermore, these antibodies were
`speci?c for a determinant of the HA of the PR8 virus.
`TABLE 4
`Anti-PR8 Antibody Produced by Clones of HK-PEG-l Cultures
`Concentration of Anti-PR8
`HA Antibody‘
`(us/ml)
`z
`L2
`24
`6:9
`
`(E) ANALYSIS OF THE IMMUNOGLOBIN
`PRODUCED BY THE HYBRID CELLS
`Culture ?uids of HK-PEG-l and P3X63 Ag8 cells 25 Culture Fluid from HK-PEG-l
`were analyzed for the presence of immunoglobulins (ig)
`Clone #
`reacting in the radioimmune assay (RIA) with PR8.
`2
`P3X63 Ag8 is known to secrete IgGl, k. As shown in
`3
`Table 2, P3X63 Ag8 cuture ?uids did not contain lg
`5
`
`
`with anti-PR8 reactivity. In contrast, large quantities of 30 g IgG anti-PR8 antibodies were produced by HK-PEG-l. 9
`
`TABLE 2
`I
`Class of Anti-PR8 Antibody Produced by HK-PEG-l Cultures
`CPM Observed in RIA with Antisera
`Labeled with 1251
`Anti-IgM Anti-IgA Anti-IgG
`
`Culture Fluid
`Assayed iii RIA' Anti-?ab’);
`
`185
`ND" ND
`44 i 24"
`P3 x 63 Ag8
`8335
`4255 i 158" 204
`26
`HK-PEG-l
`‘Medium from cultures with similar cell concentration. Analysis performed on
`replicate samples of 15 ul (P3 X 63 Ag8) or 7.5 ul (I-IK~PECi-l) of culture ?uid.
`"Mean + SE of!) detenninationes. Other entries: mean of RIA done in duplicate.
`"'ND = Not Determined.
`
`The data of Table 3 indicate that the viral antibodies
`(anti-PR) produced by the hybrid cells are speci?c for
`the antigen (hemagglutinin) of the PR8. This is evident
`from the functional assays in which the antibodies
`(roughly 40 ug/ ml) exhibited a hemagglutinin inhibition
`(HI) but no detectable neuraminidase inhibition (NI).
`TABLE 3
`Activity of Antibody Produced by HK-PEG-l in
`Functional Assays Against PR, Virus
`NI-titer'
`Antibody secreted in vitro by
`HI-titer'
`(log ID)
`HK-PEG-l and concentrated
`(log 10)
`‘1.00
`by ammonium sulfate precipitation"
`2.98
`'Hemagglutination inhibition (HI) and neuraminidase inhibition (NI) against PR8
`virus.
`"Filly ml of HK-PEG-l_culture medium was precipitated with ammonium sulfate
`at 4' C. at 41% (v/v) saturation. Prccipitate was dialyzed against PBS and concen
`trated by ultrul'lltration to 0.6 ml. This ?nal sample contained, as determined by
`RlA, 47% ofthe antiviral antibody present in the starting volume oi'culture
`and thus represents roughly a 40~fold concentration of antiviral antibodies.
`
`4.7
`10
`5.8
`II
`.002
`12
`2.8
`13
`2'7
`14“
`2-6
`14b
`'Qumlimion based on RIA done with 'zsl-lnti-Ftlb'h
`
`40 (G) TUMORIGENICITY OF THE HYBRID CELLS
`In order to determine the tumorigenicity of the hy
`brid cells, adult BALB/c mice were injected subcutane
`ously in the abdominal wall with either HK-PEG-l
`(1x107) or P3X63 Ag8 (1x107) cells.
`As shown in Table 5, ten to twelve days after implan
`tation, tumors developed at the site of inoculation of 5/ 5
`mice injected with the HK-PEG-l cells and in 3/ 5 mice
`injected with the parental P3 X 63 Ag8 cells. In a second
`experiment, 4/5 mice developed tumors after inocula
`tion with hybrid cells and 3/5 after inoculation with
`P3X63 Ag8 cells. After i.p. injection of cells (experi
`ment 3, Table 5), tumors were found to grow as masses
`in the peritoneal cavity. In order to produce ascitic
`?uid, it was necessary to inject the mice with tumor
`cells resuspended in complete Freund adjuvant. In a
`fourth experiment, pristane-primed mice developed
`ascites after i.p. inoculation of cells of the HK-PEG-l
`clones.
`
`60
`
`TABLE 5
`
`(F) CLONAL ORIGIN OF THE HYBRID CELLS
`The clonal origin of HK-PEG-l was tested by 3 inde
`pendent criteria:
`(i) Concentraged Ig obtained from HK-PEG-l mass
`cultures (see legend, Table 3) was subjected to isoelec
`
`Exp. Route of
`65 No. Inoculation
`
`l
`
`subcutaneous
`
`2
`
`subcutaneous
`
`Inoculum
`
`HK-PEG-l
`P3 X 63 Ag8
`HK-PEG-I
`P3 X 63 Ag8
`
`Ratio of Mice
`Developing:
`Sub
`cutaneous
`Tumor
`
`Ascites
`
`5/5
`3/5
`4/5
`3/5
`
`—'
`—
`—
`—
`
`Merck Ex. 1073, pg 1590
`
`

`
`TABLE 5-continued
`
`Exp. Route of
`No. Inoculation
`
`3
`
`intraperitoneal
`
`4
`
`intraperitoneal’“
`
`Inoculum
`
`HK-PEG-l
`in CFA
`I-IK-PEG-l
`I-IK-PEG-l cl 6
`HK-PEG-l cl 7
`HK-PEG-l cl 12
`
`Ratio of Mice
`Developing:
`Sub-
`cutaneous
`Tumor
`
`Ascites
`
`—-
`
`—
`—-—
`—
`—
`
`3/3
`
`0/3
`4/4
`4/4
`4/4
`
`5
`
`10
`
`‘No tumor or ascites developed 30 days after inoculation.
`"Pristane-primed mice.
`
`The results of these studies indicate that hybrid cells
`between mouse myeloma and normal spleen cells be
`have as the malignant parent without suppression of
`malignancy.
`Sera and ascitic ?uid obtained from tumor-bearing 20
`mice at various intervals after injection were tested to
`determine the concentration of anti-PR8 antibody by
`means of the Rla. The results are shown in Table 6.
`TABLE 6
`Anti-PR8 Antibodies in Sera and Ascitic Fluids Obtained
`From Mice Injected with HK-PEG-l Cells
`Estimated
`Concentration of
`Antiviral Antibody‘ 30
`mg/ml
`
`15
`
`25
`
`Type
`Material
`Obtained Days of
`After Injection
`Material
`
`Exp. No.
`
`l
`
`2
`
`28
`
`19
`
`Serum
`Serum
`Serum
`Serum
`Serum
`Serum"
`Ascites"
`cl 6
`Ascites
`cl 7
`Ascites
`cl l2
`
`1.35
`1.1
`2.3
`0.9
`1.6
`1.2
`0.5
`
`0.45
`
`35
`
`0.002
`
`40
`
`Quantitation based on RIA done with "stand-Fowl.
`"From the same mouse.
`
`Sera of mice injected subcutaneously with HK-PEG- 45
`1 cells (experiments 1 and 2) contained anti-PR8 anti
`bodies at a concentration of l-to 3 mg/ml. Anti-PR8
`antibodies were also found in the ascitic ?uid of mice
`injected i.p. (experiment 4) with hybrid cells, although
`the concentration of antibodies was 3- to 4-fold lower
`than in the serum.
`Electrophoresis of serum obtained from mice bearing
`HK-PEG-l tumors revealed the presence of 3 main
`Ig-populations: one corresponding to the parental
`(P3X63 Ag8) IgGl; one characteristic of IgG3; and a
`third intermediate between the other two. In order to
`determine with which class of immunoglobulins the
`antiviral antibody was associated, ascitic ?uid was col
`lected from 20 pristane-primed mice was pooled and the
`immunoglobulins separated therefrom. Separation was
`accomplished by using a technique as follows:
`Ascitic ?uid collected from the mice was pooled and
`the pool neutralized with phosphate-buffered saline
`(PBS). An equal volume of saturated ammonium sulfate
`65
`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 over
`
`55
`
`4,196,265
`
`6
`night dialysis, was redissolved in PBS and re
`precipitated by dialysis against 0.01 M Tris buffer at pH
`8.0 The supernatant from the original dialysate was
`absorbed on 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 in 0.01 M
`Tris, pH 8.0.
`Following precipitation of the IgG3 by dialysis
`against 0.01 M Tris buffer, the remaining supernatant
`was found, by electrophoresis, to lack one of the com
`ponents seen i the ascites pool prior to fractionation.
`Chromatography of this material on DE-52 did not
`result in complete resolution of the remaining compo
`nents, but electrophoresis of the individual aliquots
`indicated the presence of two major components: one
`corresponding to the parental (P3X63 Ag8) IgGl, and
`the other indicative of a hybrid Ig, intermediate be
`tween IgG1 and IgG3, which was also observed i the
`serum of HK-PEG-l tumor-bearing mice (see above).
`The concentration of anti-PR8 antibody was deter
`mined in the RIA using 125I-anti-F(ab')2 and the speci?c
`anti-PR8 activity was computed as ratio of anti-PR8
`(mg/ml)2Ig (mg/ml), the latter estimate being based on
`the OD 280 measurement assuming absorptivity (1%, 1
`cm) for mouse Ig of 14.0. The speci?c anti-PR8 activity
`determined in the low salt precipitate, was roughly 2
`times higher than at the peak of the anti-PR8 activity of
`the DE-52 chromatography (0.06 in fraction 21). Analy
`sis of the samples in the RIA with 123I-anti-IgGl indi
`cated that approximately 15% of the anti-PR8 antibod
`ies in the low salt precipitate expressed G1 determi
`nants. In contrast, l25I-anti-F(ab')2 was as effective in
`quantitating the anti-PR8 antibodies in the various frac
`tions of the DE-52 chromatography.
`Another series of tests were performed with rabies
`virus.
`BALB/c mice were primed with vaccine containing
`inactivated rabies virus (ERA strain) and one month
`later received an intravenous booster inoculation of the
`same vaccine. The mice were sacri?ced three days after
`the booster and hybrid cells were formed by fusion of
`the spleen cells with P3><63 Ag8 myeloma cells as
`described earlier. A large number of the hybrid cells
`produced anti rabies virus antibodies. In contrast, when
`mice were sacri?ced ten days after the booster the num
`ber of hybrid cells producing anti rabies antibodies was
`quite low. This procedure was employed to obtain the
`hybridomas employed for the tests shown in Tables 7, 8
`and 9.
`The strains of rabies virus employed in the following
`tests are known strains in the art. For example, strain
`ERA is described, inter alia, in U.S. Pat. Nos. 3,423,505 ‘
`and 3,585,266. HEP-Flury is described, inter alia, in
`U.S. Pat. No. 2,768,114 and SAD is described in Can. J.
`of Microbiology, 6, 606 (1960). Similarly the analytic]
`tests employed are well known in the art.
`Fifty-two hybridomas that produced anti rabies virus
`antibodies as determined by radioimmune assay (RIA)
`were tested against three different stains of rabies virus
`(ERA, CVS and HEP - Flury strains). Moreover, four
`different assays were used, Virus Neutralization (VN),
`Cytotoxic Test (CT), Membrane Fluorescence (M), and
`Nucleocapsid Fluorescence (NC). The results of the
`tests are shown in Table 7.
`
`Merck Ex. 1073, pg 1591
`
`

`
`4,196,265
`
`TABLE 7
`Reactivity Against Virus Strains in Different Assays
`Number
`ERA
`CVS
`HEP
`of
`Hybridomas VN CT M NC VN CT M NC VN CT M NC
`
`7
`2
`2
`
`II
`
`2
`2
`
`1
`
`2
`
`1
`
`+
`+
`+
`
`+
`
`+
`+
`
`+ + —
`+ +
`+ + —
`
`+ + —
`
`+ + +
`+ + +
`
`+
`i
`+
`
`—
`
`—
`—
`
`+ + —
`i +
`+ + ~
`
`— — —
`
`— — +
`— — +
`
`+
`+
`—
`
`+
`
`+
`~
`
`+ + —
`+ + —
`~ # —
`
`+ + —
`
`+ + +
`— — +
`
`+
`
`+ + —
`
`—
`
`— — —
`
`—
`
`— — —
`
`-
`
`i + +
`
`-
`
`- — +
`
`+
`
`+ + +
`
`—
`
`- - -
`
`—
`
`— — —
`
`+
`
`+ + —
`
`22
`
`-
`
`- - +
`
`—
`
`— — +
`
`—
`
`~ — +
`
`As Table 7 demonstrates, antibodies that reacted with
`one strain may not interact with another strain of rabies
`
`ies. In addition, however, HEP was neutralized by anti
`bodies secreted by three other hybridomas.
`Table 8
`Cross-reactivity between strains of rabies virus of various origins determined
`in neutralization test with hybridoma antibody
`Origin of Strain
`Neutralimtion Index (by Logs)
`Host Country Prototype
`194
`120
`103
`193
`
`Derivative
`
`101
`
`I10
`
`I04
`
`Dog USA
`
`Fixed Cow France
`
`SAD
`
`Pasteur
`
`ERA
`
`PM
`CVS
`
`HEP Flury
`Man USA
`Kelev
`Dog Israel
`NYC
`Dog USA
`UD
`Bat USA
`DR
`Brazil
`Street Fox France AF
`Dog Rwanda RD
`Man South
`Duvenhage
`Africa
`
`> 3.0 > 3.0
`
`33 >
`3.0
`3.0 > 3.0
`> 3.0
`> 3.0 > 3.0 > 3.0
`0
`> 3.0 > 3.0
`> 3.0
`2.0 > 3.0
`> 3.0 > 3.0
`0
`0
`0
`0
`> 3.0
`3.0
`3.0
`l.5 > 3.0 > 3.0
`> 2.0
`1.5
`> 2.0
`2.0
`2.0 > 2.0
`> 3.0 > 3.0 > 3.0
`0
`0
`1.5
`
`2.5
`
`3.0
`
`3.0
`2.5
`3.0
`1.0
`3.0
`3.0
`0
`0
`3.0
`2.0
`0
`> 3.0
`3.0
`2.0
`2.0 > 3.0
`L5
`1.0
`2.0
`2.0
`2.5 > 3.0
`1.5
`0
`
`1.5
`
`1.0
`1.0
`O
`0
`0
`0
`1.0
`1.5
`1.0
`0
`2.5
`0
`
`0
`
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`Note: lunacy“ , '
`
`' ?uor
`
`following
`
`‘ a in the presence of medium from hybridoma 104
`
`virus. As shown in Table 7, of 52 hybrid cells secreting
`rabies antibodies, nine reacted in virus-neutralization,
`cytotoxicity and membrane immunofluorescence assays
`with all three strains of rabies, ERA, CVS and HEP
`Flury; ?fteen reacted with the ERA and HEP strains;
`two with ERA and'CVS; three with ERA only and one
`with HEP virus only. Twenty-eight hybrid cultures
`produced antibodies reacting with nucleocapsids of all 3
`virus strains; and of those 23 reacted only with nucleo
`capsids and with no other viral components.
`Analysis of antigenic relationship among strains of
`street and ?xed strains of rabies virus was studied using
`additional ten strains of different geographical origin.
`The hybridomas were produced as described above
`using ERA. Results presented in Table 8 indicate that
`?xed strain Kelev virus is neutralized only by antibodies
`secreted by one hybridoma (No. 120) and the recently
`discovered South African street virus (Duvenhage)
`only by antibodies secreted by two hybridomas. In
`contrast, all street virus strains seem to be cross-reactive
`in VN except for the AF strain which did not seem to
`react with the antibody of No. 193. Hybridoma antibod
`ies were found to be quite heterogenous in VN assays
`with ?xed strains of virus. For instance, the PM and
`CVS strains, which were both derived from the Pasteur
`strain, reacted differently from the Pasteur strain and
`from each other. Conversely, the SAD virus and its
`derivative ERA reacted in an identical fashion with
`antibodies secreted by the same hybridomas. Finally,
`the non-virulent HEP was found to be cross-reactive
`with the Kelev strain in the VN with No. 120 antibod
`
`45
`
`50
`
`55
`
`65
`
`Cells infected with all viruses regardless of origin
`showed intracytoplasmic ?uorescence after ?xation and
`exposure to antibodies produced by hybridoma 104 (NC
`assay), even though none of the viruses reacted with the
`same hybridoma in VN. This con?rms that although the
`hybridomas antibodies easily distinguish strains of ?xed
`rabies virus in VN, CT and MF tests, the antibodies
`react with all strains of rabies in the NC assay.
`The results shown in Tables 7 and 8 demonstrate that
`the 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 effect of rabies
`virus. Cells of hybridoma mass cultures C-1 and B-1 and
`of clone 110-5 (see Table 9) were emplanted subcutane
`ously into 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 con
`centration of rabies antibodies than medium from tissue
`cultures of the same hybridoma. Five days after implan
`tation of hybridomas, mice were challenged intracere
`brally with a lethal doze of PM rabies virus. As shown
`in Table 9, mice that were implanted with hybridoma
`cultures that secreted VN antibody survived viral chal
`lenge, whereas mice that carried hybridomas that did
`not secrete VN antibody were not protected.
`
`Merck Ex. 1073, pg 1592
`
`

`
`9
`Table 9
`Protection of mice against challenge by rabies virus
`by hybridoma antibodies
`Number of Cells
`Number of Mice Proteeted/
`Inoculated
`Number Challenged
`5 >< 10*5
`6/6
`5 >< 105
`9/10
`s >< 104
`6/6
`5 x 106
`0/6
`none
`0/6
`
`VN
`+
`+
`+
`~—
`—
`
`Hybri-
`-doma
`c1
`110-5
`
`B-1
`-
`
`The cell line of this invention represents a hybrid
`culture displaying characteristics of both the normal
`spleen and the myeloma parental cells and appears to be
`derived from a single fusion event. The cells are hybrid
`in nature because:
`(a) The cell line has been grown for several months in
`selective HAT medium which inhibits the growth of
`the parental P3><63 Ag8 myeloma cells but not of the
`normal spleen cells which, in turn, would probably not
`survive for 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 my
`eloma parent cells;
`'
`(c) The HK-PEG-l hybrid produces not only IgGl,
`which is also secreted by P3 X 63 Ag8, but IgG3 as well,
`and probably, hybrid molecules; and
`(d) The hybrid produces antibodies with antiviral
`activity, whereas P3><63 Ag8 does not.
`Although the antibodies are best produced by propo
`gating the hybrid cell lines in vitro, antibodies can also
`be produced by injecting a histocompatible animal with
`the hybrid cell line and producing the antibodies in
`vivo. For example, mice, relatively inexpensive animals,
`may be injected with the mouse derived antibody pro
`ducing cells of this invention to produce recoverable
`antibodies in the sera and ascitic ?uids. Table 6, dis
`cussed previously, shows the amount of antibody ob
`tained under such conditions.
`The antibodies produced by the hybrids can be har
`vested using standard techniques and may be used in
`analytical medical research for identi?cation of viral
`antigens in blood samples and culture mediums. Those
`antibodies produced in vitro are homogeneous and
`highly speci?c to the viral antigen. A supply of various
`homogeneous antibodies, each highly speci?c to a par
`ticular antigen permits researchers to rapidly determine
`the speci?city of an antigen and/or characterize viral
`antigens. In addition, the antibodies can be administered
`to diseased animals to assist in combating the disease.
`We claim:
`1. A process for producing viral antibodies compris~
`ing fusing a viral antibody producing cell and a my
`
`4,196,265
`10
`eloma cell to provide a fused cell hybrid, culturing said
`hybrid and collecting the viral antibodies.
`2. The process of claim 1 wherein said hybrid is cul
`tured in vitro.
`3. The process of claim 2 wherein said hybrid is
`cloned and cultured in a medium containing hypoxan
`thine-aminopterin-thymidine.
`4. The process-of claim 1 wherein said hybrid is in
`jected into a histocompatible animal and cultured in
`VlVO.
`5. The process of claim 1 wherein said viral antibody
`producing cell is a cell selected from the group consist
`ing of spleen cells and lymph node cells.
`6. The process of claim 5 wherein said spleen is mouse
`spleen and said myeloma is mouse myeloma.
`7. The process of claim 5 wherein the said mouse is a
`BALB/c mouse.
`8. The process of claim 6 wherein said myeloma is (P3
`63 Ag8) derived from the MOPC-Zl line.
`9. The process of claim 1 wherein said viral antibody
`producing cell is obtained from an animal immunized
`with a virus.
`10. The process of claim 1 wherein said viral antibody
`producing cell is obtained from a mouse immunized
`with a virus.
`11. The process of claim 1 wherein said viral antibody
`producing cell is obtained from an aminal immunized
`with an in?uneza virus.
`12. The process of claim 1 wherein said viral antibody
`producing cell is obtained from an animal immunized
`with a rabies virus.
`13. A process for producing antibodies comprising
`injecting a BALB/c mouse with a virus to induce anti
`body formation of spleen cells of said mouse, forming a
`fused cell hybrid of said viral antibody producing
`splenic cells with a BALB/c myeloma cell clone
`(P3X63 Ag8) derived from the MOPC-2l line, cultur
`ing said hybrid in vitro in selective HAT medium and
`harvesting the antibodies produced.
`14. The process of claim 13 wherein said virus is an
`in?uenza virus.
`15. The process of claim 13 wherin said virus is simian
`virus 40.
`16. The process of claim 13 wherein said virus is a
`rabies virus.
`17. A composition comprising a continuous cell line
`which produces in?uenza antibodies in vitro in hypox
`anthine-aminopterin-thymidine medium comprising a
`fused cell hybrid of in?uenza primed BALB/c mouse
`spleen and MOPC-2l mouse myeloma and a culture
`medium therefor.
`18. The composition of claim 17 wherein said contin
`uous cell consists essentially of clone HK-PEG-l.
`# i i Q i
`
`5
`
`5
`
`30
`
`45
`
`55
`
`65
`
`Merck Ex. 1073, pg 1593
`
`

`
`0
`
`_UNITED STATES‘ PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`4,196,265
`
`PATENT N0. :
`
`DATED
`
`:
`
`. April v1, 1980
`
`I
`
`' lNVENT0R($) 1
`
`Hilary Koprowski, Walter U. Gerhard 6t Carlo M. Croce
`
`It is certified that error appears in the above—identified patent and that said Letters Patent
`is hereby corrected as shown below:
`‘
`
`. Column 1, immediately below the Title, add the following.
`
`—— The invention described herein ,was made in the course of work under a grant
`or award from The Department of Health, Education and Welfare -
`Signed and Scaled this
`Twenty-?rst Day Of‘ October 1980
`
`'
`
`[SEAL]
`
`'
`Amsu'
`
`.
`
`Arresting Oj?cer
`
`Commissioner of Parents and Trademarks
`
`SIDNEY A. DIAMOND
`
`‘
`
`‘
`
`Q
`
`Q
`
`G
`
`O
`
`Merck Ex. 1073, pg 1594
`
`Merck Ex. 1073, pg 1594