IM14 I· I :1:1'i / AA/ 41Uf•·Ul°1i :!$O:!JMl/ O
`TKANS~l.ANTATION
`( '11pyrii:h1 c:• l~!\l'i h\' Tfw \\ 'illiam< & Wilkin< ('o
`MONOCLONAL ANTIBODY THERAPY
`
`Vol.41. No. a
`/'rmto•d 111 l l. S. A .
`
`ANTl - II>I OTYPIC ANO NON-ANTl -I OIOTYPIC ANTIBODIES TO OKT:~ ARISINC: DESPITE 1NTEN3 E
`I MM UN OSUPPRESSION 1
`
`GREGORY J. ,JAFFERS, TH OMAS C. F ULLER, A. BENEDICT COSIMI, PAU L S. RU SSELL,
`HENRY J . WINN , ANO ROBERT B. C or.vrN'.!
`
`Tronspla11tati1111 -lm1111mri/11;:y 11ncl lmm11n11path11/11;:y I /nits, lJepnrlm1•11/,, 11/ S11r;:1•ry n11d Path11/11~\'. Ma.<.<achusells G1•rwrnl H11spital
`and Harvard Ml!d1cal Schwl. H11.,t1m, Ma.,,;arhu.w?ll.~ 02114
`
`nomenon has acquired new relevance with the application of
`mu rine hybridom11-derived antibodies to clinical medicine. De(cid:173)
`spite some init inl optimism, the injection of the1;e murine
`protein1; into man. a1; expected, reh'ularly provokes an immune
`response hy the host ( / - / / ) .
`We have had extensive experience wit h the use of one mono(cid:173)
`clonal ant ibody, OKT:\ ClgG" .. ~ . BALH/c), used as an adjuvant
`to azathioprine and prednisone for treatment of acute renal
`allograft rejection ( I , 2, //).This antibody, reactive with an
`invariant component (T;W of t he T cell antigen receptor com(cid:173)
`plex ( / :l l, lrns proved to he dramatically effective in reversing
`acute cPllular rejection when ndministered at doses of 1- !i mg/
`day ovt'r I ll 10 days. This report describes in detail the speci(cid:173)
`ficity of the antibody response to OKT:l in 21 renal allograft
`rel·ipients. and the implications for therapy. A preliminary
`report of the lirst 11 patient s has been presented (6, 7).
`
`The freque ncy, timing, and specificity of the humora l
`a ntibody response to a murine monoclonal a ntibod y
`(0KT3 , lgGi.) were measured in 21 consecutive re na l
`a llograft recipie nts. These patie nts received i.v. OKT3 ,
`1- 5 mg/day for J 0- 20 days as treatment for acute graft
`rejection . Maintena nce immunosuppression consisted of
`azathioprine a nd corticosteroids. Using three different
`assays, an antibody response was de tecte d in 75% of the
`20 patients with adequate samples. The ELISA assay of
`the overall JgM and lgG reactiv ity to OKT3 r evealed
`that lgM anti-OK1'3 appeared in 65% and IgG anti(cid:173)
`OKT3 in 50% of the patients, reaching a peak 20- 33
`days afte r the last dose of OKT3. The l gM preceede d the
`l gG in most cases (P<0.02) and in 8 cases was detected
`during therapy. One patient ha d high levels of IgM ant i(cid:173)
`OKT3 before therapy, yet responded no rma lly to OKT3.
`Inte rfe re nce w ith the therapeutic effectiveness was ev(cid:173)
`ident in one patie nt who developed IgG antibodies dur(cid:173)
`ing therapy. His serum bloc ked the binding of F-OKT3
`to normal lymp hocytes in the presence of norm a l
`BALB/c serum . The blocking assay, done by flow cytom(cid:173)
`etry, measured anti-idiotypic (Id) reactivity since t he
`sera did not affect the binding of OKTS (another l gG 2_)
`or anti-Le u '& (another a nti-T3), und the blocking acti v(cid:173)
`ity r emaine d a fte r a ffini ty absorptio n with normal
`mouse IgG . Using this assay, 60"/,. of the pate ints made
`an a nti-Id response. One made only anti-Id, and sever a l
`had anti-Id at times when othe r reactivities were unde(cid:173)
`tecta ble. Antibodies to non-idiotypic, presumably iso(cid:173)
`t ypic, det e rmina nts represented on OKT8 occurred in
`only 44 %, while othe r reactivity (0KT4; lgG2bK) was
`less common (12 %) a nd weake r .
`While no adverse a lle rgic reactions occurred in t his
`~roup of patie nts, t he a nit-Id a ntibodies, whic h are a
`prominent feature of the immune response to this a nd
`probably other monoclona l a ntibodies, can block the ir
`the rapeutic e ffectiveness and can a rise despite intense
`immunosuppression . This response may r equire the use
`of different idiotypes for prolon~ed or r epeated courses
`of t he ra py a nd may be the major obstacle to the use of
`huma n monoclona l ant ibodies.
`
`MATEHIALS AN D METHODS
`Pat ient.~. Thl• 1 1 con:=;ecut ivr OKT:l -treated patients studied
`were initially treated with prednisone And azathioprine after
`rt•ct'iving a cnclavl'r clonor renal alloi:raft (I. 2). They were given
`OKT :{ <Ort ho Pharmaceuticals, Raritan, N.J) when they had
`their first cpi!mde nf renal allograft rejection. The ant ihody was
`given once dnily as a 1- f>-mh' i.v. bolus. No patient manifested
`a skin react ion to intradermal OKT:I (O. l µg) prior to t reatment.
`In an attempt to modify thl' immune response to OKT3, 6
`patients ahm rerrived rydophosphamide (patients N()s. IO and
`17-~ll .
`.'frrum snmpll's. Hloo<l samples were collected in acid-cit rate(cid:173)
`dextrose (ACD) at lt>ast twicE' weekly beginninl{ at the time of
`transplantation and continuing fo r up to ten months after
`OKT:I therapy. An averagE' of' 19 samples per patient were
`analyzed in :W patients (range l:l- 14). One patient (No.;{) had
`only :1 posttreatment samples (all negAtive) and therefore was
`not induded in further analysis. The samples were centrifuged
`at I !"100 rpm and the plasma was stored at - :lD°C. Peripheral
`hlood lympl,ocytes from these samples were used fo r immuno(cid:173)
`logir moni111rin1'( as described previously (I).
`The parenteral administ ration of li>reil{n serum proteins has
`1~·11zy11w- link1•d immur10.<11rbn11 assay (f;LJSA). This assay,
`het>n known since the early days of scrot herapy to stimulate an
`performed by a sli~ht modiliC'at ion of puhlisbed met hods ( /3),
`imnn1ne response. The typical allergic mani fest at ions include
`was used to cll'tect hoth anti-Id and non-anti-Id anti-OKT:l
`f'l'ver. skin rash. anaphylaxis, and serum sickness. This phe-
`antibodies. Polyvi nyl vinyl plastic microtiter plates (imulon I,
`' This work was supported h~· lJSl'HS (;rant H L-18">41i and by funds
`• Ahhrt>\'ialion,. lht>d: Al'D. al'id 1·itralt• dexJro.-;t>: ('AF,. CHAI.HJ
`pru\'idt•cl hy llw Ori ho l'harma«C'Ut ic-al C'urpurnt ion.
`cxA/ .JJF,: El.ISA. t>niynw·linkl'd 1111munosorhen1 assay: F·. lluores(cid:173)
`: lh•print rl'qllt'"'" ,..f1011lcl ht• adrlres,..ed 111 H.B. Colvin, M .D .. Im·
`t·l'in conjui:at('(I (t'.J:., F-OKT:IJ: Id. idiulype: MFC. median lluorese!'nl'!'
`nn111u11a1holu),[y lJnil . llt'partnwnl of Pmhnlui.ty. Massal'huse11s (;<'11 ·
`<'hanilt'I; l'Bs. phosphau .. hufft•n•d ,.alirw: l'HA. phytolwmai:i:lutinin:
`t•rnl Hos11ital. Hoslon. MA 0:!1 1-1.
`Tl. 1 lw :!II :.l!"> kilodailon surfan· anl ii:C'n of millure T <·ells .
`...... ,
`., ,_
`
`1 of 7
`
`Celltrion, Inc., Exhibit 1027
`
`

`

`May, 19~
`
`JAFFERS ET AL.
`
`Dynatech, Alexandria, VA) were coated with 1 µg/ml of purified
`parenteral grade OKT3, 0.05 M NaHC0:1 buffer (pH 9.6). The
`plates were incubated overnight at 4 °C then washed with phos(cid:173)
`phate-buffered saline containing .05% (v/v) Tween-20 (0.01 Na
`phosphate, 0.14 M NaCl, (pH 7.4) (PBS-Tween). Each serum
`sample was diluted 1:240 in PBS-Tween (5 µl/1.2 ml buffer).
`200 µI of this mixture was added to the wells. The plates were
`incubated for 2 hr at room temperature, washed with PBS(cid:173)
`Tween, and 200 µI of a 1:500 dilution of alkaline-phosphatase(cid:173)
`conjugated goat antihuman IgG (Kirkegaard-Perry, Gaithers(cid:173)
`berg, MD)-or !{Oat antihuman IgM (both heavy chain specific)
`was added at a similar concentration. Anti-IgG with light chain
`reactivity was used in early studies as a screening test. These
`solutions were incubated for 2 hr at room temperature and
`washed again using PBS-Tween. Finally, 200 µl of I mg/ml p(cid:173)
`nitrophenyl phosphate in 10% diethanolamine was added. The
`resultant color change of substrate was read on an ELISA plate
`spectrophotometer (Bio-Tek Instruments) at 40fi nM. Sera
`from 15 healthy volunteers and assays without OKT3, serum,
`or anti-lg were used as controls. All assays were done in
`triplicate. Each daily run was standardized by running aliquots
`of a known post ive control. This serum had a net OD .. i:. (test(cid:173)
`blank) of about 650 and 70 for the lgG and IgM assays,
`respectively. Standardized values for IgG were obtained by
`multiplying the net OD.111. of the test sample by 650/(00,,,, of
`standard) on that day (or 70/0D.11., of standard for lgM). A
`sample was considered positive ifthe corrected OD., .... was 2 SD
`above the mean of the controls (95th percentile) in the case of
`the pretreatment samples, or if the on ....... was more than double
`the pretreatment oo •. ~. in the case of subsequent samples
`(provided the OD,,,., was >100).
`fl HA blast pr<'paration. The preparation of phytohemagglu(cid:173)
`tinin (PHA)-stimulated T cell blasts was accomplished hy
`methods previously described ( /4). Briefly, normal peripheral
`mononuclear cells were isolated on Ficoll-Hypaque gradients
`and stimu!ated with 1 µg of PHA per JO" lymphocytes in 1 ml
`of RPM I 1640 medium containing 5% normal human serum,
`antibiotics, and 5 µM mercaptoethanol. Cells were maintained
`by dilution to 101'/ml in T cell growth factor medium containing
`interleukin-2 about every third day.
`Enhancin~ assay for rnJn-Anti-ld antibodies. Using flow cy(cid:173)
`tometry, non-anti-Id human antibodies that included isotype(cid:173)
`specific reactivity to murine IgG". were detected by the binding
`to OKT8-coatE"d (lgGt.K,CAF,) PHA blasts. Antibodies not
`specific for Id or isotype were similarly measured with OKT4 -
`coated (lgG""A,CAF,) targets. OKT3-coated targets were used
`as a positive cont rol. PHA blasts (which contained both T:3•T4+
`and T:rTs+ cells) were suspended at a concentration of 10°/ml
`in RPM! 1640. Nonfluoresceinated OKT8, OKT4, or OKT3
`(0.5 µg) or PBS was added to each tube containing 50 µl of
`PHA blasts. The suspensions were incubated at 4°C for :JO min
`and washed twice with PBS. 50 µI of test or normal serum was
`added and the mixture incubated and washed as before. Flu(cid:173)
`oresceinated goat antihuman lgG (heavy and light chain reac-
`1ive) was then 11dded and the tubes were incubated and washed
`as before. The median intensity of the positive staining was
`evaluated using the Spectrum Ill flow cytometer (Ortho Diag(cid:173)
`nostics System, Westwood, MA) ( /.'}). The percentage of in(cid:173)
`crease in the median fluorescence channel (MFC, linear units)
`above uncoated blasts (no monoclonal antibody) for each sam(cid:173)
`ple was calculated. This corrects for any contribution by anti·
`T-cell antihodies that may be present in the patient's serum,
`
`sn
`e.g .. anti-HLA. Positive values were defined as 2 SD units
`above the mean of 21 normals.
`Affinity columns. OKT3 and normal mouse IgG (Miles) were
`coupled to CNBr-activated Sepharose 4B (Pharmacia) by
`standard techniques, with approximately 10 mg of protein/ml
`swollen gel. Columns were 12X0.9 cm (mouse lgG) and 4.5x0.9
`cm (0KT3). Fractions were eluted in PBS and then 3M KSCN,
`dialyzed, and reconcentrated to the starting volume by positive
`pressure filtration (Am icon XM-50).
`Anli-idiotype (Id) as.~ay. The basis of this assay is that certain
`anti-Id block the binding of the Id-bearing antibody with an(cid:173)
`tigen. The test was done by flow cytometry using fluorescein(cid:173)
`ated OKT:l (F-OKT3) as the Id and T3+ T cell blasts (or
`normal peripheral blood lymphocytes) as tbe antigen in the
`presence of the test serum. Normal BALB/c mouse serum was
`added to block non-anti-Id antibodies. The controls included
`normal human serum and fluoresceinated OKTS (F-OKT8).
`The amount of F-OKT3 required to saturate PHA blasts or
`peripheral lymphocytes was determined by titration with a
`constant number of cells, plotting the MFC-vs.-concentration
`of F-OKT:l used (Fig. 1). Saturation was achieved at 8 µg/ml
`of F -OKT:l: beyond this point no further shift in the MFC was
`achieved. A point of about 30% saturation was selected (2.5 µg/
`ml) for use in the assays to maximize sensitivity. A similar
`point was determined for F-OKT8.
`The assay was performed by adding 25 µI of test or normal
`serum to 20 µl of BALB/c serum and incubating for 30 min at
`room temperature. Subsaturating amounts of F-OKT3 or F(cid:173)
`OKT8 were added and these were incubated for 30 min on ice
`Normal buffy coat peripheral blood lymphocytes (25 µI), pre·
`viously prepared from ACD blood, were added to each tube
`with further incubation for ao min at 4°C. Residual erythro(cid:173)
`cytes were lysed with NH.Cl and the cells were washed twice
`in PBS. Samples were analyzed using the Spectrum III flow
`cytometer gated on the lymphocyte or lymphoblast cluster. The
`MFC of the posit ive cells above the positive threshold channel
`was taken as the index of staining intensity. Pretreatment
`samples had inhibition of 7±7%. Therefore a decrease in the
`MFC of greater than 20% of the normal serum control, provided
`there was not a concommitant inhibition ofF-OKT8, was taken
`as evidence for the presence of anti-Id antibody. Samples
`
`~
`~
`75
`~ -
`~
`t1
`t3
`~ :::i
`25
`~
`
`50
`
`10 20 40 eo 160 320 640 12eo 2!J60
`DILUTION
`F1<a11u: I. 1'101 of t1uore!>rence intensity (MFC, lineur unils) vs.
`dilution or F -OKT:l on l'HA lym1>hohlasts. A point of uhout :!0%
`~aturntion wns >'eler1ed for thl' anti-Id inhibition assay.
`
`2 of 7
`
`Celltrion, Inc., Exhibit 1027
`
`

`

`574
`
`TRANSPLANTATION
`
`Vol. 41. No . • 5
`
`OKT8 and OKT3 enhancing reactivity were disparate in 2
`patients. The serum from patient No. I reacted with OKT8
`coated, hut not OKT:l coated cells. She had blocking anti-Id
`antibodies and it is possible t hat these displaced the OKT3
`from the target cell, giving a false negative. A similar phenom(cid:173)
`enon may explain the negative OKT3-enhancing results in
`patients Nos. 2 und 17 who also had blocking anti-Id. A i:;econd
`patient !No. 14) had the opposite pattern, reacting with OKT3-
`coated, but not. OKT8-coated cells. The restricted reactivity in
`this case may have been due to anti-Id of the nonblocking type.
`Hloc:kin;: anti-Id assay. None of the pretreatment sera specif(cid:173)
`ically blocked the binding of F-OKT:-1 or F-OKT8 to normal
`lymphocytes (Table I). However, after the course of OKT:l
`therapy. 12 patients (60'};, ) developed antihodiei:; that selectively
`blocked the antigen-binding of F-OKT:l. The sera blocked F(cid:173)
`OKT:l in the presen('e of normal BALH/c serum and did not
`block F-OKT8 or F-anti-Leu 4. arguing that the specificity was
`restricted to idiotypic- and not allotypic or isot.ypic--deter(cid:173)
`minants on OKT:1 IFiJ!. 2).
`
`TAHU: :L Onset of anti -OKT:I tintihodies and effect on OKT:I
`response"
`
`Pa1it·nt
`
`r:LISA -
`OKT:I
`
`Hirn··
`kini:
`
`I~-<;
`
`li:M Anli·
`l<l
`
`Enhnnl'inl(
`
`Response lo
`OKT:I («ells/
`mm3>d
`
`01\T:I 01\TX OK'l't
`
`T:l THTI\
`
`containing no BALB/c serum were analyzed in parallel. F-anti(cid:173)
`Leu 4 <Becton Dickinson Monoclonal Center, Mountain View,
`CA) was used for comparison.
`Statistical analysis. Values given are mean ± SD unless
`otherwise noted. Student's t and Fisher's exact tests were used
`for evaluating statistical significance. An antibody response
`was considered positive if two or more sample values were
`above the defined threshold, either a doubling of the pretreat(cid:173)
`ment value or above the 95th percentile of normal controls.
`
`RESULTS
`
`ELISA: total Anti-OKT3 response. Using the ELISA with
`OKT:l as the antigen, the lgG and lgM antibodies of all
`specilicities to OKT:3 were determined. Except for patient No.
`18, the ELISA reactivity of the patient's pretreatment i:;erum
`was similar to that oft he If> controls. hoth for IgG and for lgM
`(Table l) and the anti-lg with light chain reactivity gave
`essentially the same result as anti-')' chain.
`Patient No. 18 had the highest pretreatment reactivity, which
`was exclusively lgM . This hinding could be completely elimi(cid:173)
`nated by preincubation of the tei:;t serum with BALB/c serum,
`and partially blocked with preincubation in normal goat serum.
`A rheumatoid factor assay with human lgG was within normal
`limits (<60 l.lJ.). The level of the lgM antimouse lgG antibody
`did not change hy more than !)";, during treatment, and the
`patient responded normally to the OKT:-1 therapy (Table 2).
`An anti-OKT:{ antibody response, defined as doubling of the
`pret reatment oo ... ! .. wns deteC'tahle at various times post treat (cid:173)
`ment in 14 (7()';;, ) oft hose individuals (Tables 2 and :H. Four
`h11d only lgM and one had only lgG. The lgG was detected
`significantly later than the lgM by an average of 10 days,
`although the time of the peak level was the same (20-2:l days)
`(Tables 2- 4). Antihorlies were detected during OKT:S adminis(cid:173)
`tration in 8 patients. All 8 had lgM antibodiei:; and 2 also had
`lgG. Only one patient (No. 4), who had lgG as well as lgM
`anti -OKT:I, showed resistance to the pharma<·ologic cfft•cts of
`OKT:l. The other 7 cleared their T C'ells normally despite the
`presence of lgM anti -OKT:l. Only JO':;, (fgG) and :l:l% <lgM)
`of the responders had persistent antibodies when checked 6
`weeks or longer after the last dose. Two had detectable levels
`14 weeks posttherapy (Nos. 7, l!J).
`Enhandn;: assay. This assay was used to detect ii:;otype and
`light chain reactivity by ass11ying T8• and T4• PHA blasts
`coated with OKT8, OKT4, or OKT:S antibodieR. Three patients
`(Nos. 10, 12. and 14) had pretreatment reactivity above the
`9f1th percentile (Tables I and 2). Of the 16 tested, 7 (44 <;;,)
`showed a two-fold or greater increase in reactivity to OKT8
`{lg(;" .. ~)-conted cells. Only two (12%) hound (both weakly) t.o
`OKT4 (lg(;,.,,)-coated cells. suggesting that the reactivity de(cid:173)
`teC'ted was primarily to isotype specificities on the heavy chain.
`Tht• timing of the anti-isotype was closer to that of the lgM
`than t he lg(; (Table 4).
`
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`I
`" Thi· clay wh1·n antibody wa" first d1•tt•ctrd (0 = last clay of OKT:l
`t h1•rap~· : rwt.:at iH• numlwrs-da~·s lwfon• I h..rap_v end('(!).
`'Onlv :1 post! rl';tt ment sampl1•s.
`' Abhrt>viations: p = prt>treot1111•111 ~ampll' positiv!'. >!lf>th per<'entile
`of normal control~: - = m·1wtiv1•: blank = not donl'.
`"Last blood values on OKT:I.
`
`TAHU: I. Pr1•treatment rPil<.'livity
`
`Enhnndng As~HY
`4(·,· i ncrea:wl
`
`Hlu.-kin,: assn~·
`( i·; inhihi1 ion l
`
`Ii:
`
`li:C
`
`ll(M
`
`OKT:I
`
`OKTX
`
`OKT4
`
`OKT:I
`
`OKTI\
`
`Pat ii·nts
`('0111rofs
`
`:16!1±:.!l!I
`:t:.t!:t I :!f,
`
`;1:1!1±141
`
`!i9±7(i
`:.!9±:10
`
`:lll±XO
`l>O±!l:.!
`J.l·l±:.!!-.17
`Wx±lf>I
`:!i±:l-1
`IH±:.!l
`·-·-- ·
`" Excludes patit>nt No. 18 whos1• n 1l1ws were t:l!q !Ii:>. ! Ill (lg(;J. and 171/l llgM) on tht> :1 ELISA assays. lg wns a scr1>ening test usinl{ goat
`anti human inununoglobulin n•m·t ivt' to ) !wavy 1·hain and hoth lit.:ht 1·hains. whill' lg(; and h:M wt-re dt'tt'l'ted hy hl•1wy-diain-specifir ant isera.
`
`7±7
`
`8±11
`
`3 of 7
`
`Celltrion, Inc., Exhibit 1027
`
`

`

`May, 191:i6
`
`.JAFFERS ET AL.
`
`575
`
`Hlocking:
`
`ELISA-OK'J':I: OD..,.
`
`TAHU: :1. Maximum antihody level"
`. -·-----·----------
`Enhancinj.! (% increase
`MFC>
`. ·-- --- ------ ---------
`Anti-Id t% In·
`OKT:I
`OKTA
`OKT4
`lgM
`hihition)
`- - - - - - - - - - - - - - - - - - - - - - -
`.59fi(.59l
`(;:14( 19)
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`1:114(14) 460( f>)
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`1971(12) 5583(12)
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`869< 1:n
`;,:l8(29)
`.'J/'i.51- J)
`
`8:!(14)
`91(/2)
`92(1-1)
`
`94( l'i)
`:l~I( 1 Ol
`
`f)69( 2)
`:158( 1)
`489( 7) 178( 7)
`1116(14) 118:1(19)
`
`640(22) 200(22)
`400113) 263(1 :1)
`
`- 94(52)
`
`89( 2)
`.')f)(l4)
`
`1'200(76) 1500(76) 67(76)
`1067( :l)
`
`81if>(:IO)
`
`.5971 II)
`
`AA!22l
`92(1G)
`
`Pt1tien1
`
`•)
`
`:1
`4
`f)
`I)
`7
`8
`9
`10
`II
`I :!
`1:l
`14
`lfi
`Hi
`17
`18
`I 9
`
`f)8(4!i)
`
`:w
`21
`• Uay of maximum !after OKT:l therapy ended) in parentheses.
`•Assays in iwlks were positive in the last sample 1.ested (more than
`6 weeks from the last OKT:I dose).
`
`TAHl.F. 4. Summary of time <'ourse of antihody respon!<e
`Numher
`Pcuk•
`Onset•
`-- - -·-·-·
`
`O±:lb
`1:l
`l!!M
`li:C
`10±:1
`10
`ldiotype
`12
`11±4
`lsotype
`4±1
`7
`• Day after last OKT:l dose. mean± SE.
`b Preceeds Ii:<:. /'<.0'2.
`
`2:1±7
`20±5
`16±:!
`2:1±9
`
`Further experiments were performed to document whether
`the effect was indeed due to anti-Id antibodies. The presence
`of residual free OKT3 in the serum was ruled out by the lack
`of detectable mouse lg on normal lymphocytes after incubation
`in the blocking serum. In all patients OKT3 antibody was
`undetectable in the serum by 2 days after therapy had ended
`(data not shown}. For this reason only sera taken at least 48
`hr after the end of therapy were analyzed (except for patient
`No. 4 who was demonstrated to clear t he OKT3 within 7 hr).
`Serum that blocked OKT3 did not block F-anti-Leu 4, an IgG1
`antibody t.o a different epitope on the same molecule (Fig. 2).
`This argues strongly against free or complexed T3 antigen itself
`as a significant source of antigenic competition in the blocking
`assay.
`Sera from two of t he patients were analyzed by affinity
`chromatography using columns with immobilized OKT3 or
`normal mouse IgG. The sera from patients Nos. 4 (Fig. 2 and
`3) and 8 (Figs. 4 and 5) were absorbed with mouse IgG on
`Sepharose. The effluent and t he dialyzed 3M KSCN eluate
`were tested for blocking activity. The eluate from normal mouse
`lgG (patient No. 8, day 5) showed equivalent blocking of F(cid:173)
`OKT3 and F-OKT8 of 88% and 83%, respectively, in t he
`
`absence of RALB/c serum. The analogous eluate from patient
`No. 4 had no blocking activity. In contrast, t he effluent blocked
`F-OKT:l completely but had little activity against F-OKT8,
`even in the absence of added BALB/c serum (Fig. 4). The
`effluent (patient No. 4) and the starting serum were then
`absorbed on an OKT3 column. This removed the blocking
`activity, which was undetectable even after reconcentration.
`Taken together, these data indicate that the blocking activity
`was due to antibodies that reacted selectively wit h OKT3 and
`not to other mouse immunoglobulins, satisfying the criteria for
`ant i-Id specificity.
`The t ime course of the anti-Id antibody production was
`variable, but generally paralleled that of the IgG (Table 4). The
`anti-Id antibodies first became detectable at an average of 11
`days (range 0-45) after therapy had ended. The most rapid
`antibody response was manifested at 12 days after the start of
`therapy in patient No. 4 (Fig. 3). At this t ime he became
`refractory to the T lymphopenic effect of OKT3. The only
`clinical signs of an allergic reaction were an eosinophilia of
`12% and a fever upon administration of the OKT3. No serum
`sickness lesions (glomerulonephrit is, vasculitis, mouse lg de(cid:173)
`posits by immunof1uorescence) were seen in renal biopsy taken
`during the OKT3 treatment. The patient suffered no adverse
`systemic effects from this response, although t he rejection
`process became reactivated and t he allograft was eventually
`rejected. T his was the only patient whose t herapy was stopped
`hecause of a nti -O KT:~ antibodies. Three other patients had
`anti-Id antihody in the first sample analyzed, 2 days a fter
`therapy ended. Anti-Id antibody persisted in 33% of patients
`after 6 weeks; the longest documented was 6 months (No. 8).
`Of t he 12 patients that made a blocking anti-Id antibody, 3
`(25%) had no IgG and 2 ( 17%) had no JgM react ivity detectable
`by ELISA. Similarly, 30% of t hose with blocking anti-Id had
`no isotype/ light chain reactivity detectable by t he enhancing
`assay. Pat ient No. 17 made exclusively an a nti-Id response,
`detectable only in the blocking assay. Furthermore, the anti-Id
`assay detected antibody on one or more occasions in 7 other
`patients when the other assays were negative. This can be seen,
`for example, in the t ime course of t he antibody response in
`patient No. 8 (Fig 4), in which the anti-Id response was persis(cid:173)
`tently positive when the ELISA and enhancing assays were
`negative. Two patients (Nos. 11 and 15) made anti-OKT3
`antibody detected on ELISA that did n'ot block antigen binding
`or have reactivity to OKT8. These antibodiel-l may include
`nonblocking anti-Id.
`To estimate the portion of t he antibody response that was
`anti- Id, serum from patient No. 8 at the peak of t he antibody
`response (day 12, Fig. 4) was analyzed by ELISA using normal
`BALB/c serum as a competitive inhibitor for non-anti· Id anti(cid:173)
`bodies. Normal mouse serum (100 µI) was able to block 69% of
`the lgG binding to OKT:l, while 100% could he blocked by 10
`µg OKT3, so t hat anti-Id constituted about 31 % of t he reactiv(cid:173)
`ity.
`Correlations with clinical cour.~e. With the exception of the
`one patient (No. 4) who developed anti-Id antibodies that
`neutralized the in vivo effectiveness of OKT3, no adverse
`effects were associated with the antimouse lg or anti -OKT3
`idiotype response. The response to therapy and outcome of the
`graft were not correlated with t he presence or absence of an
`antibody response. However, of the 6 patients who received
`cyclophosphamide (Nos. 10 and 17-21) only 173 made an lgG
`anti-OKT3 response detectable in ELISA, whereas 9 of 14 of
`
`4 of 7
`
`Celltrion, Inc., Exhibit 1027
`
`

`

`TRANSPLANTATION
`
`Vol . .//, N<>. ,5
`
`NORMAL
`
`OKT3
`
`C4/1 3
`
`OKT3
`
`576
`
`10
`
`..
`
`1· 1 · 1 1-·, ,-, - , .
`
`1a
`'"° ~ ~·~
`
`, 1 · 1 - n
`
`10
`
`1~0
`
`OKT8
`
`OKT8
`
`104
`1 ' .""r'rl
`:?O
`
`I~
`
`Leu4
`
`Leu4
`
`..
`
`ll't
`
`1.0
`
`,..
`
`l ..
`
`10
`
`..
`
`•
`
`llO
`
`I..
`
`-
`
`l'ot
`
`Poti.nt 4
`
`Skin '"'e
`
`Ba: R•i•t!lon
`
`Ba. Rei.tllon
`8Ml9G
`
`i
`
`"--11~~,.:..:.....,;:.........~~,~ .JIM!~~~~
`'
`
`FH:t11u: '2. Fluorescence histograms ofhlocking
`anti · Id assay. Normal lympho(·ytes stuined with
`fluores\'einated monoclonal antibodies in the
`prese1we of normal HALH/c serum and either
`normal human serum {left t•olumnl or serum
`from patient No.~ !right column). The patient's
`serum hlocb F'·OKT:I. hut not F·OKTR (both
`are lg(;,,.,1 or F·anti ·Leu 4 {li:<:l. reactive to the
`T:I antigen}. ('ell numher is on the y·axis and
`lluores«em·t· intensit~' is on the x·axis. The num·
`her in the lower right of ea(·h histogram is the
`median tluort'seence channel oft he positive cells.
`
`0000
`
`SO<IO
`
`a
`~ JOOO
`~
`iil ..,
`....
`~ ..
`
`4 000
`
`2000
`
`1000
`
`0
`
`lgM a0KT3
`
`ELISA aOK 13 (0040Y
`to.,. _ 'o
`lgO
`'114
`
`~Qf~!,.~G i;i~ 11
`'
`1nhol;Hhon
`
`PREOHISONE
`
`AZATHIOPRINE
`
`,.
`
`l!.tt
`
`OKT3
`
`]~omQ/d
`
`I ]~!>()mQ/d
`
`11~ J~e
`, ~, !> fJ
`
`8l
`
`4 G O
`
`181
`
`.,
`
`T3+
`
`25
`20
`15
`10
`5
`OAYS POST TRANSPLANT
`FH; l' t<~: :1. ('liniral ('ourse of pati<·nt No. 4. who developed blocking
`anti·ld during therapy and IX'came resistent to the T lymphopenic
`effects of OKT:I. Ii:<; and lgM ant i·OKT:I was detected during therapy.
`Hlo\'king anti·ld appearance was accompanied hy resistance to the T
`lymphopenic response to OKT:I (day:!:!}.
`
`the non-cyclophosphamide group made such antibodies
`(/'<0.076). Cyclophosphamide had little effect on the blocking
`anti-Id response. which still occurred in !JO'/(, of patients(cid:173)
`although somewhat later than the others, peaking at 16- 45
`days. No correlation with the recipient's HLA phenotype and
`the antibody response was found.
`
`~ ~ 300 L'.:I
`
`l!; ~
`~ ;
`100
`~
`"'
`
`100 ,.:
`
`'("'<
`
`i
`
`a~KA IC
`. " ~
`l_~ ·""""""· 1.&?Ki4
`I ,
`~iii I ~ I:: r
`
`.
`
`~ ~ 40
`iC-:
`8" 10
`:, .
`•
`'
`.
`.
`01'_ -,.---.. - ..
`~
`- 20 · •O o
`10 20 :ao co so 60 10 ao
`(0KT3l DAY AFTER tAST OKT3
`FHaai~; ·I. Time course of antibocly response in patient No. R, as
`dete\'ted hy each assay in this study. The horizontal bar is the thresh·
`hold of positivity <:! SD ahovt' tht• mean of normals). Note the early
`rise of li:M and the presence of both blocking anti· Id and anti·isotype
`<OKTH enhancini: assay). No antibodies to OKT4 were detectable.
`
`OISC'USSION
`In this series 60'};, of the patients treated with OKT3 devel·
`oped anti-Id antibodies and 44'7/, anti-isotypic antibodies, de·
`spite intense immunosuppressive treatment (Table !J). The
`
`5 of 7
`
`Celltrion, Inc., Exhibit 1027
`
`

`

`May, 1986
`
`JAFFERS ET AL.
`
`577
`
`,:
`
`~
`u
`1.\..~ .~ , . ,.,., ,., .. . '.' ....... .,
`u•
`Fu:URE 5. Rlocking assay of the mouse lgG absorbed serum from
`patient No. 8, day f>. Fluorescence histograms of T cell blasts stained
`with F-OKT:l (T~) or F-OKT8 (T8) in the presence of the effluent (E)
`that passed through the mouse IgG column or the control (C) which
`was the ultrafiltrate of the effluent. (passed through XM-50). (U)
`unstained cells. No BALB/c serum was added. The effluent completely
`hlocks F-OKT:l, in contrast to its slight inhibitory effect on F-OKT8.
`
`The nonidiotypic reactivity detected by the binding of OKT8
`was probably isotypic, because little reactivity was found to
`OKT4. We cannot exclude the possibility that a portion of the
`OKT8 reactivity may be to allotypic determinants not ex·
`pressed on OKT4. OKT3 was derived from a BALB/c mouse,
`and both OKT8 and OKT4 were obtained from (BALB/cxA/
`J) F, mice ( /6). The strains of origin of the heavy chains in
`OKT4 and OKT8 are unknown. Several lgGi. allotypic deter(cid:173)
`minants of BALB/c are expressed by A/J but none are appar(cid:173)
`ently expressed on lgG2i. (17). Anti-isotype specificities have
`been detected in other patients treated with OKT3 ( /0).
`The presence of some degree of reactivity to mouse immu(cid:173)
`noglobulin in the pretreatment sera and in normal controls was
`expected. Antibodies to equine A TG have been detected by
`passive hemagglutination in up to 50% of patients awaiting
`renal transplantation with no previous exposure to ATG (18) .
`These antihorse immunoglobulin antibodies were found to
`crossreact with goat and rabbit immunoglobulin. The specificity
`of these antibodies has not been established. They may be
`crossreactive antibodies to immunoglobulin antigens absorbed
`orally (beef, pork, etc.) or rheumatoid factors. We found no
`correlation between preexisting antibodies and the severity of
`side effects from monoclonal therapy. The one patient with a
`high titer prior to t reatment responded fully to OKT3 therapy
`for rejection. A further analysis of these antibody crossreactiv(cid:173)
`ities will be necessary to determine the importance of these
`interactions.
`Although early studies were optimistic that monoclonal anti(cid:173)
`bodies might have low immunogenicity ( /, 3, 19), following our
`initial report (6), several other studies have documented the
`presence of both non-anti-Id and anti-Id responses (4, 5, 7, 9,
`10, 14, 15). The production of such antibodies may be influ(cid:173)
`enred by the particular monoclonal antibody used and the
`immune reactivity of the individuals t reated. More than half
`the patients treated for lymphoreticular malignancies with a
`nonmitogenic antibody (anti-Leu l) produce antimouse anti(cid:173)
`body, similar to that of our transplant recipients. Although
`these antibodies do not cause immune complex disease, the
`response effectively neutralized the monoclonal antibody, as in
`our patients (15).
`OKT3 does not 1;eem particularly more or less irnmunogenic
`than other anti-T-cell or antitumor antibodies, despite its
`known mitogenic activity. The one distinctive feature of the
`immune response to OKT3 may be the predominance of the
`anti-Id response. Only 5% of the antibodies to the murine
`immunoglobulin have been to idiotypic determinants in lym(cid:173)
`phoma patients treated with anti-Leu 1 (20), whereas in one of
`our patients only anti-Id antibodies were detected, and in
`another they accounted for 31 % of the antibody response.
`Overall, 75% of our patients who had any antibody response
`made anti-Id that was able to block antigen binding. Why
`OKT3 should be particularly effective at promoting an anti-Id
`response is unknown, but one could speculate that this might
`be related to the target molecule, a part of the T cell antigen
`receptor complex. However, the strong anti-Id response is not
`unique to OKT3, as anti-Id constituted about one-third of the
`antibody response of goats immunized with myeloma prot