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Nawre V ol. 256 August 7 / Y7 5

Vol. 256 No. 5517 August 7, 1.975

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Cover picture Oeser! locusts settle for the nj~ht.

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Volume 256

Whitlam, Connor and Cameron

INTERNATIONAL l'"'EWS

NEWS AND VlEWS

REVIEW ARTICLE T he origin of nuclei and of eukaryotic cells T. Cm'filier-Smirh

ARTICLES Palaeolithic remains at the Hadur in the Afar region-G. Con•i!rus Integration of viral genomes- V. J\11. Zltdtmov

LETrERS 1'0 NATUJlE Definition of 'charge on an atom' and nuture of the inductive effect­

S. M . Dealt rmd W. G'. Richards A low velocity zone underlying a fast-spreading rise crest-J. Orcutt,

B. Ke1mett, L. Dorma11 1md W. Prothero

Mercury contamination in a 54-m core from Jake Huleh-U. M . Cowgill A resonant point absorber of ocean-wave power- K. Budar and J. Fa!J1es

Climatic reversal in northern North Atlantic- R. R. Dickson, H. H. Lamb, S.·A . Malmbgrg and J. M. Colebrook

Americium 242rn in nuclear test debris-V. T. Bowen and H. D. Livingston

Tree remuins in southern Pennine peats- J. H . Tallis

August 7

Regularities in duration of regional desert locust plagues- Z. Waloff and S. M. Gr~en Development of a desert locust plague-L. V. Bennell

Seed-borne microorganisms stimulate seedcorn maggot egg laying­C. J. Eckenrode, G. E. Harmanmtd D. R. Webb

Defensive stoning by baboons-W. J. Hamilto11 Ill, R. E. Ouskirk and W H. Buskirk Eccentricity-specific dissociation of vi~ual functions in patients wi th lesions of the

centro! visual pathways-E. Poppe!, D. vo11 CralrtOII o11d !{. Backmrmd

Evidence for visual function mediated by anomalous projection in goldfish­D. Yager a111i S. C. Sharma

Thymus rudiment of the nthyrnic nude mouse-M. Holub, P. Rossman11, H . Tfaskalova and H. Vidmarava

Stria ted muscle fibres difTerentiate in monolayer cultures of adult thymus reticulum­H. Wekerle. 8. Pttterson, U.·P. Ketelsen and M. Feldman

Continuous cultures of fused cells secreting antibody of predefined specificity­G. Koltler rmd C. Milstein

Naturally occurring cytoto.~ic tumour reactive ami bodies directed against type C viral envelope antigents-S. £. Martin a11d W. J. Martin

Antigen formation in metal contact sensit ivity-f. M. Jones and H. E. Amos

Induced thermal resistance in Hel a cells-E. W. Gerner and M. J. S ch11eider Regional turnover and synthesis of catecholarnines in rat hypothalamus­

D. fl. G. Ver.lteeg, J. wm der Gngren and J. 1\1. van Ree

Rate of nucleologenesis as a measure of g~:ne activity-C. de Ia Torre, M. £. Fel'ltlllidn-Gomez and G. G'imenez-f,<fartill

-5S RNA secondary structure- G. E. Fox and C. R. Woese Diffcrenrial effect of plasma fractions from normal and tumour-bearing rats on

nuclear RNA restriction-D. £. Schumm and T. E. Webh

Early role during r.hemical evolution for cytochrome P4SO in mcygen detoxilication R. H. lflickrvmosinglre ami C. A. Vil/ee

Human embryonic hacmoglobins including a comparison by homology of the · human ~and n chains- H. Kamu:ora tmd H. Leltmamt Lycorine as 3n inhibitor of ascorbic acid biosynthesis-a. Arrigoni,

R. A . Lisa und G'. Calabrese

lntracellular killing of Listeria monocytogenes by activated macrophagcs (Mackancss system) is due to antibiotic- ?. Cole a11d J. Brostoff.

1975

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1: \Cttw<' Vol. !j6 ...1 u~usr 7 1\175

L--· 1 i Continuous cultures of fused cells \ 5ecrding antibody of predefined specificity l fll• r .anufa~:ture of predefined specitic antibodies by means of :L ~rr.;Jnent tissue culture cell lin~s is of general interest. There

:r. a t present a considerable number of perman.:nt cultures of

I ~cloiT'a cells '· 2 and screeni:lg procedures h;>ve been used to lr~>eal antibody act ivitY. in some of them. This, however, is

:111 a satisfactory source of monoclonal anti bodies of predefined ,"ecificity. We describe here the derivation of a number of

• ,;;sue culture cell lines which secrete anti-sheep red blood ,~11 (SRBC) antibodies. The cell lines are made by fusion of a 1nouse myeloma and mouse spleen cells from an immunised dl)nor. To understand the expression and interactions of the Jg chains from the parental lines, fusion experiments between 1110 known mouse myeloma lines were carried out.

Each immunoglobulin chain result~ from the integratl!d !·.pression of one of several V and C ge.aes coding respectively iJr its variable and constant sections. Each cell expresses only on(. of the two possible alleles (allelic exclusion; reviewed in ref. 3). When two antibody-producing cells are fused, the

1 products· of both parental lines are expressed•·•, and although rh~ light and heavy chains of both parental lines are randomly JOined, no evidence of scrambling of V and C sections is Jb.ened'. These results, obtained in an heterologous system IDIO)ving cells of rat and mouse origin, have now been con· tinned by fusing two. myeloma cells of the same mouse strain,

I Chains

. !_, H(Pl)/ 1 •• ._..-~._ - H(P3)

A • ..

~ -ta=:;i; ~ . -4. ._.·, ' . < • - ...

_; ... . .. ~~ ... .,.. 1 ,_

L(P3)

495

The protein sc.:rc tcd ( M 0 PC 21) •~ ·n I gG 1 ( 1\) ·.vhich has been f~lly scQuenc.:cd' ·1

. ~qu?l nu~1bers o r cel:s from each parental line were fused usmg ma.;:uvatcd Sena.,; virus• and samples contining 2 J( 10' cells \\ere gr0 11 n in seketive medium in sep:1ratc dbhes. Four out of ren disbes shOI\•d Krowth in selective medium and these were taken as indepenue,,: "-tb~lu lines, probably derived from single fusion events. The karyotype of the hybrid cells after 5 months in culture \\US just under the sum of the two parental lines (Table !).',Figure 1 shows the isoelectric focusi ng10 {IEf) pattern of the secreted products of difl'erent lines./The hybrid cells (samples c-h in Fig. I) give a much more complex pattern than either parent (a and b) or a mixture of the parental lines (m). The important feature of the new p:lttern is the presence of extra bands (fig. I, arrows). T hese new bands, however, do not seem to be the result of diO'erences in primary structure; this is indicated by the IEF pattern of the products after reduction to separate the heavy and light chains (Fig. I B). The IEF pattern of chains of the hybrid clones {Fig. 1 B, g ) is equivalent to the sum of the lEF pattern (a and b) of chains o f the parental clones with no evidence of extra products. We conclude that, as previously shown with interspecies hybrids '·', new lg molecules are produced as a result of mixed nssociation between heavy and light chains from the two parents. This process is intracellular as a mixed cell population does not give rise to such hybrid molecules (compare m and g , Fig. I A). The individual cells must therefore be able .to ·express both isotypes. T his result shows that in hybrid cells the expression of one isotype and idiotype does not exclude the expr('SSion of another : both heavy chain

.. ;. . .. ,.... -· L(Pl) r

Fig. I Auaoradiograph of labelled compo­nents secreted by the parentalnnd hybrid cell lines analysed by IEF before (A) and after reduction (8). Cells were i n~ubatcd in the presence of 11C-Iysine11 and the supernatant applied on polyacrylamide slabs. A. pH range 6.0 (bonom) to 8.0 (top) in 4 M urea. B, pH range 5.0 (bottom) to 9.0 (top) in 6 M urea; the supernatant was incubated for 20 min at 37 •c in the presence of 8 M urea, 1.5 M mercaptoelhanol and 0.1 M potassium phos­phate pH 8.0 before being applied to the right slab. Supernatants from parental cell lines in: a, PI Bu I ; b, P3-X67 AgS ; and m, millture of equal number of PI But and PJ-X67Ag8 cells. Supemiltants from two independently derived hybrid lines are shown: .:-[, four subclones from Hy-3; g and h, two subclones from Hy-B. Fusion was carried out•·• using to• cells of each parental line and 4,000 haemagglulination units inactivated Sendai virus (Searle). Cells were davaoe<i into ten equal samples and grown separat;!y i:J selet:tive medium (HAT medium, ref. 6). Medium was changed every 3 d. Successful hybrid lines were obtained in four of the cul­tures, and all gave similar JEF panerns. Hy-B and Hy-3 were further cloned in soft agar".

I

I.

e m f m g b ~ j d provide the background for the derivation and under·

\,~~nding of antibody-secreting hybrid linc:s in whit:h one of 1:"~: parental cells is an antibody-producing spleen ct:ll.

f,vo myeloma cell lines of BALB/c origin were used. PI Bul 11 resi:;tant to :i-bromo-2'-dcoxyuridine•, does not grow in !elective medium (HAT, ref. 6) and secretes a myeloma protein,

1

.\•li PCS, which is a n lgG2/\ (K), (ref. 1). Synthesis is not ~lan~~d and free light chains arc also secreted. The wcond ~~·1 line, PJ-X6JAg::l, prepan:d from P3 ccJb!, i~ resis tant to ) ) ~1g mt - • S-aw guani ne and ttoc~ not grow :n HI\ T medium.

. ' ' -- ' -L, Light ; H, heilvy . .....

a h b g {l

:sotypes (yl and y2a) and both V" a nd bo th VL regions ·(idiotypes) are expressed. There are no allotypic markers for the C K region to provide direct proof for the expression of both par.::ntal C K regions. Out this is indicated by the phenotypic lia.k between the Vand C regions.

Fi11ure lA shows that clones derived from different hybridi­satio~ experiments and from subclones of one line are indistin· guishable. This has also been observ.:d in other experiments (data not shown). Variants \\ere. however, found in a survey of JOO subcloncs. The dill'.:rence is o ften associated with changes

PFIZER EX. 1522 Page 3

49h

Ji\ In the ratios of the different chains and occasionally with the

llotaf d isappearance of one or other of the chains. Such eve~ts are best visualised on IEF analysis of the separated chams (fo1'example, Fig. lh , in which the heavy chain of P3 is no lon er obser ved). The important point that no new chains a re c!et ·-::ted by IEF complements a previous study' of a rat-mouse

5 hybrid line in which scrambling of V and C regions ~rom the a light chain' of rat and mouse was n:>t observed. 'n this study, d ... .,th light chains have identical c. regions and therefore 1 scrambled VL-CL molecules would be undetected. On the other p hand, the heavy chains a re of different subclasses a nd we p exp:ct scrambh:d V11-CH to be detectable by IEF. They were li not observed in the clones studied and if they occur rnust do

so at n lowet frequew:y, We conclude that in syngeneic cell ~' hybrid> (as well a .; in interspecies cell hybrids) V-C integration is

not th ~.csu1 , C.>IOplasrnic events. Integration as a result of ;";'~A ::·11nslocation or rearrangement during transcription is also suggested by the presence of integrated mRNA molecules11

tmd by the existence of defective heavy chains in which a deletion of V and C sections seems to take place in a lready •:l'mmitted ceJisH.

The cell line P3-X63Ag8 described above dies when exposed to HAT medium. Spleen cells from an immunised mouse also die ln growth medium. When both cells are fused by Sendai " !rus and the resulting mixture is grown in HAT medium,

' :>urviving clones can be observed to grow and become estab­hsh~d after a few weeks. We have used SRBC as immunogen, \\·hil;h enabled u~. after culturing the fused lines, to determine the ~presence of specific antibody-producing cells by a plaque un~!\} technique13 (Fig. 2a). The hybrid cells were cloned in soft agar'~ and clones producing <~ntibody were easily detected by an overlay or SR BC and complement (Fig. 2b). Individual dt•nes were isolated and shown to retain their phenotype as ;(lr'lost all the clones of the derived purified line arc capable of 1:/~·ng SRBC ( Fig. 2c·). The _Iones were visible to the: naked c.'"~~ \for example, Fig. 2d). Both direct and indirect plaque

NoturC' Vol. 256 All;i lllt 7 l'ii.'

Fig. 2 Jsolatiun of an ami-SRBC antibc~v. secreting cell cl(lne. Activity wns re' ea:cct 6, a halo of hacmolyscd SRBC. Dir.:ct plaq~~ given by: a. 6,0ll0 hybrid cells Sp-l ; 1>, cl<n.., grown in soft agar from an inoculum or 2,111..-.: Sp-1 cells: c. recloning of one of the positi~t clones Sp-1/7; d, higher magnification or a positive clone. Myeloma cells (10' P3-X67A g8) were fused to 10' spleen cells from 'In immunised BALB/c mou;e. Mice were im­munised by intraperitoneal injection of0.2ml packed SRBC diluted 1:10, boosted afler 1 month ami the spleens collected 4 d lat~r. After fusion, cells (Sp-1) 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 week1 with hypoxanthine and thymidine. Forty day~ after fusion the presence of anti-SRBC act. ivity was revealed as shown in a. The rotio o( plaque forming cells/total number of hybrid cells was 1/30. This hybrid cell population was cloned in soft agar (50% cloning ef. ficiency). A modified plaque assay was used to reveal positive clones shown inb-das follows. When cell clones had reached a suitable site, they were overlaid in sterile conditiom witll 2 ml 0.6% agarose in phosphate-buffeR(} saline containing 25 111 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 pick.ed out and grown in suspension. Th.is clone was called Sp-1/7, and was recloned as shown inc: over 90:1. oft he clones gave positive lysis. A second • experiment in which 106 P3- X67Ag8cellswcre fus~d with 10• spleen cells was the source or 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,

assays13 have been used to detect specific clones and represenla· tive clones o f both types have been characterised and studied.

T he derived lines (Sp hybrids) are hybrid cell lines for the following reasons. T hey grow in selective medium. Their karyotype after 4 months in culture (Table l) is a little .nailer !han 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. Io addition the lines contain a metaccntric chromosome also present in the parental P3-X67Ag8. Finally, the secreted immunoglobulins contain MOPC 2 L protein in addition to new, unknown components. T he lat ter presumably represent the chains derived from the specific anti-SR BC antibody. Figure JA stwws the JEF pattern of the material secreted by rwo such Sp hybrid clones. The IEF bands derived from the parental Pl line are visible in the pallern of the hybrid cells, althougb obscured by the presence of a number of new bands. TJ:c pattern is very complex, but the complexity of hybrids of thiS type is likely to resul t from the random recombination or chains (see above, Fig. 1). Indeed, IEF patterns of the reduced material secreted by t he spleen- PJ hybrid clones gave a simpler pattern of lg chains. The heavy and light chains o f the P3 pa rental line became prominent, and new bands were apparent.

Th<! hybrld Sp-1 gave direct plaques and this suggested t~al it produces an lg M antibody. This is confirmed in Fig. 4 whiCh shows the inhibition of SRBC lysis by a specific anti-IgM

Table I Number of chromosomes in parental and hybrid cdl lines ! I Cell line Numbet' of chromo;,on1es per cell M~lll l

P3-X67Ag8 66,65,65,65,65 655

5 PI Bul Ref. 4 Mou~espleen cells 1~ I Hy-B(PI - PJ) 112,110,104,1U4, i01

9r :

Sp-1/7-2 9J.YU.1!9.t:9.~7 ~~ Sp-Z/ l-3 97,n ,1!5.96,\14,X~ _ , ·- - · •. l

PFIZER EX. 1522 Page 4

.,1

.J11tlll,,dy. I D ' t~<.:hniques u~uu l ly do not reveal 19~ l gM .. ,,•c~llks. lgi\1 I'> th.:r..:fo re unlil-..;ly to be pr<:'i<!nl 111 the

11 '1rc.ltaceu ~<llllpk 11 (Fig. J [ I) bur p <:hllins sh<~ttld <:Ontribute o1 .~, rhc patter11 o bta ined a fter rcdu-: tio n (.;ample u, Fig. 3 .'1).

1 · Tho:: abo,·<: re;ulls $hvw that <.:dl fu>io n techniques are u I' ;,~1\crful tool to produce ~pecific antibody directed a);ainst a 1 'fctkt•;rminc:d antigen. It further shows that it is possible to r ·vl~ te hybrid lines producing different antibodies directed

·Jinst the same anti!;en and carrying d lffcrent eflector func-·"' (direct and indirect plaque). *

;I r :1c uncloned population of PJ- spleen hybrid cells seems n ,uirc heterogeneous. Using suitable detection procedures it T. ;hould be possible to isola te tissue c ulture cell lines making it Jiil'aent classes of a ntibody. To fac ilitate our studies we have lt Jl~d a myeloma parenta l line which itself produced an lg. re vari.llltS in which one of the parental chains is no longer ex­:d ore.s;ed seem fairly common in the case of PI- PJ hybrids ri (fig. lh). Therefore selection of lines in whic h only the specific · mdb!Jdy chains are expressed seems reasonably simple. g~~ltcrnatively.' ~on-producing variants ~r myeloma lines could :i. be us.:d for fuston. e We used SRBC as antigen. Three different fusion experiments u~ were successful in producing a large number of antibody­N' proJ .tcing cells. Three weeks after the initial fusio n, 33/ 1,086 :r.: ·d

A B

1 I

,. -· t .• , ;

'

...... ....... c a c b c

Fill. 3 Autmadiograph of labetl.:ll co~1ponents sccr~teJ by anti­SR BC' specific hybrid lines. Fractional tOn before ( 8) anti after (A)

l redt1ction was by IE F. pH gradient was 5.0 (h<lttom) to 9.0 (top) in th~: pres~nce of 6 M urc:a. Oth~r <.:Ontli lillnS as in Fig. I. S\tper­natants fro m: u, hybrill clune Sp-1/7·2: h, hybrid clone Sp-2/J-J:

r, myeloma line P:l-X67A~:8.

497

fig. 4 Inhibition of haemolysis by antibody secreted by hybrid clone Sp-1/7-2. The reaction was in a 9-cm Petri dish with a layer of 5 ml 0.6% agarose in phosphate-buffered saline containing 1/80 (v/v) SRBC. Centre well contains 2.5 ~tl 20 times concentrated culture medium of clone Sp-1/7-2 and 2.5 111 mouse serum. a, Sheep specific anti-mouse macroglobulin (MOPC 104E, Dr Feinstein); b, sheep anti-MOPC 21 (P3) lgG I 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 diluted I :10 in Dulbecco's

medium without serum.

clones (3 %) were postttve by the direct plaque assay. The cloning efficiency in the experiment was 50%. I n another ex peri· ment, however, the proportion of positive clones was con· siderably lower (about 0.2%). In a third experiment the hybrid population was studied by limiting dilut ion analysis. From 157 independent hybrids, as many as 15 had anti-SR.BC activity. T he proportion of positive over negative clones is remark­ably high. It is possible that splt!en ce lls which have been tr iggered during immunisation a re particularly successful in giving rise to viable hybrids. It remains to be seen whether similar resu lts can be obta ined using other antigenes.

The cells used in this study are all of BALB/c origin and the hybrid clones can be injected into BALB/c mice to produce solid tumours a nd serum having anti-SRBC activity. It is possible to hybridise antibody-producing cells from different origins•·•. Such cells can be grown in l'l"tro in massive cultures to provide specific antibody. Such cultures could be valuable for medical and industrial use. ~ 1

M RC Laboratory of Molecular Biology, Hills Road, Cambridge C812QH, UK

Received May 14; accepted June 26, 1975.

G. K oHLER C. M ILSTEIN

• Poner,. M .. Phy>iol. Rtv., 52, 631-719 (1972). 1 Ho ribolo, K., and Harris. A. W., Expl Ctll Ru., 60. 61-70 11970). J Mil,lein. C .. onu Munro, A. J., in Dt/tn« and R.cognlflon(edit. by Porter. R. R.),

199·228 (1\tTP Int. Rev. Sci., Buncrworth, London, 1973). • Conon, R. G. H., and Milstein. C. , Naturr, 2~4. 42-43 (197)). 'Schwobcr, J ., and Cohen, E. P., Prar. natn. Ar11d.Sd. U.S.A .• 71,2203· 2207 (1974) • • Lillleftclu, J . \V., SciM«, 145,709 (1964). 7 Svo.ti, J., and Ml15tcin, C .. Divrhtm. I ., 128. 427-444 (1972). A Mlllo.tcin , C .• Adctu~bo. K., Co wan, N. J. , and $c:,:h~r. D . S .. Prf$.r~$S in lmmuno­

/t11fy, II , 1 (edit. by Bren l, L., and Ho lborow. ).), I S7-168 (Nonh·Hollond, Am\lerdom. 1974).

• Horri•, II., anu Watkins, J. F .. Nawrt, l OS, 640.646 ( 196~). 10 A"dch. A. L .. Willi•m•on. A. R., and Asko nos, B. A .. Nawr<. 21~. 66-67 ()968), 11 Mil\tci•t. C .. Brownlee, C . G., C3rtwright, E.. M .. J11n·is. J. M., and Proudroot,

f', J., Nultlft, 152, 3S4-J59 (19741. ll l'rongionc. B .. and Mll>tcin, C., Nu/11,., H~ • . ' Q7-S99 (1969). t ) Je• ne, N. K., and Nordin. A. A .. Scirtt<,, 1~0.~(15 (1 963). 14 Cuuon. R. G. H., Sech<r, 0 . S., and MilStein, C. , Eur. J. fmrmm., l, t3S·I40

( 19131. '

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