The Journal of Immunology
`
`2453
`
`Reprinted with permission from Nature 256 (5517): 495– 497 (1975). Copyright © 1975 Macmillan Publishers Ltd.
`
`Nature Vol. 256 August 7 1975
`
`495
`
`Continuous cultures of fused cells
`secreting antibody of predefined specificity
`THE manufacture of predefined spcciftc antibodies by means of
`permanent tissue culture cell lines is of general interest. T here
`are at present a considerable number of permanenl cu ltures of
`myeloma cells'·' and screening procedures have been used to
`reveal antibody activity in some of them. This, hov.ever, is
`not a satisfactory source of monoclonal antibodies of predefined
`specificity. We describe here the derivation of a number of
`tissue culture cell lines which secrete anti-sheep red blood
`cell (SRBC) ant ibodies. T he cell lines are made by fusion of a
`mouse myeloma and mouse spleen cells from an immunised
`donor. To understand the expression and interactions of the
`lg chains from the parental lines. fusion experiments between
`two known mouse myeloma lines were carried out.
`Each immunoglobulin chain result~ from the integrated
`expression of one of several V and C geues cod ing respectively
`for its variable and constant sections. Each cell expresses only
`one of the two possible alleles (allelic exclusion; revie"ed in
`ref. 3). When two antibody-producing cells are fused, the
`products of both parental lines are expressed'·', and although
`the light and heavy chains of both parental lines are randomly
`joined, no evidence of scrambling of V and C sec tions is
`observed'. T hese results, obtained in an heterologous system
`involvin,g cells of rat and mouse origin, have now been con(cid:173)
`firmed by fusing two myeloma cells of the same mouse strain,
`
`The protein sccreled (MOPC 21) is an JgG J (K) which has been
`fully sequenced""· Equal numbers of cell s from each parental
`line were fused using inactivated Sendai virus• and samples
`contining 2 10' cells were grown in selective medium in
`separate dishes. Four OUI or ICn dishes shO\\Cd growth in
`selective medium and these "ere taken as independent hybrid
`lines, probably derived from single fusion events. The karyotype
`of the hybrid cells after 5 months in culture was just under the
`sum of the 1wo parental lines (Table 1). Figure I shows the
`isoelectric focusing'° (IEF) pattern of the secreted products of
`different lines. The hybrid cells (samples c-li in Fig. I) give a
`much more complex pattern than either parent (a and b) or a
`mix ture of the parental lines (m). The important feature of the
`new pa11ern is the presence of exlra bands (Fig. I, arrows).
`These new bands, however, do not seem to be the result of
`differences in primary structure; this is indicated by the lEF
`pattern of the products after reduction to separate the heavy
`and light chains (Fig. IB). The IEF pattern of chains of the
`hybrid clones (Fig. IB, g) is equ ivalent to the sum of t he IEF
`pattern (a and b) of chains of the parental clones with no
`evidence of extra products. We conclude 1hat, as previously
`shown with interspecies hybrids ... , new lg molecules arc
`produced as a result of mixed association betv.ecn heavy and
`light chains from the two parems. This process is intracellular
`as a mixed cel l popu lation does not give rise to such hybrid
`molecules (compare 111 and g, Fig. I A). T he ind ividual cells must
`therefore be able to express both isotypes. Th is resu lt shows
`that in hybrid cells the expression of one isotypc and idiotype
`does no1 exclude the expression of another: both heavy chain
`
`H(Pl)f I
`
`Chains B
`
`H(P3)
`
`L(P3)
`
`L(Pl )
`
`Fig. l Autoradiograph of labelled compo(cid:173)
`nents secreted by the parental and hybrid cell
`lines analysed by IEF before (A) and after
`reduction {8). Cells were incubated in the
`presence of "C-lysine" and the supernatant
`applied on polyacrylamide slabs. A, pH range
`6.0 (bottom) to 8.0 (top) in 4 M urea. 8, pH
`range S.0 (bonom) to 9.0 (top) in 6 M urea;
`the supernatant was incubated for 20 min nt
`37 •c in the presence of 8 M urea, 1.5 M
`mercap1oe1nanol and 0.1 M potassium phos(cid:173)
`phate pH 8.0 before being applied to the right
`slab. Supcrna1an1s from parental cell li nes
`in: a, Pl Bui; b, P3-X67Ag8; and m, mixture
`of equal number of Pl Bui and P3-X67Ag8
`cells. Supernatants from two independently
`derhed hybrid lines are shown: c-f, four
`sub<:lones from Hy-3; g and h, 1wo subclones
`from Hy-B. Fusion was carried out"' using
`10' cells of each parental line and 4,000
`haemagglu1ina1ion units inactivated Sendai
`virus (Searle). Cells were divided into ten
`in
`equal samples and gro\\ n separa1ely
`s<:lttthe medium (HAT medium, ref. 6).
`Medium was changed every 3 d. Successful
`hybrid lines were obtained in four of the cul-
`1ures, and all gave similar IEF pallerns. Hy-B
`and Hy-3 were funher cloned in soft agar".
`L, Light; H, heavy.
`
`. ' ' .
`--
`
`a
`
`b cd em f mg b
`
`a / i b g t 1
`
`and provide the background for the derivat ion and under(cid:173)
`standing of antibody-secret ing hybrid lines in which one of
`the parental cells is an antibody-producing spleen cell.
`Two myeloma cell lines of BALB/c origin "ere used. Pl Bui
`is resistant to 5-bromo-2'-deoxyuridine', does not gro" in
`selective med ium (HAT, ref. 6) and secretes a myeloma protein,
`Adj PCS, which is an lgG2A (K), (ref. I). Syn ihcsis is not
`balanced and free ligh t chains arc also secreted. The second
`cell line, P3-X63Ag8, prepared from P3 cells•, is resistam to
`20 µg ml-1 8-azaguanine and <lOl.'S not grow in HAT medium.
`
`·sotypes (yl and y2a) and both V " and bo th VL regions
`(idiotypes) are expressed. There are no a llotypic markers for
`the C" region 10 provide direct proof fortheexpression of both
`parental C,. regions. But this is indjca1ed by the phenotypic
`link between the V and C regions.
`Figure l A shows that clones derived from different hybridi(cid:173)
`su1ion experiments and from subclones of one line are indist in(cid:173)
`guishable. This has also been observed in other experiments
`(data 001 sho"n ). Variants were, however, found in a survey of
`100 subclones. The difference is often associated with changes
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`2454
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`496
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`PILLARS OF IMMUNOLOGY
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`Nature Vol. 256 August 7 1975
`
`Fig. 2 Isolation of an anti-SR BC antibody(cid:173)
`secreting cell clone. Activity was revealed by
`a halo of haemolysed SRBC. Direct plaques
`given by: a, 6,000 hybrid cells Sp-1; b, clones
`grown in soft agar from an inoculum of 2,000
`Sp-I cells; c, recloning of one of the positive
`clones Sp-1/7; d, higher magnification of a
`positive clone. Myeloma cells (107 P3-X67A
`gS) were fused to 108 spleen cells from an
`immunised BALB/c mouse. Mice were im(cid:173)
`munised by intraperitoneal injection of 0.2 ml
`packed SRBC diluted I :JO, boosted after l
`month and the spleens collected 4 d later.
`After fusion, cells (Sp-I) were grown for 8 d
`in HAT medium, changed at 1- 3 d intervals.
`Cells were then grown in Dulbecco modified
`Eagle's medium, supplemented for 2 weeks
`with hypoxanthine and thymidine. Forty days
`after fusion the presence of anti-SRBC act(cid:173)
`ivity was revealed as shown in a. The ratio of
`plaque forming cells/total number of hybrid
`cells was 1/30. This hybrid cell population
`was cloned in soft agar (50% cloning ef(cid:173)
`ficiency). A modified plaque assay was used to
`reveal positive clones shown in b-d as follows.
`When cell clones had reached a suitable size,
`they were overlaid in sterile conditions with
`2 ml 0.6 % agarose in phosphate-buffered
`saline containing 25 µI packed SRBC and
`0.2 ml fresh guinea pig serum (absorbed with
`SRBC) as source of complement. b, Taken
`after overnight incubation at 37 °C. The ratio
`of positive/total nwnber of clones was 1/33. A
`suitable positive clone was picked out and
`grown in suspension. This clone was called
`Sp-1/7, and was recloned as shown inc; over
`90% of the clones gave positive Jysis. A second
`experiment in which 10• P3-X67Ag8cells were
`fused with 108 spleen cells was the source of
`a clone giving rise to indirect plaques (clone
`Sp-2/3-3). Indirect plaques were produced by
`the addition of I :20 sheep anti-MOPC 21
`antibody to the agarose overlay.
`
`in the ratios of the different chains and occasionally with the
`total' disappearance of one or other of the chains. Such events
`are best visualised on IEF analysis of the separated chains
`(for example, Fig. !h, in which the heavy chain of P3 is no
`longer observed). The important point that no new chains are
`detected by £EF complements a previous study• of a rat-mouse
`hybrid line in which scrambling of V and C regions from the
`light chains of rat and mouse was not observed. ~n this study,
`both light chains have identical c. regions and therefore
`scrambled VL- CL molecules would be undetected. On the other
`hand, the heavy chains are of different subclasses and we
`expect scrambled VH-CH to be detectable by IEF. They were
`not observed in the clones studied and if they occur must do
`so at a lower frequency. We conclude that in syngeneic cell
`hybrids (as well as in interspecies cell hybrids) V- C integration is
`not the result of cytoplasmic events. Integration as a result of
`DNA translocation or rearrangement during transcription is
`also suggested by the presence of integrated mRNA molecules11
`and by the existence of defective heavy chains in which a
`deletion of V and C sections seems to take place in already
`committed cells12•
`The cell line P3-X63Ag8 described above dies when exposed
`to HAT medium. Spleen cells from an immunised mouse also
`die in growth medium. When both cells are fused by Sendai
`virus and the resulting mixture is grown in HAT medium,
`surviving clones can be observed to grow and become estab(cid:173)
`lished after a few weeks. We have used SRBC as immunogen,
`which enabled us, after culturing the fused lines, to determine
`the presence of specific antibody-producing cells by a plaque
`assay techniquei3 (Fig. 2a). The hybrid cells were cloned in
`soft agar14 and clones producing antibody were easily detected
`by an overlay of SRBC and complement (Fig. 2b). Individual
`clones were isolated and shown to retain their phenotype as
`almost all the clones of the derived purified line are capable of
`lysing SRBC (Fig. 2c). The clones were visible to the naked
`eye (for example, Fig. 2d). Both direct and indirect plaque
`
`assays13 have been used to detect specific clones and representa(cid:173)
`tive clones of both types have been characterised and studied.
`The derived lines (Sp hybrids) are hybrid cell lines for the
`following reasons. They grow in selective medium. Their
`karyotype after 4 months in culture (Table I) is a little smaller
`than the sum of the two parental lines but more than twice the
`chromosome number of normal BALB/c cells, indicating that
`the lines are not the result of fusion between spleen cells. In
`addition the lines contain a metacentric chromosome also
`present in the parental P3-X67Ag8. Finally, the secreted
`immunoglobulinscontain MOPC 21 protein in addition to new,
`unknown components. The latter presumably represent the
`chains derived from the specific anti-SRBC antibody. Figure 3A
`sh.ows the lEF pattern of the material secreted by two such
`Sp hybrid clones. The lEF bands derived from the parental P3
`line are visible in the pattern of the hybrid cells, although
`obscured by the presence of a number of new bands. The
`pattern is very complex, but the complexity of hybrids of this
`type is likely to result from the random recombination of
`chains (see above, Fig. !). Indeed, lEF patterns of the reduced
`material secreted by the spleen- P3 hybrid clones gave a simpler
`pattern of lg chains. The heavy and light chains of the P3
`parental line became prominent, and new bands were apparent.
`The hybrid Sp-I gave direct plaques and this suggested that
`it produces an IgM an tibody. This is confirmed in Fig. 4 which
`shows the inhibition of SRBC lysis by a specific anti-lgM
`
`Table 1 Number of chromosomes in parental and hybrid cell lines
`Number of chromosomes per cell
`Cell line
`Mean
`P3-X67Ag8
`66,65,65,65,65
`65
`Pl Bui
`Ref. 4
`55
`40
`Mouse spleen cells
`106
`Hy-B (Pl- P3)
`Sp-1 /7-2
`90
`95
`Sp-2/3-3
`
`112,l 10.104,104,102
`93,90,89,89,87
`97 ,98,96,96,94,88
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`The Journal of Immunology
`
`Nature Vol. 256 August 7 1975
`
`anti body. IEF techniques usually do not reveal J9S lg M
`molecules. 1gM is therefore un likely to be present in the
`unreduced sample a (Fig. 38) but µ c hains should contribute
`to the pattern obtained after reduction (sample a, Fig. 3A).
`The above results show tha t cell fus ion techniques are a
`powerful tool to produce specific an tibody directed against a
`predetermined a ntigen . II further s hows tha t it is possible to
`isolate hybrid lines prod ucing di fferen t a ntibodies d irected
`against the sa me antigen and carrying different effector func(cid:173)
`tions (direc t and indirect plaq ue).
`The un cloned population of P3- spleen hybrid cells seems
`q uite heterogeneous. Using s uitable detection procedures it
`should be possible to isolate tiss ue c ulture cell li nes making
`d ifferent classes of antibody. To facilitate our studies we have
`used a myeloma parental line which itself produced an lg.
`Variants in which one of the parental chains is no longer ex(cid:173)
`pressed seem fairly common in the case of P J- P3 hybrids
`(Fig. lh). Therefore selection of lines in which o nly the specific
`an ti body chains are expressed seems reasonably si mple.
`Al ternatively, non-producing varian ts ~f myeloma lines could
`be used for fusion.
`We used SRBC as antigen. T hree different fusion experiments
`were successful in producing a la rge n umber of a ntibody(cid:173)
`prod uci ng cells. Three weeks after the initial fusion , 33/ 1,086
`
`A
`
`.B
`
`J
`
`,.
`
`·.
`
`c
`
`a
`
`c
`
`b
`
`c
`
`a
`
`c
`
`c
`
`Fig. 3 Auiorad iograph of labelled components secreted by anti(cid:173)
`SR BC specific hybrid lines. Fractionation bcfore(B)and after (A)
`reduction was by IEF. pH gradient was 5.0 (bollom) to 9.0 (top)
`in the presence of 6 M urea. Other conditions as in Fig. I. Supcr(cid:173)
`natants from: a, hybrid clone Sp-1/7-2; b , hybrid clone Sp-2/3-3;
`c, myeloma line P3-X67Ag8.
`
`2455
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`497
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`Fig. 4 Inhibition of haemolysis by antibody secreted by hybrid
`clone Sp-J/7-2. The reactioo was in a 9-cm Petri dish with a layer
`of5 ml 0.6 % agarose in phosphate-buffered sali ne containing I /80
`(v/v) SRBC. Cenire well contains 2.5 µI 20 li mes concentrated
`culture medium of clone Sp-1/7-2 and 2.5 µ I mouse serum. a,
`Sheep specific anti-mouse macroglobulin ( MOPC 104E, D r
`Feinstein); b, sheep ant i- MOPC 21 (P3) lgGl absorbed with Adj
`PC-5 ; c, sheep anti-Adj PC-5 ( lgG2a) absorbed with MOPC 21.
`After overnight incubation at room temperature the plate was
`developed with guinea pig serum dilu ted I :10 in Dulbecco's
`mediwn without serum.
`
`clones (3 %) were positive by the direct plaq ue assay. T he
`cloning efficiency in the experiment was 50 %. In another experi(cid:173)
`ment, however, the proportion of positive clones was con(cid:173)
`siderably lower (about 0.2 %). Jn a th ird experiment the hybrid
`populat io n was s tud ied by limiti ng dilution analysis. From 157
`independent hybrids, as many as 15 had a nti-S RBC activity.
`The proportion of positive over negative clones is re mark(cid:173)
`ably high. It is possible that spleen cells which have been
`triggered during immunisation are pa rt icularly successful in
`givi ng rise to viable hybrids. l t remains to be seen whet her
`s imilar results can be obtained using other antigenes.
`T he cells used in this s tudy are all of BALB/c origin and the
`hybrid clones can be injected into BALB/c mice to produce
`solid tumours and serum having an ii-S RBC ac tivity. It is
`possible to hybridise ant ibody-producing cells from different
`origins•·•. Such cells can be grown i11 vitro in massive cultures
`to provide specific antibody. Such c ultures could be valuable
`for medical and indus trial use.
`
`M R C Laboratory of Molecular Biology,
`Hills Road, Cambridge C 82 2QH. UK
`
`G. KOHLER
`C. MtLSTEIN
`
`Received May 14 ; accepted June 26, J97 S.
`I Polter. M .• Phy.,iol. R ..... 52. 63 1-719 (1 972).
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`199·228 tMTP Int. Rev. Sci., Bu1tcrnorth, London , 1973).
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`5 Schwabcr, J ., a nd Co hen. E. P •• Proc. 11arn. Ac11d.Sci. U.S.A .• 7 1, 2203-2207(1974).
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`7 Svas1i, J .. and Milsiein, C., Biocliem. J .• 128, 427-444 {1972).
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`10 Awdch, A. L .. Wi lliamson. A. R., a nd Asko nas, B. A .. N ature. 219, 66-67 0 968).
`11 Milstein, C .. Brownlee. G. G .. Cartwright, E. M .. Jar\'Js, J. M ., and Proudfoo1,
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`11 Frongione, B., an d Milstein, C ., Naftlrc, 244, 597-599 ( 1969).
`IJ Je1 ne, N. K .. a nd Nord in. A. A .• Sc/Pnc'e. 140. 405 (1963;.
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`t 1973).
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