effect of the extent of chelate substitution on the ...lym-1, with 84 lysineresiduesand2...

[CANCERRESEARCH55,878â€”884,February15,1995J

ABSTRACT

Trial therapy for lyphoma with the radlolabeled chelate-antibody conJugate 67Cu-2IT-BAT-Lym-1 has been promising. It Is desirable to delivertherapeutic dosesof radlometal using a minimum amount of 2IT-BATLym-1 to minimize the risks of adverse patient reaction and antigenicresponseto antibody. This is readily accomplishedby IncreasIng thenumber of metal-bInding sites (Le., chelating agents) conjugated perantibody, but the abifity of the antibody to bind antigen and target tumorcellsin vivo must not be impaired by the conjugation reaction.To determine the maximum chelator:antibody ratio (c/a) of 2IT-BAT-Lym-1 atwhich functional Integrity Is preserved, Immunoconjugates with a c/a of1.3-23 were prepared and examIned by radlohnmunoassay and competltive antigen binding versus lightly IOdinated Lym-1. The biodistributlon intumored mice of conjugates with c/a of 2.1, 43, &4, and 11.4 also wasexamined. Conjugates with c/a up to S exhibited no loss of Immunoreactivity, and conjugateswith c/a up to 11 retained 75% or greater Immunoreactivity relative to unmodified Lym-1. All conjugatesexaminedcornpetedlesseffectively than did unmodified Lym-1 for antigenbinding, butthe effect at c/a 5 was sllght Tumor uptake declined with Increasing c/a,but the effectwasinsignificantat c/a 2.1 and4.3. ConJugatesof c/a4â€”Swerefoundtobeoptimalfor thepreparationofradloimmunocoujugateofhigh specificactivity with minimal, if any, lossof functional integrity.

INTRODUCTION

67Cuhas excellentphysicaland biochemicalpropertiesfor radioimmunotherapy. Its half-life of 62 h is comparable to the uptake andresidence time of antibodies on the tumor (1). The radionuclide emitsabundant 13particles of moderate energy, useful for therapy, and @yphotons, useful for pretherapy imaging studies. 67Co is a long range,sparsely distributed radiation source in tissue, with microdosimetrycharacteristics similar to those of â€˜@â€˜I(2). 67Cohas a stable daughterand, unlike many other radiometals, has no known biological pathways for deposition in bone (3). For these reasons, 67Co is beingactively investigated by several groups as a radioimmunotherapeuticagent (4â€”7).

In our group, these studieshave progressedto the clinical trialphase(8). The macrocyclicchelatingagentTETA3, conjugatedto themurine anti-lymphoma IgG2a mAb Lym-1 (9), is used as a earner for67Cu(10, 11). To preparethe immunoconjugate21T-BAT-Lym-1, thebifunctional TETA derivative BAT is conjugated to Lym-1 via 2ff(12). As seenin Fig. 1, conjugation occurs at the amino groups locatedat lysine side chains and the NH2 terminus of the light chain ofLym-1; the NH2-terminal aminogroupof the heavychain is blockedto conjugation by the cyclization of terminal glutamine.

Received8/10/94;accepted1217/94.Thecostsof publicationof thisarticleweredefrayedin partbythepaymentof page

charges.This articlemustthereforebe herebymarkedadvertisementin accordancewith18U.S.C.Section1734solely to indicatethis fact.

1 Supported by Department of Energy Grant DE-F003-84ER-60233 and the National

Cancer Institute Grant CA 47829.2 To whom requests for reprints should be addressed, at Molecular Cancer Institute,

1508 Alhambra Boulevard, Sacramento, CA 95816.3 The abbreviations used are: TETA, 1,4,8,11-tetraazacyclotetradecane-N,N',IVâ€•,A?â€•-

tetraaceticacid; BAT, 6-[p-(bromoacetamido)benzyl)-TETA2FF,2-iminothiolane;c/a,chelator:antibodyratio; CAE, celluloseacetateelectrophoresis;LEF,isoelectricfocusing;% ID/g, percentage of injected dose per gram of tissue; Vh, volthours.

Under optimized radiolabeling conditions, 2IT-BAT-Lym-1 hasbeenshownto bind 67Corapidly, selectively,quantitatively,andwithextraordinarykineticstability(13, 14). This hasled to the preparationof 67Cu-radiolabeledimmunoconjugateswith high radioactiveyieldfor therapeuticuse(3, 8).

There is evidence that the likelihood of a reaction to a foreignprotein, e.g., Lym-1, and the development of human anti-mouseantibody to murine antibodies, is related to the amount of antibodyadministered. To minimize the risks of patient reaction and immunogenic responseto 67Cu-2IT-BAT-Lym-1, it is desirable to delivertherapeutic doses of radiometal using a minimum of antibody. Reducingthe amountof antibodyper doseand improving the recoveryof radiometalalsoreducesthecostof therapy.The specificactivity of67Cu-2IT-BAT-Lym-1 (mCi 67Cu/mgLym-1) varies directly with thespecific activity of the radiometalitself (mCi 67Cu4@gtotal Cu) andwith the averagenumberof metal-bindingTETA sitesconjugatedperantibody (c/a). Due to contamination by nonradioactive copper, thespecific activity of 67Cois relatively low and variable (13, 15), but itis adequatefor the routine and dependablepreparation of pharmaceutical quality 67Cu-21T-BAT-Lym-1 given a sufficiently high c/a.

The ability of Lym-1 to bind to antigen must not be impaired by theconjugationreaction. One binding characteristicof an antibody orantibody conjugate preparation is the immunoreactivity, the fractionof the antibody molecules able to bind antigen. The c/a at which theimmunoreactivity is significantly reduced has been observed to varywith the type of constituentantibody,chelatingagent,or linker, andwith conjugationreactionconditionssuchas pH (16â€”19).The inactivation of large fractionsof someantibodyconjugateswith a c/a aslow as 2 or 3 has been reported (20, 21), while some conjugates witha c/a of 7â€”9have exhibited full retentionof immunoreactivity(22â€”24). Modification of 12-20 amino groups hasbeen reported with 70%retentionof immunoreactivity(25). The secondbindingcharacteristicis the affinity, or â€œtightness,â€•of binding of antibody to antigen. Asingle â€œfunctionalaffinityâ€•as measuredby conventional methods hasbeen found to be an inadequate description of the bivalent bindinginteractionof IgO to cell surface antigens(26, 27). However, theeffect of chemical modification on the binding affinity of an antibodymay be inferred from the effectivenesswith which the modifiedantibody competes with unmodified antibody for antigen binding.

The ability of the antibody to target tumor cells in vivo also mustnot be impaired by the conjugationreaction. The biodistribution ofantibodies can be profoundly influenced by conjugation tochelates, even when no effect on immunoreactivity is observed(28â€”35).

Lym-1, with 84 lysineresiduesand2 availableNH2-terminalaminogroups (36, 37), has abundant sites for conjugation via 2ff. Lym-1conjugates have demonstrated high retention of immunoreactivitywhen modifiednearthe bindingsite(38) andwhen conjugatedwith ac/a of up to 4.2 (12). In this study, the effect of varying the c/a of2IT-BAT-Lym-1 on the ability of the conjugate to bind antigen andtarget tumor cells is investigated in order to optimize the preparationof 67Cu-21T-BAT-Lym-1 radioimmunopharmaceutical.

878

Effect of the Extent of Chelate Substitution on the Immunoreactivity andBiodistribution of 21T-BAT-Lym-1 Immunoconjugates1

David L. Kukis, Gerald L. DeNardo,2 Sally J. DeNardo, Gary R. Mirick, Laird A. Miers, Douglas P. Greiner,and Claude F. Meares

Department ofinternal Medicine, University of Cal(fornia Davis Medical Center, Sacramento, California 95816 (D. L K.. G. L D., S. I. D.. G. R. M., L A. MI, and DepamnentofChemistry, Universityof Cal(fornia Davis, Ca1@fornia95616 ID. P. G., C. F. M.]

Research. on August 18, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

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IMMUNOREACI1Vfl@YAND BIODISTRIBUIIONOF 21T-BAT-LYM-1

conditions. All conjugations were conducted in 0.1 M tetramethyl ammoniumphosphate, pH 8.8â€”9.1,for 30 mm at 37Â°C.The concentrations of Lym-1, 21T,andBAT were12.0â€”12.9mg/mI,0.5â€”25.0mM,and1.0â€”50mM,respectively;in all conjugations,theconcentrationof 211'wasone-halfof theconcentrationof BAT. All conjugateswerepurifiedandtransferredto 0.1 M ammoniumcitrate, pH 5.0, by centrifuged column filtration (44).

Metal-binding Assay of the Chelator:Antibody Ratio. The c/a of everypreparationof 2IT-BAT-Lym-1 was assayedin triplicate by cobalt metalbinding asdescribedpreviously(45) with the following modifications.Standardized, trace 57Co-radiolabeled (ICN Radiochemicals, Irvine, CA) CoCl2(aqueous)was addedin excessto an aliquot of 2IT-BAT-Lym-1 in 0.1 Mtetramethylphosphatebuffer, pH 8.0.The solutionwas incubatedfor 30 mmat roomtemperature.EDTA (aqueous)(FisherScientific,Fair Lawn,NJ) wasaddedto a final concentrationof 10m@ito complexanynonspecificallyboundcobalt ions. The solution was incubatedfor 15 min at room temperature.Trace-radiolabeled Co-21T-BAT-Lym-1 was purified by centrifuged columnfiltration. Theconcentrationof cobaltchelatein purified Co-21T-BAT-Lym-1solution was determined by radiation counting versus a known quantity of thestandardizedtrace-radiolabeledCoCI2solution.The concentrationof Lym-1was determined by absorbance at 280 nm. The c/a was calculated as the ratioof the molar concentrations of cobalt chelate and Lym-1.

IEF. The isoelectric points of unmodified Lym-l and 21T-BAT-Lym-1conjugatesof c/a2.1,4.3,8.4,and11.4weredeterminedby IEF (Phastsystem,Pharmacia). Nonradioactive copper nitrate (aq) was added to 21T-BAT-Lym-1in 0.1 M ammoniumcitrate, pH 5.0, such that the final concentrationofimmunoconjugate was 10 mg/ml and copper was present in 2 times excessrelative to metal-bindingsites.Carrier ampholytes(Pharmalyte,Pharmacia)were prefocused on precast 5% polyacrylamide (3% crosslinking) IEF media(Phastgel, Pharmacia) for 75 Vh at 2000 V, 2.5 mA to form a pH gradient.Lym-1 and Cu-21T-BAT-Lym-1 (5 @.&g)and broad range p1 standards (Pharmacia)werefocusedfor 15Vh at 200V, 23mA, then410 Vh at 2000V, 2.5mA. Thegelwasmaintainedat 15Â°Cthroughout.After IEF, thegelwasstainedwith Coomassieblue.

Radiolabellng 2lT-BAT-Lym-1 was radiolabeled with â€œCo(ICN Radiochemicals),67@ (BrookhavenNational Laboratory,Upton, NY) and @â€œCu(University of Missouri, Columbia, MO) as follows. Radiometal in 0.1 M HGwasbufferedin radiolabelingbuffer,thenaddedto conjugatesuchthatthefinalconcentration of conjugate was 1.0â€”10.0mg/nil and the final radiometal:conjugateratio was 1.0â€”10.0mCi/mg. @@Colabelingwasconductedin 0.1 Mtetramethyl ammonium phosphate, pH 8.0; 67@ and @Culabeling was conducted in 0.5 M ammoniuin citrate, pH 5.0â€”5.5.The radiolabeling solution wasincubated for 30 mm at room temperature. EDTA was added to a finalconcentration of 10 mM to complex any nonspecifically bound radiometal ions.

The solution was incubatedfor 15 mm at room temperature.Radiolabeledconjugatewaspurifiedandtransferredto 0.9%(w/v) NaG (BaxterHealthcare,Inc., Deerfield, IL) or 0.9% (w/v) NaCI-lO [email protected], pH 7.4 by centrifugedcolumnifitration. All productswereexaminedby HPLC.To verify theabsenceof nonspecificallyboundradiometal,EDTA wasaddedtoa smallaliquottoafinal concentration of 10 mM, and the aliquot was examined by TLC. @Cu21T-BAT-Lym-1 injected into mice was also examined by CAE. â€˜@I(ICNRadiochemicals) iodination of Lym-1 and L6 was performed by well describedmethods (46) using chloramine-T (Sigma) as the oxidant. The reaction wasperformedusing molar ratios of 1.0 mAb:0.2 â€˜@I:70chloramine-T.Theimmunoreactivity of 1@'I-Lym-1 prepared in this way is equal to that of theunmodifiedantibody(47).

57Cowaschosenfor thec/aassaysandsolidphaseRIA of 21T-BAT-Lym-1becauseit is readilyavailableandit is suitedfor thepreparationof standardized solutions due to its long half-life. Parallel solid phase RIA of â€œCo-versus

67Cu-labeled 21T-BAT-Lym-1 produced similar results (data not shown). @Cuwaschosenfor thebiodistributionstudyon thebasisof availability.67Cowasusedasthe radiolabelfor all otherradioactiveexperiments.

Solid Phase RIA. Solid phase RIA was used to measure the relativeimmunoreactivities of all preparations of 21T-BAT-Lym-1 versus the immunoreactivity of a 1@'I-Lym-1 standard. The assays were performed in triplicate

asdescribedpreviously(47) againstpartiallypurifiedmembranefragmentsfrom Raji cells.The conjugateswere radiolabeledwith @Coat specificactivities of 0.5â€”1.0mCi/mg for the assays.To measure nonspecific binding,57Co-2rr-BAT-Lym-1 and â€˜@I-Lym-1were assayed against partially purified

879

Lym-1

r@o 21T

@O@KIN DBAT

c0@

21T-BAT-Lym-1@ 4:?@o-

Fig. 1. Conjugationof 2IT-BAT-Lym-1. Lym-1 has84 lysineresiduesand 2 availableterminalaminogroupsaspotentialsitesof conjugation.

MATERIALS AND METHODS

General Method& HPLC (Beckman 332; Beckman, San Ramon, CA) wasperformedusinga sizeexclusioncolumn(BeckmanSEC.3000)elutedin 0.1M sodium phosphate-0.1 M potassium sulfate-0.025% (w/v) sodium azide, pH

7.1. The flow ratewas 1.0 mI/mm.Radiolabeledantibodiesandconjugateswere detectedby @Nabsorbanceat 280 nra (Beckman160 detector)andradioactivity (Beckman 170 detector).

TLC wasrun on plastic-backedsilica gel plates(EM Science,Gibbstown,NJ) using 10% (w/v) ammonium acetate-methanol (1:1, v/v) as the eluent. Inthissystem,unchelatedcopper,unchelatedcobalt,andconjugatesremainattheoriginwhilefreechelatesmigrateto RF> 0.5.

CAE (Gelman Sciences, Inc., Ann Arbor, MI) was performed using 0.05 Msodiumbarbitalbuffer,pH 8.6.A currentof 5 mA perstripwasapplied.Samples were electrophoresed for 11 and 45 mm. At 11 mm, free chelates wereresolved from immunoconjugates. At 45 mm, monomeric immunoconjugateswereresolvedfrom aggregatedspecies.

Absorbanceat 280 nm were measured(BeckmanDU64) on dilutionssuitable to give absorbance readings of 0.1â€”1.0.

Wholebodyactivitiesof miceweremeasuredwith abalanced,standardizeddualsodiumiodidecrystalprobesystem(PickerNuclear,NorthHaven,Cl')(39).All othersampleswerecountedin awell counter(PharmaciaLKB 1282;Pharmaci,Piscataway,NJ) with decaycorrectionfor esCu(half-life, 12.7h)and67@(half-life, 62 h) whenappropriate.

Antibodies. Lym-1 (Techniclone,Inc., Tustin,CA), a murineIgO2amAbgeneratedusingRaji cellsasanimmugen(40),reactswith amembraneantigenfoundon malignantcells of mostpatientswith B-cell lymphoma.L6 (Oncogen, Seattle,WA), a murine anti-adenocarcinomaIgG2a mAb, targets amembrane-bound antigen found on human adenocarcinoma cells of the lung,colon,ovary,andbreast(41, 42).

Determination of the Molar Extinction Coefficient of Lym-1. Aminoacid analysis was performed on three aliquots of stringently measuredvolumefrom a solutionof Lym-1. The aminoacid compositionof theantibody (36, 37) was usedto calculatethe concentrationof Lym-1 in thesolution. Three dilutions of varying Lym-1 concentration were preparedfrom the sample,andtheir absorbanceat 280 nm wasmeasured.The molarextinction coefficient of Lym-1 was determinedby linear regressionof aplotof absorbanceversusLym-1concentrationdata(Statviewversion4.0;AbacusConcepts,Berkeley,CA).

Preparation of 2IT-BAT-Lym-1 Immunoconjugate@ BAT was prepared

as describedpreviouslyand conjugatedto Lym-1 via 2ff (SigmaChemicalCo., St. Louis, MO) by standardmethods(12, 43) under the following



IMMUNOREACTIVITY AND BIODISTRIBUTIONOF 21T-BAT-LYM.1

membranefragmentsfrom theirrelevantacutelymphoblasticleukemiaCCRFCEM cell line.

Cell-binding Assays for Lineweaver-Burk Analysis. Immunoreactivity is

commonly determined by Lineweaver-Burk analysis of cell-binding assaysofantibody (48). Briefly, based on the law of mass action by a derivationdescribedin detailby Lindmo a al. (49):

[7'J@ 1 1 1[B] nC0 [F]@ r

where[T] is the concentrationof total antibody,[B] is the concentrationofbound antibody, [F] is the concentration of free antigen, Ka is the affinityconstant, and r is the immunoreactive fraction of the antibody. [TJ/[B] plottedas a function of 1/[F] yields a straight line; r is taken as the inverse of the Yintercept.

Binding assays for Lineweaver-Burk analysis are conducted under conditions of great antigen excess so that the concentration of free antigen isapproximated by the concentration of total antigen. Because antigen concentration is directly proportionalto cell concentration,binding assaydataareusuallyplottedas[TJ/[B] versusthe inverseof cell concentration.

Theinadequacyof asingleaffinity constantK0to describeantibody-antigenbinding was noted previously. The implications of the presence in the equationof a valueKahavebeenexaminedelsewhere,andtheability of a LineweaverBurk plot to correctlypredictr hasbeensupportedbothby experimentandontheoretical grounds using a bivalent binding model (50).

Preparations of 21T-BAT-Lym-1 of c/a 2.1, 4.3, 8.4, and 11.4 were examined by cell-bindingassayfor Lineweaver-Burkanalysisas follows. Assaysolutionswere preparedin duplicatein 1.0%BSA in PBS.67Cu-2IT-BATLym-1 (8.0 ng) was added to Raji cells (0.13, 0.25, 0.50, 1.0, 2.0, or 4.0 X 106)in a totalvolumeof 0.30ml andgentlyvortexed.Thesolutionswereincubatedfor 60 mm at room temperature with gentle vortexing every 15 min. Thesolutions were centrifuged, and the supernatant and pellets were separatedandcounted.â€˜@â€˜I-L6wasassayedin thesamewayto measurenonspecificbinding;1@'I-Lym-1 was assayed as the standard.

Thesuitabilityof thebindingassayconditionshadbeenverifiedby previousexperiment.The loss of cell viability during the 60-mm incubationwasminimal: from 90 to 87%viable,measuredby trypanblue exclusion.It wasalso shown that the 60-mm incubation period was sufficient for maximumuptakeof â€˜@â€˜I-Lym-1by Raji cells.

Competitive Cell-binding Assays. The ability of preparations of 2ffBAT-Lym-1 of c/a 5.4, 13.9, and 19.1 to compete with lL@I@Lym@1for bindingto Raji cells was assayedas follows. Assay solutions were preparedinduplicate in 1.0% BSA in PBS. @I-Lym-1(10 ng) was added to varyingamounts of unlabeled immunoconjugate (100, 250, or 500 ng); then Raji cells(106) were added to a final volume of 0.15 ml. The solutions were gently

vortexed and incubated at room temperature for 60 mm. The solutions werecentrifuged, and the supernatant and pellets were separated and counted.Unmodified Lym-1 was assayed for comparison. To measure nonspecificbinding, similar assays were performed in which CEM cells were substituted

for Raji cells.Biodistributlon Studies. Adult female athymic nude mice (Harlan Spra

gue-Dawley, Frederick, MD) bearing s.c. Raji tumor xenografts received iv.injectionsof MCu@2IT@BAT@Lym@1preparationsof varying c/a. L6, an isotype-matchednonspecificmAb, wasaddedto eachdoseto increasethe totalprotein content to >100 @gto promote uniform blood clearance rates within

groups(51,52).In group1,eachof 11miceweregiveninjectionsof 83 @CV19ILg MCu@2lT@BAT@Lym@1(c/a 2.1) plus L6 in 100 @lsaline. In group 2, 10

mice were given injections of 92 @Ci/34jtg MCu@2IT@BAT@Lym@1(c/a 4.3)

plus L6 in 100 @tlsaline. In group 3, 12 mice were given injectionsof 92@CV34@gMCu@2IT@BAT@Lym@1(c/a 8.4) plus L6 in 100 p1 saline. In group

4, 12 mice were given injections of 92 @tCi/34 @.tgMCu@2IT@[email protected] (c/a

11.4) plus L6 in 100 p@lsaline. Whole body counts were measured at 0, 4, 24,48,and72 h. Blood sampleswerecollectedat 5 mm and1,4, 24,48, and72h. One-halfthe mice in eachgroupweresacrificed24 h after injection,andorgansandtissueswereharvested,weighed,andcounted.Theremainingmiceweresacrificed72 h after injectionandhandledsimilarly.

25

20

15

SS

S

SS

10

5

0

[BAT](mM)Fig.2. 2IT-BAT-Lym-1 conjugationreactionconditionsand final chelator:antibody

ratios (c/a). The c/a of the purified immunoconjugates were plotted as a function of theconcentrationof BAT in thereactionsolutions.Theconcentrationof 2IT wasone-halftheconcentrationof BAT in every reaction.The reactionconditions neededto prepareimmunoconjugateof a desiredc/a may be interpolatedfrom this plot. The apparentdivergenceof dataat high concentrationof BAT is discussedin the text.

RESULTS

Extinction Coefficient of Lym-1. The extinction coefficient ofLym-1 at 280 nm was determinedto be 224,400 M1 cm', equivalent to an E'@@ of 14.4, usinga molecularweight of 155,000. Thisvalue is consistent with the E1@@ of 13.5 reported for murine IgGantibodies (53); the value 14.4 was used for calculations in this studybecauseit is specificto Lym-1.

Conjugation of2IT-BAT-Lym-1 and Radiolabeling. Conjugateswith a c/a of 1.3â€”23were prepared.The relationship of c/a of the finalproductto the concentrationof BAT in the conjugationreactionwasexamined (Fig. 2). The resultant graph has subsequently been usefulfor the determinationof conditionsfor the preparationof 2ff-BATLym-1 of desired c/a by interpolation.

It is of note that the data points in Fig. 2 define two apparentlydivergent curvesat high concentrationsof BAT. These data pointsrepresent two sets of conjugation experiments performed at differenttimes.Prior to conductingeachsetof conjugationreactions,BAT hadbeenconcentratedundera vaccuumto achievehigh final concentrationsof BAT in the reactionsolution.It is likely that BAT sufferedaloss of reactivity during the concentration step. The divergent curvesprobably represent different extents of loss of reactiviity of BAT inthe two experiments.

In everypreparationof radiolabeled2ff-BAT-Lym-1, 95% or moreof the radiometal was associated with monomeric antibody by I{PLC,TLC, and CAE, where applicable.

Isoelectric Focusing. IEF was performed on Lym-1 and Cu-21T-BAT-Lym-1 of c/a 2.1, 4.3, 8.4, and 11.4. An anodal shift in the p1ofLym-1 with increasing conjugation was observed. Multiple bandsover a range of p1 were observed for each 2ff-BAT-Lym-1 preparation, indicating a heterogeneity of conjugation products. The p1 ofLym-1 was measured as 7.3; of 2ff-BAT-Lym-1, c/a 2.1, 6.3â€”6.9;c/a4.3, 6.0â€”6.4;c/a 8.4, 4.5â€”6.2;and c/a 11.4, 4.1â€”6.0.

Immunoreactivity of 2IT-BAT-Lym-1. The solid phase RIA pro

vides a relative immunoreactivity value by comparison to anLym-1 standard, which has been shown to behave like unmodifiedLym-1. Lineweaver-Burk analysisis designedto determinethe immunoreactivityof an antibodyor immunoconjugatepreparationas anabsolute value by extrapolation of binding data to infinite antigen

880

0 10 20 30 40 50



IMMUNOREACIIVITY AND BIODISTRIBUTIONOF 21T-BAT-LYM-1

25

5

02.0 2.5c/a

Fig. 3. The effect of c/a on the immunoreactivity of 2IT-BAT-Lym-1. The relativeimmunoreactivity of each conjugate versus lightly iodinated Lym-1 was measured bysolidphaseradioimmunoreactivityassay.Every conjugatewith c/a of 11 or lessexhibited75% or greater relative immunoreactivity; there was no apparent diminution of immunoreactivity amongconjugateswith c/a of 5 or less.Assayswere conductedin triplicate;Points, mean; bars, 1 SD.

0.0 0.5 hO 1.5

[ceII]1 (mVl 06 cells)Fig. 4. Lineweaver-Burk determination of the immunoreactivity of Lym-1 and

21T-BAT-Lym-1 immunoconjugates. Lineweaver-Burk analysis allows the determination of the immunoreactive fraction as an absolute, rather than a relative, value.Binding assaysof radiolabeled Lym-1 and immunoconjugates were conducted in 6concentrations of live Raji cells. The binding data were plotted as a double inverseplot according to the equation (see â€œMaterialsand Methodsâ€•).The immunoreactivefraction r was given by the inverseof the Y intercept.The immunoreactivefractionsof Lym-1 and 2IT-BAT-Lym-1 of c/a 2.1, 4.3, 8.4, and 11.4were found to be 0.75,0.82, 0.79, 0.58, and 0.45, respectively. The assays were conducted in duplicate.Points, mean; bars, range of values.

80

70

@50@

csJ1@

40 Â°c/a13.9U c/a19.1

0@ _ U0 100 200 300 400

Competitor (ng)Fig. 5. Competitive binding assayof 2lT-BAT-Lym-1 immunoconjugates versus

Lym-1.AssaysolutionscontalnedRaji cells,a traceramountof @â€˜I-Lym-1andrelativelylarge and varying amountsof competitor,21T-BAT-Lym-1or unmodifiedLym-1. Thepercentageof â€˜@I-Lym-1bound were plotted versus the amount of iinmunoreactivecompetitor assayed. As indicated by lower â€˜@â€˜I-Lym-1binding, unmodified Lym-1competedmoreeffectively thanall immunoconjugates,andcompetitivenessamongimmunoconjugateswas observedto decline with increasingc/a; in fact, adding more andmore 2IT-BAT-Lym-1 with c/a of 19.1 had no observableeffect on the binding ofâ€˜@â€˜I-Lym-1.All assayswereconductedin duplicate.Points,mean;bars, rangeof data.

881

>

I 1@00C

EE 0.5a)>

@ 0.0

excess. In this experiment, the relative immunoreactivities of allpreparations of 21T-BAT-Lym-1 versus â€˜@I-Lym-1were measuredby solid phase RIA; then the immunoreactivity of the 1251-Lym-1standard was measured by the Lineweaver-Burk method. Some immunoconjugates were assayedby both methods to test the comparabilityof themethods.

By solid phaseRIA (Fig. 3), therewas no apparentlossof immunoreactivity at c/a of 5 or below. Even up to a c/a of 11, everyconjugate exhibited 75% or greater relative immunoreactivity; however, relative immunoreactivity declined to levels not suitable forimmunotherapy with further increases in c/a. The relative immunoreactivities (Â± 1 SD of the mean) of the four immunoconjugates withc/a 2.1, 4.3, 8.4, and 11.4, subsequently assayedby the LineweaverBurk method, were 109 Â±9, 104 Â±10, 80 Â±5, and 43 Â±4%,respectively.

By Lineweaver-Burk analysis(Fig. 4) the immunoreactivefractionof Lym-1 (Â±1 SD) was found to be 75 Â±6%, and the immunoreactive fractions of 2ff-BAT-Lym-1 with c/a 2.1, 4.3, 8.4, and 11.4were 82 Â±2, 79 Â±15, 58 Â±9, and 45 Â±1%, respectively. The SDswere basedon the 67% confidence interval of the Y intercept given bylinear regression of the data (Statview). For comparison to solid phaseRIA data, the relative immunoreactivity of, e.g., 21T-BAT-Lym-1

with c/a 2.1 was calculated to be 82 Â±2%/75 Â±6% = 109 Â±9%.Similarly, the relative immunoreactivities of the conjugates with c/a4.3, 8.4, and 11.4 were calculated to be 106 Â±22, 78 Â±13, and60 Â±5%, respectively,in good agreementwith the solid phaseRIAresults.

Competitive Cell-binding Assays. The assaytested the effectivenesswith which relatively largeandvarying amountsofunlabeled Lym-1and 2ff-BAT-Lym-1 immunoconjugates competed with tracer amounts

of â€˜@I-Lym-1for bindingto Raji cells. Because only the competitiveability of reactive antibody was of interest, the data were corrected toexcludeunreactivespecies.For example,Lym-1 had beenfoundto be75% immunoreactive by Lineweaver-Burk analysis; hence, 500 ng ofunmodified Lym-1 represents 0.75 X 500 ng = 375 ng of reactive

antibody. The immunoconjugate 21T-BAT-Lym-1 with a c/a of 13.9 had

beenfound to be 64% immunoreactive relative to unmodified Lym-1 bysolid phaseRIA hence,500 ng of the immunoconjugaterepresents0.64 X 0.75 X 500 ng = 240 ng of reactive conjugate.

@:io

0 5 10 15 20 25

Lym-1

21T-BAT-Lym-1:



IMMUNOREACTIVITY AND BIODISTRIBU11ON OF 21T-BAT-LYM-1

thebiologicalhalf-livesof radioactivityinthewholebodywere7.6,8.7,6.5, and 5.1 days for groups 1, 2, 3, and 4, respectively.

In a previous5-day study, the maximum uptake of radiolabeled2ff-BAT-Lym-1 in tumorin % ID/g wasobserved72 h postinjection(10). The biodistributiondatafrom the currentstudyat the 72-h timepoint suggesteda negative correlation between c/a of 2ff-BATLym-1anduptakeintumorandapositivecorrelationbetweenc/aanduptake in liver; however, these effects do not appear to be significantat c/a 4.3 or below (Fig. 7). Uptakeby othertissuesandorgansdid notappear to be affected by c/a. Linear regression was performed on thedata (Statview) to substantiatethe apparentcorrelations. In the caseof% ID/g tumor versusc/a, r@was 0.53 and the slope was negative overa 95% confidence interval; in the caseof % ID/g liver, r@was 0.62 andthe slope was positive over a 95% confidence interval (Fig. 8).

Mice receiving92 @Ciof @Cu-2ff-BAT-Lym-1,with themaximum uptake by tumor of radiolabeled conjugate observed in this

- U - study, received a dose of 32 rads to the tumor over the 72-h period of

72 study.Thisisequivalenttothedosetotumorthatwouldbedeliveredoverthesameperiodfrom a 1.8-@aCidoseof lZ@I@[email protected] higheractivitiesof â€˜@â€˜I-labeledantibodiesare routinelyusedfor biodistribution studieswith no observedtherapeuticeffect. It is concludedthatMCu@2ff@BAT@Lym@1administered to tumored mice at the levelsused in this study had no therapeutic effect.

DISCUSSION

The purposeof thisstudywasto determinethe maximumc/a atwhich the functional integrity of 2ff-BAT-Lym-1 is preserved. Therewas no detectible loss of immunoreactivity at c/a 5 or less, and 75%or more retentionof immunoreactivityup to c/a 11. Conjugatewithc/a 5.4 exhibited a small but observablelossof competitivenessforbinding to Raji cells. With increasing c/a, uptake of radiolabeledconjugateby tumor decreasedanduptakeby liver increased,but thiseffect was insignificant at c/a 4.3 or less.

The purposein increasingthe numberof metal-bindingsitesperantibody is to decrease the amount of immunoconjugate, in mg,required to deliver a therapeutic dose of radiometal. With increasingc/a, a smalleramountof immunoconjugateis requiredto carrya given

ci

U0 c/a I 1.4

24 h2520@15@10

50

30

25

,@3 20

Q150 10

50

72 hD Groupi

@ Group 2 (c/a 4.3)a Group3(c/a8.4)6 Group4(c/a11.4)

Tumor Liver Lung Muscle@ Kidney Brain Spleen HeartStomachBone

Fig. 7. Biodistribution of radiolabeled 2IT-BAT-Lym-1 of varying c/a in Raji-tumored mice 24 and 72 h postinjection. Higher c/a was associatedwith decreasing uptake in tumorand increasinguptake in liver. There was no observableeffect on the uptake into other organsand tissues.Columns,mean of 5 or more data points;bars, 1 SD.

882

100

70

504030

20

,â€˜@â€˜@t::@@

â€¢c/a2.1Oc/a4.3

0 12 24 36 48 60

Time (h)Fig. 6. Whole body ( ) and blood ( ) clearance of radiolabeled 2fF

BAT-Lym-1 of varyingc/a in Raji tumoredmice. In general,higherc/a wasassociatedwith more rapid clearance. The half-time for the first phase of blood clearance wasapproximately4 h for all species;thehalf-timesfor thesecondphaserangedfrom 2.8(c/a11.4) to 5.5 days(c/a 4.3). The half-timesfor whole body clearancerangedfrom 5.1 (c/a11.4) to 8.7 days(c/a 4.3).

Unmodified Lym-1 was observed to compete more effectively forRaji cell binding than were all 21T-BAT-Lym-1 immunoconjugatesthat were examined. The ability of 21T-BAT-Lym-1 immunoconjugates to compete effectively declined with increasing c/a, although theeffect at c/a 5.4 was slight (Fig. 5).

Biodistributlon Studies. Higher c/a was associatedwith fasterratesof blood and whole body clearance of 2ff-BAT-Lym-1 from mice (Fig.6). One-half of the injecteddoseclearedthe blood in every groupinapproximately 4 h. A slower phaseof clearancefollowed, with half-livesof 4.0, 5.5, 2.6, and 2.8 days for groups 1, 2, 3, and 4 (c/a 2.1, 4.3, 8.4,and 11.4), respectively.Whole bodyclearanceswere moreexponential;

10@



Table1 Total mCi of 67Cuand mg of 21T-BAT-Lym-1of varyingc/a requiredtodeliver 10 mCi/g tumor, 72 h post injection.

The calculationswere basedon a specific activity of 67Cu of 3.5 mCilug. Uptake intumor at 72 h wastakenfrom the mousebiodistributionstudy.

c/amCi/mg% ID/g tumorTotalmCi to deliver

10 mCi/g tumormgto deliver

10mCiJgtumor2.13.021.7103344.36.120.2111188.412.113.91611311.416.39.324115

IMMUNOREACI1VITY AND BIODISTRIBUTION OF 21T-BAT-LYM-1

activity of 67Cu;however, a smaller fraction of radiolabeled conjugateis delivered to the tumor, mitigating the advantage of higher specificactivity of radiolabeled conjugate. In Table 1, 2ff-BAT-Lym-1 immunoconjugates of c/a 2.1, 4.3, 8.4, and 11.4 were compared on thebasis of which immunoconjugate can deliver a specified dose of 67@to tumor using the minimum amount of antibody, based on theobserved tumor uptakes in mice. The dose was arbitrarily specified as10 mCi 67Cu/gtumor, 72 h postinjection. The calculations were basedon a typical 67(@ specific activity of 3.5 mCi/pg Cu. It is readilycalculated that 21T-BAT-Lym-1 of c/a 2.1 is radiolabeled with 67Cuwith a specific activity of 3.5 mCi4tg Cu to yield 67Cu-2ff-BATLym-1 with a specific activity of 3.0 mCi/mg Lym-1. At 72 h, theuptake of this conjugate in tumor was 21.7% ID/g; hence, a totalinjecteddoseof 103 mCi would be requiredto deliver 10 mCi/g tumor72 h later, after correction for decay. At 3.0 mCi/mg, 34 mg ofimmunoconjugate would be required to deliver this dose. Similarcalculations were performed for the other immunoconjugates examined in the biodistributionstudy.

The advantageof 21T-BAT-Lym-1 of c/a 4.3 versus2.1 is readilyapparent.Becausea smaller fraction of the c/a 4.3 immunoconjugate isdistributed to the tumor, slightly more 67(@(111 mCi) is required todeliver the desireddose.However, becauseof thegreaterspecific activityof 67Cu-2ff-BAT-Lym-1 of c/a 4.3, much less conjugate is required todeliver thedose(18 versus34 mg). Even lessconjugateofc/a 8.4 (13 mg)isneeded;however,dueto appreciablyloweruptakeby tumor,a muchhigher initialdose ofradionucide (161 mCi) is required.Due to the wasteof radionucide and higher radiation levels in the liver, a c/a of 8.4exceedstheoptimumratio.Theuptakeof conjugateof c/a11.4by tumorissolowthatmore67()@andmoreimmunoconjugate(versusc/a8.4)arerequired to deliver the desired dose.

ci

The effectiveness with which 21T-BAT-Lym-1 conjugates of increasing c/a competed with â€˜@I-Lym-1for binding to Raji cells wastaken to indicate the effect of chemical modification on the bindingaffinity of the antibody. Scatchard analysis is often used to makecomparative assessmentsof this type, but the validity of the method,which has no rigorous mathematical basis when applied to a bivalentsystem, is disputed (26, 27, 50). In this study, binding competitivenesswas observed to decline with increasing c/a. This may be attributed toan increasing fraction of immunoconjugate with one inactivated binding site (26). The increased fraction of immunoconjugate with bothbinding sites inactivated (i.e., nonimmunoreactive) contributes to thedecline in tumor uptake with increasing c/a (54). Strong anodal shiftsin p1 of immunoconjugates have been implicated elsewhere in lowertumor uptake (55), consistent with the observations in this study. Thissuggests a possible advantage in a 67Cu chelate conjugation systemwith a net neutral effect on antibody charge.

This effort to optimize the conjugation of 21T-BAT-Lym-1 coincided with efforts at BrookhavenNational Laboratoryto improvethespecific activity of 67Curadionuclide (56). The mean specific activity(Â± 1 SD) among 8 shipments of 67Cu received in 1992 was0.44 Â±0.36 mCi 67Cu4tg Cu; the comparable value for 6 shipments

received in 1994 was 3.8 Â±2.2 mCi/pg. To illustrate the practicalsignificance of the improvement in the quality of the radiometal andthe radiolabeling capacity of the immunoconjugate, using 67Cuwith atypical specific activity of 3.5 mCi/p.g, 2IT-BAT-Lym-1 with c/a 5.0,and allowing 24 h decay for preparation,a therapeuticdose of 100mCi 67Cu can be delivered on 26 mg of immunoconjugate.Previously, using 67Cuwith 0.5 mCi4i.g and 21T-BAT-Lym-1 with c/a 2.0,450 mg would be required to deliver the therapy dose. Optimizationof the c/a of 21T-BAT-Lym-1, coupled with significant improvementin the quality of 67Cu,represents important advances in 67Cutherapyby facilitating the dependable and routine preparation of 67Cu-2ITBAT-Lym-1 for therapeuticuse.

ACKNOWLEDGMENTh

We gratefullyacknowledgeDr. LeonardF. Mausner(BrookhavenNationalLaboratory) for helpful discussionsand supplying 67Q@;[@ Kurt Zinn (University of Missouri) and Carolyn A. Anderson (Washington University) for kindlysupplying64Cu;Dr.JustinMoran(UniversityofCalifomiaatDavis)for synthesisof BAT; Jim Schiffing (Protein Structure Laboratory, University of California atDavis) for performing amino acid analyses; and Dr. Rosemary A. Marusak(Universityof Californiaat Davis)for helpfuldiscussionsandassistance.

REFERENCES

1. DeNardo, 0. L., and DeNardo, S. J. Perspectiveson the future of radioimmunodiagnosisandradioimmunotherapyof cancer.In: S.W. BurchielandB. A. Rhodes(eds.),Radioimmunoimagingand Radioimmunotherapy,pp. 41â€”62.New York: ElsevierSciencePublishingCo., Inc., 1983.

2. Jungerman,J. A., Yu, K-H. P.,andZanelli, C. I. Radiationabsorbeddoseestimatesat thecellular level for someelectron-emittingradionuclidesfor radioimmunotherapy.Int. J. AppI. Radiat. Isot., 35: 883â€”888,1984.

3. DeNardo,0. L, DeNardo,S.J., Kukis, D. L, Dliii, H., Suey,C., andMeares,C. F.Strategiesfor the enhancementof radioimmunotherapy.NucI. Med. Biol., 18:633â€”640,1991.

4. Smith-Jones,P. M., Fridrich, R., Kaden, T. A., Novak-Hofer, I., Siebold, K., Tschu

40

30

20

10

0

10

2

00

c/aFig. 8. Tumor and liver uptake of MCu@2IT@BAT@Lym@1as a function of c/a, 72 h

postinjectionin Raji-tumoredmice.Linearregressionof thedatawasstronglyindicativethat c/a had a negativecorrelationto uptakein the tumor and a positive correlationtouptake in the liver: the regressionslopeswere nonzero in the 95% confidenceinterval(

883

2 4 6 8 10 12



IMMUNOREACFIVITY AND BIODISThIBUTION OF 21T-BAT-LYM-1

din, D., andMaecke,H. R. Antibody labelingwith copper-67usingthebifunctionalmacrocycle4-[(1, 4, 8, 11-tertaazacyclotetradec-1-yl)methyljbenzoicacid. BioconjugateChem.,2: 415â€”421,1991.

5. Morphy, J. R., Parker, D., Kataky, R., Harrison, A., Eaton, M. A. W., Millican, A.,Phipps,A., and Walker, C. Towards tumor targetingwith copper-radiolabelledmacrocycle-antibody conjugates. J. Chem. Soc. Chem. Commun., 792â€”794,1989.

6. Mercer-Smith,J. A@,Cole, D. A., Roberts@J. C., Lewis, D., Behr, M. J., andLavallee,D. K. The biodistributionof radiocopper-labeledcompounds.Adv. Exp. Med. Biol.,258:103â€”121,1989.

7. Roberts,J. C., Newmeyer,S. L, Mercer-Smith,J. A., Schreyer,S.A., andLavallee,D. K. Labeling antibodieswith copper radionucides using N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl)porphine.mt. j. Appl. Radiat.Isot.,40:775â€”781,1989.

8. DeNardo,0. L, DeNardo,S.J.,Meares,C. F., Kukis,D. L, Dull, H., McCall,M. J.,Adams,0. P.,Mausner,L F., Moody,D. C.,andDeshpande,S.V. Pharmacokineticsof copper-67 conjugatedLym-1, a potential therapeuticradioimmunoconjugate,inmice and in patientswith lymphoma.Antibody, Immunoconjugates,and Radiopharmaceuticals, 4: 777â€”785,1991.

9. Epstein,A. L, Zimmer, A. M., and Spies,S. M. Radioimmunodetectionof humanB-cell lymphomas with a radiolabeled tumor-specific monoclonal antibody (Lym-1).In: F. Cavalli, 0. Bonadonna,andM. Rozencweig(eds.),Malignant LymphomasandHodgkin's Disease:Experimentaland TherapeuticAdvances,pp. 569â€”577.Boston:MartinusNijhoff PublishingCo., 1985.

10. Deshpande,S. V., DeNardo, S. J., Meares, C. F., McCall, M. J., Adams, G. P., Moi,M. K., and DeNardo, 0. L Copper-67 labeled monoclonal antibody Lym-1, apotential radiopharmaceutical for cancer therapy: labeling and biodistribution in Rajitumoredmice. J. Nuci. Med., 29: 217â€”225,1988.

11. Moi, M. K., DeNardo,S.J.,andMeares,C. F. Stablebifunctionalchelatesof metalsusedin radiotherapy.CancerRes.(Suppl.),50: 789sâ€”793s,1990.

12. McCall, M. J., Diril, H., and Meares,C. F. Simplified methodfor conjugatingmacrocycle bifunctional chelating agents to antibodies via 2-iminothiolane. Bioconjugate Chem., 2: 222â€”226,1990.

13. Kukis,D. L, Li, M., andMeares,C. F.Theselectivityof antibody-chelateconjugatesfor binding copper in the presence of competing metals. Inorg. Chem., 32:3981â€”3982,1993.

14. Kukis,D. L, Diril, H., Greiner,D. P.,DeNardo,S.J.,DeNardo,0. L, Salako,Q.A.,and Meares,C. F. A comparativestudy of copper-67radiolabelingand kineticstabilities of antibody-macrocycle chelate conjugates. Cancer (Phila.), 73 (Suppl.):779â€”786,1994.

15. Mausner,L. F., and Srivastava,S. C. Selectionof radionuclidesfor radioimmunotherapy.Med.Phys.,20: 503â€”509,1993.

16. Yin, 0., Narula, J., Nossiff, N., and Khaw, B. A., Correlationof immunoreactivityandpolymerformationto DTPA modificationof a monoclonalantibody.NucLMed.Biol., 18: 859â€”864,1991.

17. Sinkule,J. A., Rosen,S. T., and Radosevich,J. A. Monoclonalantibody44â€”3A6doxorubicinimmunoconjugates:comparativein vitro anti-tumorefficacyof differentconjugationmethods.TumourBiol., 12: 198â€”206,1991.

18. Reilly, R., Lee,N., Houle,S.,Law,J., andMarks,A. In vitro stabilityof EDTA andDTPA immunoconjugatesof monoclonalantibody203 labeledwith indium-ill.AppI. Radiat. Isot., 43: 961â€”967,1992.

19. Slinkin, M. A., Klibanov, A. L, Khew, B. A., and Torchilin, V. P. Succinylatedpolylysine as a possible link betweenan antibody moleculeand deferoxamine.BioconjugateChem.,4: 291â€”295,1990.

20. Paik, C. H., Ebbert, M. A., Murphy, P. R., Lassman, C. R., Reba, R. C., Eckelman,w. C.,Pak,K.Y.,Powe,J, Steplewski,Z.,andKoprowski,H.FactorsinfluencingDTPA conjugationwith antibodiesby cyclic DTPA anhydride.J. Nucl. Med., 24:1158â€”1163,1983.

21. Carney,P.L., Rogers,P.E.,andJohnson,D. K. A dualisotopestudyof 1@@Iand @â€˜@Inlabeledantibodyin athymicmice.J. Nucl. Med.,30: 374â€”384,1989.

22. Sumerdon,0. A., Rogers,P.E., Lombardo,C. M., Schnobrich,K. E., Melvin, S.L,Hobart, E. D., Tribby, I. I., Stroupe, S. D., and Johnson, D. K. An optimizedantibody-chelatorconjugatefor imagingof carcinoembryonicantigenwith indium111. NucI. Med. Biol., 17: 247â€”254,1990.

23. Visser, 0. W., Gerretsen,M., Herscheid,J. D., Snow, 0. B., andvan Dongen,0. Labelingof monoclonalantibodieswith rhenium-186usingtheMAG3 chelatefor radioimmunotherapyof cancer@a technicalprotocoLJ. NucLMed.,34: 1953â€”i%3,1993.

24. Bedel-Cloutour,C. H., Maneta-Peyret,L., Pereyre,M., andBezian,J.H. Synthesisofa monoclonalantibody-indium-ill-porphyrin conjugate.J. Immunol. Methods,144:35â€”41,1991.

25. Law, K. L, Studtmann, K. E., Carlson, R. E., Swanson, T. A., Buirge, A. W., andAhmad, A. Tumor reactivecis-aconitylatedmonoclonalantibodiescoupledto daunorubicin througha peptidespacerareunableto kill tumorcells.AnticancerRes.,10:845â€”852,1990.

26. Ong,0. L, andMattes,M. J. Re-evaluationof theconceptof functionalaffinity asapplied to bivalent antibody binding to cell surfaceantigens.Mol. ImmunoL, 30:1455â€”1462,1993.

27. Kyriakos,R. J., Shih,L B., Ong,0. L, Patel,IC, Goldenberg,D. M., andMattes,M. J. The fate of antibodiesboundto the surfaceof tumorcells in vitro. CancerRes.,52: 835â€”842,1992.

28. Li, M., Meares, C. F., Thong, G-R., Micra, L, Xiong, C-Y., and DeNardo, S. J.Labeling monoclonal antibodieswith @Â°ytttiumand 111indium-DOTA chelates:asimpleandefficient method.BioconjugateChem.,5: 101â€”104,1994.

29. Meares,C. F., McCall, M. J.,Deshpande,S.V., DeNardo,S.J.,andGoodwin,D. A.Chelateradiochemistry:cleavablelinkerslead to alteredlevelsof radioactivityin theliver. mt. J. Cancer(Suppl.), 2: 99â€”102,1988.

30. Beatty, B. G., Paxton, R. J., Hawthorne, M. F., Williams, L E., Rickard-Dickson,K. J., Do, 1., Shively, J. E., and Beatty, J. D. Pharmacokinetics of an anti-carcinoembryonic antigen monoclonal antibody conjugated to a bifunctional transition metalcarboranecomplex(venusflytrap cluster)in tumor-bearingmice.J. Nucl. Med.,34:1294â€”1302,1993.

31. hard, M. E.,Boniface,0. R.,Hardiman,K. L, Brchbiel,M. W., Gansow,0. A., andWalkers,K. 1 An improvedmethodfor labelingmonoclonalantibodieswith samarium-153:useof thebifunctionalchelate2-(p-isothiocyanatobenzy)-6-methyldiethylenetriaminepentaaceticacid.BioconjugateChem.,3: 346â€”350,1992.

32. Subramanian,R., Colony, J., Shaban, S., Sidrak, H., Haspel, M. V., Pomato, N.,Hamia, M. 0., Jr., and MCCabe, R. P. New chelating agent for attaching indium-illto monoclonalantibodies:in vitro and in vivo evaluation.BioconjugateChess.,3:248â€”255,1992.

33. Weber, R. W., Bourn, R. H., Nedelman, M. A., Lister-James,J., and Dean, R. T.Enhancedkidney clearancewith an ester-linked @Tc-radioinbeledantibodyFab'-chelatorconjugate.BioconjugateChem., 1: 431â€”437,1990.

34. DeNardo, 0. L, Kroger, L A., DeNardo, S. J., Micra, L A., Salako, 0. A., Kukis,D. L, Fand, I., Shen,S., Renn,0., andMesses,C. F. Comparativetoxicity studiesofyttrium-90 MX-DTPA and 2-fl-BAD conjugated monoclonal antibody (BrE.3).Cancer (Phila.), 73 (Suppl.): 1012â€”1022,1994.

35.Mathias,C.J.,Sun,Y.,Welch,M.J.,Connett,J.M.,Philpott,G.W.,andMattel,A. E. N@'-bis2-hydroxybenzyl@l@bromoacethmidobenzyl)-1,2-ehtylenedismineN,N'-diacetic acid: a new bifunctional chelate for radiolabeling antibodies. BioconjugateChem.,1: 204â€”211,1990.

36.Weilman,A.A.,andMeares,C.F.SequencesoftheLym-1antibodyheavyandlightchainvariableregions.NucleicACIdSRes.,18:5281,1990.

37. Kabat,E. A., Wu, T. T., Reid-Miller, M., Perry,H. M, and Gottesman,K. S. In.@Sequencesof proteinsof immunologicalinterest,Ed. 4. Washington,DC: UnitedStatesDepartmentof HealthandHumanServices,1987.

38.Rana,T.M.,andMeares,C.F.N-terminalmodificationOfiInmUnOgIObUIinpolypeptidechains taggedwith isothiocyanatochelates.BiOconjUgateQieia. 1: 357-362 1990.

39.DeNardo,0. L, KmhnK A., andDeNardo,S. J. Comparisonof oncophilicradiopharmaceuticals, *I@@ge@, 67@ft@ta, â€œâ€˜In-bleomycin,and eIbl@y@in tumorbearingmice.Cancer(Phila.),40: 2923-2929,1977.

40. Epstein, A. L, Marder, R. J., Winter, J. N., Stathopoulos, E., (len, F.M., Parker,J.W.,andTaylor,C.R.Twonewmonoclonalantibodies,Lym-landLym-2,reactivewith humanB-lymphocytesandderivedtumors,with ImmUnOdiagnOstiCandimmunotherapeuticpotential.CancerRca.,47: 830â€”840,1987.

41. Hellstrom, K. E., Yelton, D. E., Fell, H. P., Beaton, D., Gayle, M., MacLean, M.Kahn, M., and Hellstron, I. Epitope mapping and use of anti-idiotopic antibodies tothe L6 monoclonal anticarcinoma antibody. Cancer Rca, 50: 2449-2454, 1990.

42. Hellstrom,I., Horn,D., Linsley,P.,Bmwn,J.P.,Brankovan,V., andHellstrom,K. E.Monoclonalantibodiesraised againsthuman lung carcinomas.CancerRae.,46:3917â€”3923,1986.

43. Moi, M. K., Meares, C. F., McCall, M. J., Cole, W. C., and DeNardO,S. J. Copperchelates asprobes ofbiological systems: stable copper complexes with a macrocyclicbifunctionalchelatingagent.Anal. Biochem.,148: 249â€”253,1985.

44. Penefsky, H. S. A centrifuged-column procedure for the measurement of ligandbinding by beef heart F1. Methods Enzymol., 56: 527â€”530,1979.

45. Meares,C. F., McCall,M. J.,Reardan,D. T., Goodwin,D. A., Diamanti,C. I., andMcTigue,M. Conjugationof antibodieswith bifunctionalchelatingagents:iaothiocyanateandbromoacetamidereagents,methodsof analysis,andsubsequentadditionof metalions.Anal. Biochem.,142: 68-78, 1984.

46. DeNardo, 0. L, DeNardo, S. J., Lamborn, K. R., van Hoosear, K. A., and Kroger,L A. Enhancement of tumor uptake of monoclonal antibody in nude mice with PEGIL-2. Antibody, Immunoconjugates, and Radiopharmaceuticals, 4: 859â€”870,1991.

47. DeNardo,S. J., Peng,J-S. B., DeNardO,G. L, Mills, S. L, and Epstein,A. LImmunochemicalaspectsof monoclonalantibodiesimportantfor radiopharmaceutical development. Nucl. Med. Biol., 13: 303â€”310,1986.

48. Lineweaver, H., and Burk, D. The determination of enzyme dissociation constants.J.Am.Chem.Soc.,56:656-666,1934.

49.Lindmo,T.,Boyce,E.,CUttitta,F.,Fedorko,J.,andBurnt,Jr.,P.A.Determinationofthe immunoreactivefractionof radiolabeledmonoclonalantibodiesby linearextrapolationto bindingat infinite antigenexcess.J. ImmunoLMethods,72: 77â€”89,1984.

50. Kaufman,E. N., andJam,R. K. Effect ofbivalent interactionuponapparentantibodyaffinity: experimental confirmation of theory using fluorescence photobleaching andimplicationsfor antibody-bindingassays.CancerRca.,52: 835-842, 1992.

51. Adams,0. P., DeNardO,S. J.,Deshpande,S. V., DeNude, 0. L, Meares,C. F., McCall,tvL J., and Epstein,A. L Effect ofmass of 111In-bcnzyl-EDTA monoclonal antibody onhe@c uptake and processingin mice. Cancer Rca.,49: 1707-1711, 1989.

52. DeNardo,0. L, DeNardo,S.J.,O'Grady,L F.,Levy,N. B.,Mama, G.P.,andMills,S. L Fractionatedradioimmunotherapyof B-cellmalignancieswith 131I-Lym-1.CancerRca.(Suppl.),52: lOi4sâ€”1016s,1990.

53. Harlow, E., and Lane, D. In: J. Cuddihy (ed), Antibodies:a LaboratoryManual, p.673. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988.

54.Yokoyama,K., Reynolds,J.C.,Paik,C.H.,Sand,V. K., Maloney,P.J.,Larson,S. M., andReba,R. C. Immunoreactivitiyaffectsthebiodistributionandtumortargeting of radiolabeled anti-P97 Fab fragment. I. Nucl. Med., 31: 202â€”210,1990.

55. Slinkin,M. A., Curtet,C.,Faivre-Chauvet,A., Sai-Maurel,C.,Gestin,J.F.,Torchilin,V. P., andChatal,J. F. Biodistributionofanti-CEA F(ab')@fragmentsconjugatedwithchelatingpolymers:influenceofconjugateelectronchargeontumoruptakeandbloodclearance.NucL Med. Biol., 20: 443â€”452,1993.

56. Kolsky, K. L, Joshi,V., Meinken, G. E., Sweet, M., Mausner,L F., and Srivastava,S. C. Improvedproduction,andevaluation,of Cu-67 for tumor radioimmunotherapy(abstract).J. Nucl. Med. (SuppL),35: 259P,1994.

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1995;55:878-884. Cancer Res David L. Kukis, Gerald L. DeNardo, Sally J. DeNardo, et al. ImmunoconjugatesImmunoreactivity and Biodistribution of 2IT-BAT-Lym-1 Effect of the Extent of Chelate Substitution on the

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