I
`
`Tu~: JouRNAL ot' Bmt.OGtCAL CtmMtSTHY
`@ 1985 by The American Society of Biological Chemists, Inc.
`
`VoL 260, No. 22, Issue of October f>, pp. 1203fJ-12041, !985
`Printed in U.S.A.
`
`Purified Immunotoxins That Are Reactive with Human Lymphoid
`Cells
`MONOCLONAL ANTIBODIES CONJUGATED TO THE RIBOSOME-INACTIVATING PROTEINS GELONIN
`AND THE POKEWEED ANTIVIRAL PROTEINS'
`
`(Heceived for publication, February 25, 1985)
`
`John M. Lambert, Peter D. Senter, Annie Yau-Youngt, Walter A. Blattler, and
`VictorS. Goldmacher
`From the Dana-Farber Cancer Institute, Divt~'lion of Tumor Immunology, Boston, Massachusells 02115
`
`Seven different monoclonal antibodies of the lgG
`class that are reactive with four different antigens on
`human lymphoid cells were utilized to form immuno(cid:173)
`toxins with the ribosome-inactivating proteins gelonin
`and the three known pokeweed antiviral proteins.
`Thirteen different immunotoxin combinations were
`prepared. The ribosome-inactivating proteins were
`modified with 2-iminothiolane. The sulfhydryl groups
`so introduced were reacted with maleimido groups or
`with dithiopyridyl groups that had been introduced
`into the antibodies. The toxin-antibody conjugates so
`formed were purified by affinity chromatography on
`protein A-Sepharose CL-4B, ion exchange chromatog(cid:173)
`raphy, and by gel filtration and were characterized by
`polyacrylamide-dodecyl sulfate gel electrophoresis.
`The purified immunotoxins were free of nonconjugated
`monomeric proteins and aggregates of very high mo(cid:173)
`lecular weight. All the immunotoxins showed the spe(cid:173)
`cific binding of the component antibody as measured
`by indirect immunofluorescence binding assays. The
`activities of the ribosome-inactivating proteins were
`unaffected by conjugation where the cross-link to the
`antibody contained a disulfide bond and when assayed
`after reductive cleavage of the linker. Disulfide-linked
`immunotoxins with six of the antibodies were highly
`cytotoxic for the target cells. However, immunotoxins
`containing an anti-Bl antibody showed no cytotoxic(cid:173)
`ity.
`
`The possible use of antibodies to target pharmacologic
`agents, such as toxins, was first proposed by Ehrlich (1).
`Research to exploit this idea has developed rapidly in the last
`decade, owing much to the ability to produce pure highly
`specific monoclonal antibodies using the hybridoma technol(cid:173)
`ogy (2). Recently, monoclonal antibodies have been developed
`that recognize tumor-associated antigens (3, 4, 11, 12), and it
`is the hope that such antibodies can be exploited to deliver
`toxic agents to particular types of tumor cells in order to kill
`them selectively. The ribosome-inactivating proteins (5, 6)
`seem to be ideal toxic agents for this purpose. Most effort has
`been directed toward using ricin (extracted from castor beans,
`Ricinus communis) which consists of two nonidentical sub-
`
`*This work was supported by a grant from ImmunoGen Inc. The
`costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby
`marked "advertL~ement" in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`:j: Present address: Liposome 1'echnolO{,')' Inc., 1050 Hamilton
`Court, Menlo Park, CA 94025.
`
`units (A- and B-chains) that are joined by a disulfide bond
`(6). The B-chain has the property of binding to cell-surface
`carbohydrates and promotes the uptake of the A -chain into
`cells (6). Entry of the A-chain into the cytoplasm of a cell
`then results in the death of the cell by catalytic inactivation
`of its ribosomes. Immunotoxins have been made by conjugat(cid:173)
`ing intact ricin to antibodies (7-9). Such immunotoxins ex(cid:173)
`hibit specific toxicity only in the presence of lactose which at
`high concentration competes with the cell surface carbohy(cid:173)
`drates for the ricin B-chain binding site(s}. In vivo, these
`immunotoxins are expected to be nonspecifically toxic, as is
`ricin itself, and are, therefore, unlikely to be of therapeutic
`value, although they may have limited use in the in vitro
`treatment of bone marrow for transplantation (9, 10),
`There is a class of ribosome-inactivating proteins that have
`properties and characteristics similar to those of ricin A-chain
`alone (5). Gelonin (20) and the three known pokeweed anti(cid:173)
`viral proteins (21} are examples of such proteins. They are
`basic proteins, of M. about 30,000 (5). These proteins have
`several advantages over ricin A-chain in the preparation of
`immunotoxins: they are extremely stable proteins, they do
`not bind to cells and so are nontoxic to intact cells (except at
`very high concentrations), and they are safe to purify and
`manipulate in the laboratory without the extreme precautions
`necessary for work with ricin (5), These proteins are good
`candidates for the preparation of immunotoxins since, at least
`in principle, such immunotoxins will only bind to the cells
`selected by the antibody. Immunotoxins have been made using
`gelonin and PAP, 1 and in general they showed specific cyto(cid:173)
`toxicity similar to immunotoxins prepared with ricin A-chain
`(13-19, 22-25).
`There is an important caveat in the interpretation of the
`experiments that have been reported using immunotoxins
`made with ricin A-chain, gelonin, or PAP. There is not a
`single example of an immunotoxin that was completely puri(cid:173)
`fied from nonconjugated antibody, This is an important ob(cid:173)
`stacle to the proper interpretation of these reports and ham(cid:173)
`pers understanding of the mechanisms involved in the cyto(cid:173)
`toxicity of such immunotoxins. We describe here the prepa(cid:173)
`ration of highly purified immunotoxin conjugates using seven
`different monoclonal antibodies and using the ribosome-in-
`
`1 The abbreviations used are: PAP, pokeweed antiviral protein;
`PAP II, pokeweed antiviral protein type II; PAP-S, pokeweed anti(cid:173)
`viral protein from seeds; SPDP, n-succinimidyl 3-(2-pyridyldi(cid:173)
`thio)propionate; SMCC, succinimidyl 4-(N-maleimidomethyl)cyclo(cid:173)
`hexane·l-carboxylate; bis-tris, 2- [bis(2- hydroxyethyl)amino-2-(hy(cid:173)
`droxymethyl)-propane-1 ,3-diol; HEPES, 4-(2- hydroxyethyl) -1-piper·
`azineethanesulfonic acid; CALLA, common acute lymphoblastic leu(cid:173)
`kemia antigen; RPMI, Roswell Park Memorial Institute.
`
`12035
`
`IMMUNOGEN 2098, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`12036
`
`Purified I mmunotoxins
`
`activating proteins gelonin, PAP, PAP II, and PAP-S. The
`principle of the purification methods should be generally
`applicable. The immunotoxins reported here were prepared
`both with a cleavable linker containing a disulfide bond and
`with a noncleavable linker, in order to evaluate the impor(cid:173)
`tance of release of the toxic agent from the antibody. All the
`immunotoxins were analyzed for their ribosome-inactivating
`capacity, their ability to bind to cell-surface antigens, and
`their in vitro cytotoxic potency toward lymphoid cell lines.
`
`l':XPE:RIMENTAL PROCEDURES AND RESULTS2
`
`DISCUSSION
`
`We have developed methods for the preparation of purified
`immunotoxin conjugates in high yield, utilizing the techniques
`of affinity chromatography, gel filtration, and ion exchange
`chromatography with buffers of carefully defined composi(cid:173)
`tion. Thirteen different immunotoxin conjugates were made
`using seven different monoclonal antibodies and four different
`ribosome-inactivating proteins. The immunotoxin prepara(cid:173)
`tions contain no nonconjugated antibody, no nonconjugated
`ribosome-inactivating proteins, and no aggregates of very high
`molecular weight. These purified and well defined immuno(cid:173)
`toxins made it possible for the first time to perform quanti(cid:173)
`tative binding studies and to perform cytotoxicity tests with(cid:173)
`out fear of the effect of competition by nonconjugated anti(cid:173)
`body on the experimental result, for example, by blocking
`antigens or saturating the internalization pathways. Also, the
`purity of the immunotoxin preparations permitted a careful
`comparison to be made of the biological activities of the
`component proteins with their nonconjugated counterparts.
`Gelonin and the pokeweed antiviral proteins were not af(cid:173)
`fected by modification with 2-iminothiolane in their ability
`to inhibit protein synthesis. The ability of the modified toxins
`to inactivate ribosomes in a cell-free system of protein syn(cid:173)
`thesis was indistinguishable from that of the native proteins.
`This is in contrast with the results of earlier work using N(cid:173)
`succinimidyl 3-(2-pyridyldithio)propionate to modify gelonin
`(22); the gelonin was inactivated by about 90%. It is possible
`that 2-iminothiolane and N-succinimidyl 3-(2-pyridyldi(cid:173)
`thio)propionate each react preferentially with different amino
`groups of the gelonin molecule. Another factor is the preser(cid:173)
`vation of the positive charge at amino groups upon reaction
`with 2-iminothiolane that may account for this difference.
`Our results suggest that 2-iminothiolane is the reagent of
`choice for modifying ribosome-inactivating proteins, in order
`to introduce sulfhydryl groups while preserving their toxic
`activity.
`When the modified toxins were covalently linked to an
`antibody, the ribosome-inactivating activity of the toxins was
`reduced by about 70%. When the covalent link between the
`toxin and the antibody included a disulfide bond, then the
`full ribosome-inactivating activity as measured in a cell-free
`system could be restored by reductive cleavage of the linker.
`This may account, in part, for the observation that the im(cid:173)
`munotoxins J5-gelonin and J5-PAP-S exhibited greater cy(cid:173)
`totoxicity (about 10"-fold) on cultured CALLA-positive cell
`
`2 Portions of this paper (including "Experimental Procedures,"
`"Results," Figs. 1 ~5, and Table 1) are presented in miniprint. at the
`end of this paper. Miniprint is easily read with the aid of a standard
`magnifying glass. Full size photocopies are available from the Journal
`of Biological Chemistry, 9650 Hockville Pike, Bethesda, MD 20814.
`Request Document No. 85M-546, cite the authors, and include a
`check or money order for $9.20 per set of photocopies. Full size
`photocopies are also included in the microfilm edition of the Journal
`t.hat is available from Waverly Press.
`
`lines when the linker included a disulfide bond than when the
`linker was noncleavable. There is one claim in the literature
`that immunotoxins containing pokeweed antiviral proteins
`are more cytotoxic when linked by a noncleavable linker than
`when linked by a disulfide linker (25). However, we could not
`confirm this result, and our observations with purified im(cid:173)
`munotoxins were consistent with previous findings with im(cid:173)
`munotoxins containing ricin A-chain made with cleavable and
`noncleavable linkers (17). A cleavable linkage between the
`toxin and the antibody presumably allows the toxin to escape
`more easily into the cytoplasm from the membrane-bound
`antibody/antigen complex (48).
`Binding studies showed that the immunotoxins made with
`all seven antibodies showed specific binding to antigen-bear(cid:173)
`ing cells. Cytotoxicity experiments in vitro showed that im(cid:173)
`munotoxins made with six of the seven antibodies showed
`considerable enhancement of the toxic effect of the ribosome(cid:173)
`inactivating proteins, with ID60 concentrations that were
`about 104-fold lower than that of the native ribosome-inacti(cid:173)
`vating protein. The enhanced toxicity was completely specific
`for cells bearing the cognate antigen. lmmunotoxins made
`with gelonin or with the pokeweed antiviral proteins gave
`qualitatively similar results. However, none of the immune(cid:173)
`toxins made with anti-Bl showed any more toxicity than that
`exhibited by the native ribosome-inactivating protein, even
`at concentrations that were near the saturation of antibody/
`antigen binding. This result was obtained on several Bl
`antigen-positive cell lines, including some that were also
`positive for CALLA and for Ia antigens, and which were
`sensitive to immunotoxins made with the antibodies J5 or I-
`2, respectively.
`Receptor-mediated endocytosis of an antigen/immunotoxin
`complex may be essential for cytotoxicity. This has been
`suggested on the basis of experiments with lysosomotropic
`agents which raise the pH of intracellular acidic vesicles (51)
`and which increase the potency of immunotoxins (52, 53).
`There is some evidence to suggest that the Tll surface antigen
`(29) and the CALLA (49) can be internalized, and perhaps
`these antigens utilize the mechanisms involving coated pits,
`coated vesicles, and endosomes (48). Our preliminary results
`suggest that the Ia antigens can also be internalized carrying
`the I-2 antibody. However, the current evidence suggests that
`the Bl antigen (50) remains firmly on the cell surface and
`shows no tendency to be internalized. 3 Thus, the Bl antigen
`may be excluded from the coated pits involved in receptor(cid:173)
`mediated endocytosis (48). This suggests a possible explana(cid:173)
`tion for the lack of cytotoxicity of immunotoxins made with
`anti-Bl; that is, that the Bl antigen does not transport the
`immunotoxin complex inside the cell. Further evidence will
`be required to assess the relationship between endocytosis
`and the cytotoxicity of the immunotoxins made with ribo(cid:173)
`some-inactivating proteins. However, it is clear from our
`present results that the properties of the target antigen require
`careful consideration when designing immunotoxin conju(cid:173)
`gates.
`The availability of pure immunotoxin conjugates will allow
`us to examine in greater detail than hitherto the effect of
`various parameters on the cytotoxicity exhibited by the im(cid:173)
`munotoxins. For example, the antibody-binding affinity of
`the immunotoxins, the number of cell-surface antigens capa(cid:173)
`ble of binding the immunotoxins, and the properties of differ(cid:173)
`ent antigens once bound by an immunotoxin such as in
`receptor-mediated endocytosis may all influence the degree of
`cytotoxicity of the immunotoxins. We are now employing
`
`3 L. M. Nadler, personal communication.
`
`IMMUNOGEN 2098, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Purified I mmunotoxins
`
`12037
`
`several different in vitro cytotoxicity assays, including those
`that directly measure cell survival, to investigate these param(cid:173)
`eters and to study potentiators of cytotoxicity such as ade(cid:173)
`novirus (54) and lysosomotropic agents (53). These experi(cid:173)
`ments may help to determine how to improve the efficacy of
`the immunotoxins. Also, little is known about the biological
`properties of immunotoxins in vivo. Purified conjugates using
`different anti-Tll antibodies, three of which are described
`here, may be useful biological reagents for this purpose since
`the antibodies react with a surface antigen found on T cells
`of various species of monkey that is analogous to the human
`Tll surface antigen (34).
`
`Aclmowledgments-We wish to thank Christina Doyle, Susan Bro·
`deur, Nancy Tinnel, and Jean Anderson for skilled technical work
`and Diana Sam for excellent typing.
`
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`
`Continued on next page.
`
`IMMUNOGEN 2098, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

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`~£!
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`
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`
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`'!l!l!~
`
`~!m¥~~[
`
`2!~ ~~~~
`
`IMMUNOGEN 2098, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`~ "" 0 w
`
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`J
`i1?i~ ~ 2.[
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`[~~~~~
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`d .. dH
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`
`IMMUNOGEN 2098, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Purified I mmunotoxins
`
`(a )
`
`(b)
`
`( c )
`
`12040
`
`( a )
`
`E
`c
`~ 0.4
`N
`
`w
`u
`z
`<:(
`al
`a::
`0
`(f)
`al
`<:(
`
`r0
`
`' 0
`><
`
`0.2
`
`0
`
`\
`
`60
`
`'
`80
`( b ) FRACTION (3m L)
`
`100
`
`120
`
`'
`
`FRACTION NUMBER
`75 80 84 88 92 94 96 98 100102 104
`
`-~
`
`Top
`
`22 I
`190
`160
`
`~
`
`~
`
`93
`
`53
`
`30
`
`Figure 1. Gel filtrati<>n of A JS/golonln conjugation reaction 11ixture after
`purification on n protein A colut~n, no>J andyHo of the elution profile by
`polyacryU.otl.do/dod~eyl aulhtc gol clcctrophorcoia.
`JS and gcloola wore conjugAted u
`duerlb<>d In tlu! Hethodo ocction to font conjugatoa contsln1"8 disulfide links nnd tl•cn
`non-conjugated gelonJ.n wu r.,.ovcd fr<a the 11ixturc by purificoUO<l through protein A
`colUDna so deocribod in Hcthodo. Panel (4), clut1on profile of the ponlnUy purified
`la::mnotoxin llizturc (H mg prot<>in in 0 ml) outw.ttcd to gel HltUt!on thr<mgh a colutul
`(99 em x 2.6 c,.) of Scphs.cryl S-300 equilibrated with 10 <t!! Kl't buffer, pR 7,0,
`contalniu,g HnCl (H~ llfi'l), Po.nel (b), 5-lOl (ll/V) ~olyacryLo.<Udc/dodccyl sulfate gal of
`aa11plea talu!n frO<O !roctioM of the elution pr<>filo aho1111 in panel (a). The gol vao run
`undor non-rnduciu,g conditions oo described in Hcthodo, and tho calibration of !!_r >149 fr<>:>
`the ..,bUity of proteina of l«lovn !!_r, Ig(; (160,000), phoophorylaae b (93,000), glut.....,to
`dehydrogenase (53,000) and carbonic ~~nhydraoc (30,000).
`
`2 3 4
`
`5 6 7 8
`
`Top
`
`221
`190
`160
`
`93
`
`"'
`'
`0
`
`X
`
`:i
`
`12
`
`figure 2. Polyae<ylatoide/dodccyl oulhtc gel analyoio of purHicd hmunotoxins.
`Panel (a), 5-lOl (v/v) polyacryla<rldc gel under non-rcduclng conditions of J}/gelonin
`conjU$at1on containing a dioulfido link:
`lane 1, antibody J5; l.nno 2, J5/gclonin
`conju,gation <>i~turc aftH purification through protein A-Sepharoac CL-4B; lano 3,
`J5-golottln conjugate >fter funhor purificnUon by C~rccllulose chr(K:latog<a)>hy; lane o,
`J5-gelonln conju,g~te aftcr gel filtration through Sopho.cryl S-300. Panel (b), 5-10% (~/v)
`p<>ly~cr~lnl>ido gel under non-rcdueing condiUot\0 of J5/gelonln conjugntion eontninlu,g a
`thioether link:
`l.nne ~. JS/gclonin conjugation rcact1on .Uxture bcforc any purl!!cation;
`lane />, J5/gel.onin conjugation l>ixture dtor pu<ificatlon through protein A-Sepharo•e
`CL-4B; lane ), J5-gclon.\n conjugate after further purification by Cu-cellulooo
`chr(K:lntogrophy; lane 8, J5-gclonin conjugate ~fter gel filtration througlo Sephacryl s-JOO.
`hncl (c), 11)-ZO% (u/v) polyncryla,.ide gel under reducing conditions of J5-gelonin
`conjugatea:
`)ane 9, sanple Identical to lone 8; bnc lO, Mnplc identical to lsne 4; laM
`11, golonln; !nne 12, J5 ontibcdy. Tho callbrulon of !!r woo froo. the oobllity of lgG
`(160,000), pho•pl\oryln•c b (9),000), b-ovine Mro:>. albunin (68,000), aldolnoe (40,000),
`corbonic anl,ydrooe (30,000) ~nd oytocloro"" C (11,700).
`
`w
`
`~.
`
`~
`0
`
`~
`
`~ 0
`~ 0
`0
`0
`~
`
`•oo
`
`50
`
`/•--.---·
`:-a--Q--•
`
`0
`
`100
`
`(d)/'__....
`.~
`
`50 1-0--o
`t=-·-·--·
`
`10
`
`30
`
`50
`
`"'--,~oc-"-3~o~~~5o~
`
`"' u
`" w ~
`4oo (c) I
`. -
`:I: 0
`0 .
`z .
`"' ~
`,_ E
`200· ;;_._J
`fi-;-a--'
`
`0 u
`
`"'
`
`TIME (min)
`
`Inh1bition of ptotein synthcoia in a eoll-froc oyot""' fr"" rabbit
`Figure 3,
`reticulocytco by gelonin, PAl'-S or 1w:l.unotodna. Protein oynthcoio "'"" ""aourcd by tho
`Incorporation of 13HJ-louolno into protein precipitable by triohloro<oeetic acid an
`deacribcd In Methodo. The iueorporntJ.on of udioaetivlty in the controb within 50 .uu wu
`In the r~nse no,ooo to 600,000 cp~ por .. aay (vol,.,., 27 )11) In different expcrl,.cnta with
`different blltcheo of lyoato ond of u!UlA., and uu counted with on efUcieney of 21)-Ht
`(Packard Tti~Carb liodol 4530 scintillntion counter).
`llhCTe indicated, prc-treatfl(tnt of
`oo<>.pleo llith dithloe<ythritol (20 I<M) \148 for 30 <>in at 300 C. Panel (a), (0) control
`ouny; (II, D ,()) noooyo th<ot contained 20 pg of purified gdonln, 20 PS of gclonin
`pre-treated vtth dithioerythritol and 20 pg of golonin t><>dificd uith 7.-io.inotMol.nno and
`pre-treated uith dithioerythritol, roopeetively.
`(A ,6) aaoayo th<tt contained on mwunt
`of the J5-gol;onin conjugate vith a non-cl.eavoble link tluo.t contained 20 pg of gclonin,
`without (A), and "lth (A), prc-trent,..ot of thO: conjugate with dlth!oHythrltol. Pond
`(b), (0) control """"Y; (a) assay tlwt contained ,20 pg of gelonin: (A,ll.) aoooyo thAt
`contained an nowunt of the J5-gelonin conjusotn 1.1!th a disulfide Unk that contained 20 pg
`of gelonin, vlthout (.6,), nod v1th (ll.), pre-trcat.,nt llith ditbioerythritol. Panel (c),
`(e) control aooay; <•l aoooy that contained 20 pg of gelonin; (A,.a) oasnyo tbs.t
`oontoined an ocount of the outi Bl-gclonin conjugate with a dhulflde link that contni01ed
`20 pg of gclon.J.n, vithout (A), <1nd vith (Ll), pra-troatnent with dith!oorythritol.
`fanel
`(d), (0) control 8Uay; (0) asooy that contained 20 pg of Plo.P-S; (D,•) ooooyo thst
`contained an o!lount of the J5-?!J'-S conjuaatc with o disulfide link that contained 20 pg of
`PllP-S, without (0), •nd vlth (D), prc-treat<>Onl uith dithioerythritol.
`
`IMMUNOGEN 2098, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Purified I mmunotoxins
`
`12041
`
`Til~ C~TOTOXIC!TY OF GELON!N /iliO lTS OlSUI,f'lOE \,JIIK£0 H~UNOTOX!N
`
`COSJUGAnS TOIIARlJS l.YH~IIOBU.STOlD C~LL LlN~S•
`
`Gdontn
`
`'"'
`
`000
`
`>).00
`
`1000
`
`''00
`
`,,
`
`>OO
`
`J>f8cJ.oMn
`
`0.4(+)
`
`O,OJ.(<·)
`
`0.08(+)
`
`0,2{+)
`
`>100(-)
`
`ro~o (n.'Q
`
`l-2/zclonin
`
`O.Ol(+)
`
`o.OOJ.(+l
`
`0.03(+)
`
`>100(-)
`
`JlO/gdonln
`
`0.04(+)
`
`>)00(-)
`
`il•\ti-TUtA/gclonln
`
`> 100 (-)
`
`~nt 1-T lljg/ gclonin
`
`> 100 (-)
`
`antl-1llJ.cfgclonl»
`
`> 100 (-)
`
`8.0(+)
`
`0.3(+)
`
`2,0(+)
`
`antl-Bl/gdonln
`
`'100(+)
`
`'100(<·)
`
`>)00(-)
`
`• C)·totodcity ~ao ccoourcd ~ftcr 3 dnyo ""I'"""'" of th" c.dlo to the gclon!n or
`l=unotoxtn, cca"urln& {31!)-thycldinc incMpor~tion i~tto D~A during a 2 h pulse os an
`1ndcx of cytotoxlcHy. The lD50 vol<•~• (ni-l) arc the ~c>n <>f ~t lcaot three Jndcpcn~cnt
`cxpcricc•H•, sud\» thonc ;ho~n In t'igurc ), "here each point is the <:.oon oi trlpHcotc
`oJctcrOI!Mt!on•. Tho lD50 of J)-r,clonln on iiolt>-6 cello uat 0,02 + 0.01 n.'! (n•S). The
`tht• relcv,,nt >l>tlgcn Ol\ the cell "urfaoc, 'to wl,!ch the 1>.utlculor
`prcocnoc or ahocncc M
`,,.\tibod)' Wo\Old ~!nd, io 1ndic.1tcd by(+) or(··), rc;pcct!volr.
`
`·----~
`
`(b)
`
`•
`
`I
`I
`
`0
`
`•
`
`•
`I
`
`/'
`
`(c) t' ( d )
`
`,..
`•
`
`I
`
`0
`
`~
`(h> .......
`•
`I
`
`100
`
`50
`
`~~
`
`X 0 . " ~r z
`
`"0 ou
`z" 20
`1- a!' 100
`
`• " 0 " " 0
`
`u
`z
`
`50
`
`:~
`\ -
`
`(d)
`
`PROTEIN CONCENTRATION {M)
`
`Ylgurc S, Cytotoxicity of !=.unotoxins and rll>oo"""-lnoctivotl<>s protolno on hu:"m
`lya.phol~ cell lineo, Tloo lnhiblUOll of DliA oyotheoio oo !lcaourcd hy tho inhibHion of the
`lncorporatloo of [lH]-th)'l'lidlne rcl<otivc to control-s """ uocd oo a <>MOure of
`cytotoxicity •• deocribod ln ltcthodo. Panel, (a), Noho-6 cel.l.o grovo. for 46 h in tho
`prceonoc of gdonin (IB), Jl--gcl-onln oontaini<>g n thlocthcr link (0) nnd JS-gcJ:onln
`containlns n dioulfidc Hnlt (0), Pnncl (h), )!.o.Jn .. 6 cello gro"" in the preoencc of
`I-1-gclonin for 24 h (A), 46 h (A) ond 72 h (0): cell.n "ere nloo gro>ln for 72 h in the
`prcocncc of &Clonln (IQ). Panel (c), 1!nllr6 cello gro"'' J.n the proscncc of ricin for l h
`(8), 31, (L'I), 5 h (A) and 2/o h (0), Pnncl'(d), BJAB cello gro"" for n l• in tho
`prcocncc of gelonln (Ill), nnti-Bl-gelonin (D.) nnd JS•gclonin (0),
`
`(a )
`
`eo
`
`/
`
`0
`
`I
`40 J
`
`6
`
`;
`
`w
`u
`z-
`w~
`u"
`~-
`wz
`""
`0 o>
`~· "" "
`w"
`::ro
`"" eo
`•• ~~
`w
`"
`
`I
`•
`)
`1•1 'l "f
`I'(
`J
`40 ~
`.l
`'}
`~~ L
`
`I o·<O
`
`10-6 10·10
`
`_,
`10-$ 10
`
`10 -· 1o·•o
`
`10- 6
`
`ANT 180DY CONCENTRATION IMI
`
`figure<.. Co~p.u!son of the binding of 1'"'"""'""1"' wah that of Mt!vo antlbodr
`~:caourcd by Indirect l=unofluoro"ocncc. The rclotlvo iluorcoconcc In arbitrary unlto "·'"
`taKen no the ~ld-polnt of (luorcoccncc hl$lOgr.,M dotcr<>lncd uolng ,, fluorcoccncc-actlvatcd
`ccH oortcr, and"·'~ plotted ogainot antll>:>oJr concentration of the Mtlve ant!l>o~y (0), or
`Of tho l=unotox!n (0). Pone\ (o), JS and J'>-gclo\\ln cO\>t~!nlng tho Moulfldc lin~.
`?,;nd (b), J'> and JS-gclonln <ontainlng the thlo...ther H11L All of the re<>olol<>g pone!• (c
`to h) >~ere of oonjugatco contoin!ng dJsulf!do l!nko. Pond (c), JS and J'l-?AP-s. hnel
`(d), JS o>~d J'>-gclonln containing c•actly l Mlocul-co of gclonln por JS <>Oloeulc. Pnnel
`(o), anti-Bland anti-Bl-gclonln. fond(!), ant1-Tll 1A ~nd anU--rll}A-geloMn. Panel
`(g), ontl-nllB and anti·Tll)g-&clonln. Panol (h), ont!-Tlltc and
`ont!-T llJ.c-&clonln.
`
`I
`"
`
`IMMUNOGEN 2098, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676