PHIGENIX
`PHIGENIX
`Exhibit 1009
`Exhibit 1009
`
`

`
`Vol 290 N0 5802 12 March 1981 £1
`
`.25 $3.00
`
`m.
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`..A
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`25....3
`
`PHIGENIX
`
`Exhi
`
`it 1009- O1
`
`
`
`
`

`
`Nature Vol. 290
`
`I2 1'VIr1rc}2
`
`I 981
`
`iii
`
`
`
`Volume 290 No.5802
`
`12 March 1981
`
`How true is the theory of evolution?
`’{\}1’i§izi77‘ei'elcon1e for genes on Wall Street: Genentech’s
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`75
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`79
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`Chemical warfare: US prepares?
`W 79
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`r;i;e;rm..nszarsaigscseii¥;T<,ii;;;“‘”“ “”“e“""“” 71
`eat.-ttzpordeig;-melt.1Q i;;st.r;c’fr2f§mna;; Dt;;vi,;;;'su;m.i
`(H.W. Ball er a/.); Conservation sites (C. Muir); Origin of
`cancer (D.l. Edwards; M. Woodruff)
`82, 173
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`NEW/SANDViEW5
`_ _
`The intergalactic medium: Joseph Silk on the search for
`Hytlrothermal activity at mid-ocean ridge axes: J.M. Edmond
`83
`evitlerrce ofits existence
`describes the results of deep-sea searches
`8"?
`to cancericetlsi-gritir Olsnes assesses progress-H
`Klitloititttrinp, Mount HwclettsV:VM.--lvleéormicls;‘reports on two
`84
`recent meetings
`-Will-the real Crertville Orogeny please stand iipi’:
`Bell on the Proterozoic in Canada
`
`88
`
`89
`
`
`
`ield
`inthi
`The worltlsivoir-st vveeds: Rovber-t”l\/l.All/lzty describes a
`pro mote to prevent; accidental introduction
`Eltetazrtzsgttetic liieltlurriodelling
`satellite data:
`86
`Barrzuzlough reassesses the present. models
`"'"'
`““"““""”"""'
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`REVIE W AR filth
`
`85
`
`R."
`
`Textoiis, the elements of
`texture perception, and their
`interactions
`
`91
`
`R.D. Evans, .I.H. Harley,
`W. Jacobi, A.S. McLean,
`W.A, Mills and CG. Stewart
`
`Estimate of risk*from
`environmental exposure to
`radon-222 and its decay
`products
`
`98
`
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`
`Structure, strength, and
`polarization changes in radio
`source S8433
`Structure of the hydrophobic
`protein crambin determined
`directly from the anomalous
`scattering of sulphur
`Ill"!
`
`
`ltlll
`
`.l.R. Nevins
`and tvt.C,‘. Wilson
`
`Regulation of adenovirus-2 gene
`expression at the level of
`transcriptional termination and
`RNA m°°eSS‘"g
`113
`
`RM l-ljellming
`and R35 . Johnston
`
`W..«\. Heudrickson
`and MM. Teeter
`
`
`
`M ‘ Wm“ ""
`
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`
`"'“_”"" ~ t""7i§”“'”"""'—"””“““"““"'"""‘"”"‘”“""'""“”"""""‘"””""""
`LET ERS
`
`UV observations of X-ray
`{Zoe
`sources 2Atl31l~227 and
`and D31". Wickramasing.he
`H9.
`—.e......._
`_____
`_-
`_ 328 ,,
`_.,_._.__
`L. Blitz. .l.W.—K. Mark
`Vi/hy is the central gas disk of the
`KP. Sinha
`Galaxytiltetl?
`12%
`G. Bonnie‘, PP. Lombardini, Radio acoustic measurement of
`A. Longltetto
`fog~capping thermal
`and ti‘. Trivem
`inversions
`
`12t
`
`EA. Shillington
`
`R.S. Harmon
`and H.P_ Schwarcz
`
`A,(}. Baer
`
`Low frequency 0.045-l-lz swell
`123
`from the Sonthem Ocean
`Changes of ‘H and “*0 enrichment
`of meteoric water and
`Pl istocene glaciation
`Two orogenies in the Grenville
`Belt?
`
`125
`
`129
`
`Cover: The three textures on the cover appear very distinct even
`ll’10ng,h.they lli1V€‘1d9nuCa-1 th1rd‘0rde]' St-flushes and thus ldenncal
`5900nr.l«0t'der statistics; and Fourier power spectra. Experiments
`reviewed on pages 91 to 97 show that ‘instantaneous’ texture
`di_:,g1~im,'naU‘0n Cannot be explained solely in terms of global
`pro 71.
`‘
`1.
`1
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`I
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`local, conspicuous features.
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`
`
`Content.-;continued overleaf’
`(ISSN 002.8-t)83(i) l€ pnblislied we-skly, except the last week in Dr-cernber, by
`Nalunfl
`,\lat:mill:An murimit Ltd. .~\nnnul stlbacnplioll for US.‘\ mutt:-.tnaua US 3.173. Orders
`(withI::miIt:«tuce):utd change ot':iddrcs~,luhelsto:Macmillan _luurnal=.l,ul,l3runclRd,
`Basingsiokc RG21 2XS, UK. Second class postage paid at New York NY lt)()t0 and
`cidrlitiomzlm-ailingoffices.USl’ostni:1sterseitd i’orm.lS79to:Nmu1e, Sliastzfufitrcct.
`New York, N\'lt)()l(l. '-?..'l9lll Macmillan Journals Ltd.
`
`5~’~,: l‘}8l 3\‘l:«ctmll:m J0ttrnalsl.td
`
`PHIGENIX
`
`Exhibit 1009-02
`
`
`
`

`
`,=‘v'.rI.l‘r4re Vol. 290 12 M'arc/1 198]
`
`may have special significance at a particular time during the
`course‘ of disease. The elucidation of these mechanisms could
`provide targets for development of a potential ‘malaria vaccine’.
`We acknowledge the financial support of the UN DP/ World
`l-Bank/WHO Special Programme for Research and Training in
`’l."m;)ical Diseases and the US PHS research grant Al-12710-4,
`
`i<':‘.cmvcd 6 October; Ctcccntcd 17 December 1980.
`
`1. Cohen, S. Prnc. R. Soc. B2l).'l, 223--345 (1079).
`Rilllk. R. G. S; Weirlanz, W. P. Pmc, Soc.
`(.'.\'[). Biol. M’:-‘ii. 151. 357~—259 (1976).
`A Roberts. D. W., Rank, R, G., Weidanz, W. l’. 8.: Finc.rl_v. I. F. I/zfrr. Immtmhy 16, li2i~-£426
`(1977).
`t{ol1crts, D. \\’. Sc \Veidan7,, VV 1'’. Am. J. imp. A-Ind. Hyg. 23. i—.\ (1979).
`E. Wembaunt, F l.., Evans. C. B. & Tigclaar. R. 13.! lmnmn. 117. ll99—20()5 (1976).
`-‘/lanning, D. D. J. 7:’!l(?l(i0('Il‘r[0lhE1l()l501?. til, 63426 «I975».
`2'».
`‘I. Dxvyer. J. M.. Roscnbaum, J. T. & Lewis. S. J. cxp. Med. 143, 7tll—-790 (19763.
`Cox, F. E. (3. Bull. WM HM: Org, 53. 325~3.'l6 (3970).
`9. Allison, A C. & Clark, I. A. Am. J. trap. Mari. Hyg. 26. 2l6—22l (1977).
`1-‘)_
`.'3.llison, A. C., Christensen, .l., Clark, l. A., Elford. B. C. & liugui, E. M. in The Spleen and
`[1rRo[.: irifarrzritiir Irzfcrtiuus (ed. Torrigiani, G.) l5l—l77 (Kurgcr, Basel. 1978).
`_ Eogui, 13. M. &' Allison. A, C, Hall. W/ll Hlrlx Org. 57. Suppl. 1, 231438 H973‘).
`‘,3, Shear, H. L., Nusscnzwcig. ll. 5. & Bianca, C. J. exp. /tied. 1419. 138314298 (1979).
`3. Clark. J. A. Pirrnxile Immiur. ‘I, l’/9-196 ( l9"/9).
`
`145
`
`After incubation of 2 X 105 WEHI-7 cells for 16 h with
`different irnmunotoxin concentrations, a 50% inhibition of "C-
`leucine uptake (P150) was obtained with a 10" ‘" M concentration
`(Fig. la). Free A-chain was much less active, needing a concen-
`tration 5,0()0 times higher (P150: 5 X 10” M) while ricin itself
`was about 25 times more active than imrnunotoxin (P150 = 2 X
`‘l.()““ M). A nonspecific immunotoxin directed against dextran,
`an unrelated antigen. behaved as free A-chain. The specificity of
`iinrnunotoxin calculated as the increase of concentration needed
`to kill the same cells by the A—chain alone corresponded to a
`factor of between 2,000 and 6.000 in different batches tested
`(Fig. lb). The antibody alone without complement showed no
`significant effect (Fig. Iv). Unconjugated antibody mixed with
`free A-chain behaved as free.A-chain (Fig. 1c). No effect was
`found with an anti-Thy 1.2 immunotoxin on Thy 1,2-negative
`cells (Fig. 1d), although these cells had a similar sensitivity to
`ricin and A-chain. Complete inhibition of immunotoxin activity
`Specificity factor
`CZ°.:..._______....___:Il'
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`
` nrnnxrotoxins: hybrid molecules of
`
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`onoclonal antibodies and a toxin
`szaburiit specifically hill tumour cells
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`Fig. 1 Specific cytotoxic activity of anti—Thy l,2 immunotoxin. a,
`2 X l05 WEHl—7 cells (a mouse T leukaemia expressing Thy l,2
`antigen) were incubated for 16h with different concentrations of
`the test substances and then pulsed with MC—leucinc for 2 h (for
`more details see ref. 8). @, Ricin; A, A~chain-, ><, anti~Tliy l, 2
`immunotoxin; E, anti-dextran innnunotoxin. h. Specificity factors
`demonstrate
`the
`specific
`cytotoxicity of
`immunotoxins
`as
`compared with their toxic subunit, the A-chain alone, and were
`drawn as arrows with the tail at the concentration (M) of free
`A~chain which induces 50% protein synthesis inhibition and with
`the head at the concentration for specific protein synthesis inhibi-
`tion by imrnunotoxin. The specificity factors of three batches of
`anti-Thy 1,2 inununotoxin are compared. C. Thy l,2—positive cells
`(WEHl—7) were used to establish specificity controls for IgM alone
`without complement
`(—~»®--——),
`lgM mixed with free A-chain
`l~—vO~-—), A—chain (-~~~-A-4) and ricin (-——®‘——). (1, Thy l,2--negative
`cells (BC—3A) served as controls for the specificity of the ‘Thy 1,2
`immunotoxin (~—«><~—) as well
`sensitivity to ricin (-®——) and
`A~chain (—~A-).
`
`*t..\6/8l/ I l(ll45—-(l?.Stll .00
`© 1981 Macmillan Journals Ltd
`
`his material may be prote
`
`PHIGENIX
`
`Exhibit 1009-03
`
`
`(ff.-,..
`Proteinsymiiesis
`
`
`
`
`50 -
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`10*" 1040109 i0”8
`10“7
`i0‘5
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`[Free A~chain] (M)
`
`E-Eiildur E. Blythman, Pierre Casellns, Olivier Gros,
`i’ic.rrc Gros, Franz K. Jansen, Francis Pnolucci,
`llearnard Pan & Hubert Vidal
`
`Centre de Recherches Clin Midy, Rue du Professe-ur Joseph Blayac,
`.1/-.U'_l‘€'.7. Montpellier Cedex, France
`
`S4?"c’§EZ'£ll attempts to attack tumours in experimental systems
`lraa,-<2 been made using conjugates of chemotherapeutic agents or
`}}t.'<.€Ii.$.?li
`toxins with antibodies (imrnunotoxins)"”. In oirro stun
`dies; have been highly successful, showing target specificity of a
`high order in some cases“‘. However, so far, such conjugates
`lnsves been inadequate in viva, probably for two main reasons.
`"irsit, conventional heteroclonal antibodies are perhaps inapn
`progsriate, because purification by biochemical methods leaves a
`large amount of non-antibody ‘y-globulins. The use of mono-
`clonal antibodies may overcome this problem. Second, when
`utsoie toxins have been conjugated to antibodies there has been
`a stroiig residual nonspecific cytotoxicity due to the binding
`capacity of a subunit, the B=-piece of the toxin. (Diphtheria toxin
`or ri<:in consist of two polypeptide subunits. The A-piece is
`rcsponsilile for inhibition of protein synthesis on ribosomes, and
`the _‘i~Z»piecc binds to galactoso residues on the cell membrane
`and tscilitutes the trunsmcrnbrane passage of the Aupiece.) In
`the present work the problem of nonspecific binding by the
`B-piece has been circumvented by using the A-piece only as the
`tordn component of immunotoxins;
`these immuuotoxius are
`active both in vitro and in vivo.
`
`A monoclonal lgM antibody directed against the Thy 1,2
`difi-zxireaiitirition antigen of mouse T cells, also present on mouse
`letxluicniia cells (WEHL-7), was used. After precipitation of the
`monoclonal lgM antibody with ammonium sulphate, the anti~
`body was purified with successive filtrations on Sepharose 68. It
`W‘2i:»; then modified with 3~12-pyridyldithio)propionic acid in the
`p1'cs<~:rice of water~solnble carbodiimide and coupled to highly
`piirriiieci A-chain of ricin“. The conjugate was purified on a
`SEf';"«l}.Etil€X G-200 column from unconjugatcd A--chain. The
`resulting iminunotoxin contained on average eight A»chains per
`riioh',-'cule, about three of which were active in an acellular
`protein synthesis system. The lgM component of immunotoxin
`retained about 70% of its activity in a complement~dependent
`Cytoioxirtity test. Inhibition of protein s_vnthe.sis (PD of viable
`Cells; in Ultra was used to evaluate the biological activity of the
`con_;'z.=gates".
`
`
`
`

`
`146
`
`1\/'aIure Vol. 290 12 Marrrh 1.98.1"
`
`oooaaoovoalnnoooinwwooelovoe
`
`mo
`
`The high specificity of imniunotoxins consisting of mono-«
`clonal IgM antibodies (specificity factors of 2,ll00*6,000) niay
`be attributed to the lack of non»autibody molecules or to the
`higher avidity of IgM over IgG antibodies. The nonspecific,‘
`activity of imrnunotoxius on antigenmegative cells is very low
`and can be explained by the high purity of the A~piece replacing
`whole ricin. Such an effect was not convincingly deinoristrateti
`by Masuho at at’. because their mouse cell system is insensitive to
`whole diphtheria toxin and cannot reveal contamination by the
`B-piece or whole toxin”. Specificity factors were not reported in
`the recent coniniunications by Gillilancl at :21.“ and Krolick «st
`al.” because 50% nonspecific toxicity was not indicated.
`2?.'4
`Although a significant prolongation of survival
`time w
`achieved in viuo with ourapproach.
`the effect seemed it
`impressive when compared with the high potency of immunt:«~-
`toxin in vitro. This difference can perhaps be explained by the
`fact that the injected leukaemia cells represented less than ‘l ‘ll,
`of all normal host cells expressing the Thy 1,2 antigen and thus
`the imrnunotoxin could have been absorbed by them. With
`repeated daily injections of irnrnunotoxin compensating for
`degradation or loss, better results might be expected. Altliotiglz.
`the immediate i.p. treatment of injected leukaemia cells with
`immunotoxin cannot be regarded as a model system of leull-,~
`aernia treatment. it represents a highly sensitive test system.
`Whereas, in vitro, 50% survival of tumour cells is measured, in
`viva, even single cell clones escaping the treatment will indut:=::
`tumours some time later.
`fixing
`their complement
`Immunotoxins retained most of
`activity but, as human complement is thought to be relatively
`inethcient
`in mo for the destruction of
`tumour cells,
`the
`complernent—independent
`activity
`of
`immunotoxirrs
`rti:z-.3‘
`represent a promising means of passive immunotherapy of
`cancer.
`
`
`
`
`
`
`
`-
`This work W’ s supported in part by a grant from the Déléj. '
`tion Ge'né.ralc. a la Recherche Scientilique et Technique, l"a1..-.
`Frarice. WEI-lI—7 and BC—3A cells were obtained from the Cell
`Distribution Center, the ff-Salk institute.
`
`Received 10 October W81): accepted 7 January 198i.
`
`.‘i‘c'mzc. Acail. é«'ci.. Paris 246. l62t§~~l4=.":>:
`
`l.
`
`3v.:.A'.uto
`
`.'vlMhé. G.. Loc, T B & Bernard, J. C‘ Cr. helm’.
`H958).
`Ghose, T. A2. Blair. A. H. J. nuln. £."z1nr:cr Inst. ()1, (i:'i'7~(i72 (N78),
`Davies, D. A. l.. & Cl'l\leill, G .l. Hr. .7. Crzmrcr Zll. Suppl.
`l. 285-298 t_l‘?73>.
`. Arnon, R. Prat‘. Sermm Syrirji, 16, 237--319:1 (1979).
`Y. .-tcrul. Sci. .2’7'7,(i‘)r)~N)(l l'l'»?':'t'v).
`Moolxcn, E3. 1... Zajdel,
`.8: (fooperliand. S. R. Ami.
`. Thorpe, l’.
`cl ul. Nalmr 271, '7
`S til)"/Si.
`Youlc. R. J. In <.\'eviile, D W. Jr Pr: .. mun. .~\crZd. Sci. US..»\. '77, 54ii3—51l8ti (1930).
`
`
`. 1ansen,l’.K.e(al. Immzm. Lair. 2, 07-102 liililtllz Frenchl’atcnt78.27.8.l81'19‘/8);
`.4
`C(V-.‘
`Patent Application 7944} ill)‘/9).
`9. Finney, D. .l. in l’r¢1bi'r.4ixrzlyi*is l9~8l) r(.“arnbriri;3.e llriiversity Press. 197 l).
`10. Masuho, Y.. Hara, T. & Nouuchi, T. l’}i(n:/rem. hiiipiiys. Res Commmz. 90, .'i'.l.0—3'3l3 fl?"/‘-‘i.
`ll. (iilliland, D. G. at (11. Pmr. mil/r.Acar1.St'i. US./\. 77. 4539-~-$5-'13 il980l.
`I3 Krolick, K. A., \’i)icn)eI. (3.. lsakson, P., Ultr,}. W. A‘; Vitetta,
`5. Proc. num. Ama’. ti-::'.
`US A. '77. 5-'ll<)AS4.‘r.3 tl9t~‘0l.
`
`
`
`l.i‘orthe:r rnnlecnlazr complexities
`of E-E32 E»
`.§L}‘««region antigens
`
`E’. Tikéinant 8: Eieaginar lvanyi
`
`Division of Genetics. Antoni van Leeuwenl1oel«; Huis, The
`Netherlands Cancer Institute, Amsterdam, The l‘~lctherlands
`
`The K and 1) regions of the H1»? gene complex are higtilgi
`polymorphic” and control cell-surface structures involveerl in
`the }§»~2 restriction of cytotoxic 'l‘ lymphocytes“. Originally if
`was assumed that each of these regions controlled only one type
`
`of antigenic molecule, 1-E---2K and R---21), respectivelym”. "'1.‘l?é<:‘-
`finding that the D region controls E-‘i-2E.”“” and H-QM“, as well
`as H-~21), inolecnles suggests that the system of I-il——2 antigens $3
`more coniplicated. We demonstrate here previously unknown
`
`© 198! Macmillan Journ:\l:~‘
`
`
`
`
`PHIGENIX
`
`Exhibit 1009-04
`
`
`
`"P
`
`so
`
` 3.
`
`I/rr 5.7 x l(t'”M
`
`'""“"'.""""""""'""T“".."'“""M"W"“w""“"w”"
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`10''
`m—'-
`Antibody conszentrution (M)
`
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`
`10'
`
`inhibition of imrriunotoxin activity by competition with
`Fig. 2
`unconjugated antibody.
`Increasing amounts of unconjugated
`antibody were added to a constant concentration of immunotoxin
`(5.7 X 10"‘) M) which in standard tests gave l5% viability (dashed
`line). A threefold increase of unconjugated antibody (1.8 X
`ltlmg M) was needed to obtain 95% viability of target cells.
`
`on Thy l,2~positive cells (PIES) could be obtained with a
`threefold molar excess of unconjugated antibody, suggesting
`that the binding capacity of immunotoxin is similar to that of
`unmodified antibodies (Fig. 2). Filter-sterilized conjugates
`stored in phosphate—bufiered saline at ~-~20 °C for more than 6
`months did not precipitate on thawing nor did they lose their
`immunotoxin activity.
`In a lirst in vivo trial, WI-EH1-7 cells were injected intraperi—
`toneally (i.p.) into BALB/c mice and each mouse was treated
`l h later with an i.p. immunotoxin injection of 30 ng conjugated
`A—chain, repeated 7 days later. This dose corresponded to about
`l/10th of the LD5,, of free A—chain, which was 20 mg per kg
`(LD50 of ricin was about 3,000 times lower, 7.5 pg per kg). With
`6x105 cells, 9 of
`i0 control animals died within 39 days.
`Treatment with immunotoxin led to a significantly prolonged
`survival, as verified with the method of probitsg. 50% survival
`was attained in the control group after 35 days and in the treated
`group after 44 days (Fig. 31)). l-Iowever, with 2.4 X 10“ cells no
`significantly prolonged survival could be obtained (Fig. 3(1).
`
`3..Survival
`
`9
`
`#10
`
`20
`30
`40
`90
`50
`Days after tumour cell injection
`
`100
`
`immunotoxin activity. Groups of
`for
`test
`In vivo
`Fig. 3
`10 BALB/c mice (Bomlioltgaard) were injected ip. with 2.4 X105
`WEHL7 cells (a) or fix l0” WEHI-7 cells (b). After iii and 7
`days, filling of A—chain (bound to anti-Thy 12 antibody) were
`injected i.p. Survival was followed for 100 days.
`
`002841836/8 l /1 10146-—04SOl.()0