(CANCER RESEARCH 51.676-681, January 15. 1991|

I86Re Radioimmunotherapy of Small Cell Lung Carcinoma Xenografts

in Nude Mice

Paul L. Beaumier,1 Prasanna Venkatesan, Jean-Luc Vanderheyden, William D. Burgua, Lawrence L. Kunz,

Alan R. Fritzberg, Paul G. Abrams, and Alton C. Morgan, Jr.

NeoRx Corporation, Seattle, Washington 981/9

ABSTRACT

A '"Re-labeled monoclonal antibody (MAb), NR-LL'-10, was used for

the radioimmunotherapy of a subcutaneous human small cell lung carcinoma xenograft, SI 11-I, in nude mice. Biodistribution with specific andirrelevant labeled MAb demonstrated peak tumor uptake of 8% and 3%of the injected dose/g at 2 days, respectively. Dosimetry analysis predictedtumor:»hole-bodyradiation-absorbed dose ratios of 2.43:1 for NR-LU-10 and 0.62:1 for irrelevant MAb. Single-dose toxicity screening estimated a 50% lethal dose within 30 days of 600 ,/( i (880 cGy of whole-body radiation). As anticipated, a multiple-dose regimen of 490 nCi infour doses over 10 days (720 cCy of whole-body radiation, eight of eightsurviving >30 days) was less toxic than a single bolus dose of 430 fid(644 cGy of whole-body radiation), six of eight surviving >30 days). Amultidose radioimmunotherapy regimen was initiated in nude mice bearing 06-111111'tumors (total dose, 500 to 600 nCi). Complete remissions

(>140 days) were achieved in three of 16 mice, and the remainder showeda mean tumor growth delay of 53 days. Matched doses with irrelevantMAb produced one remission, one treatment-related death, and a meangrowth delay of only 20 days in six of eight mice. Thus, in this nonoptimalradioimmunotherapy model, significant antitumor responses were observed using a mildly toxic multiple dosing regimen.

INTRODUCTION

Several recents reports (1-4) have demonstrated the efficacyof radioimmunotherapy using radiolabeled antibodies against avariety of experimental human tumors including hepatoma aswell as colon, small cell lung, and breast carcinoma. '"I and90Y are the most frequently cited /a emitters incorporated inradioimmunoconjugates. "'I offers advantages of familiarityand relatively simple labeling, comparatively long physical half-life, appropriate particle energy and path length (r«,2= 0.83

mm) (5), and acceptable postmetabolic biodistribution. Its disadvantages include the metabolic lability of the o-iodotyrosyllabel, a problem under active investigation (6, 7), and y emissions that account for two-thirds of its released energy producing an undesirable penetrating absorbed dose to the whole bodyin patients and exposure of medical personnel. 9"Y offers the

advantages of strong particle energy, comparatively long pathlength (rw = 5.34 mm) (5), and an appropriate physical half-life. Its disadvantages include chelate instability, an area underactive investigation, ferric ion-like postmetabolic distributionto radiosensitive marrow space, and the absence of imageable7 emissions helpful for monitoring tumor targeting and developing dosimetry estimates (8).

Received 6/12/90: accepted 10/26/90.The costs of publication of this article were defrayed in pan by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at NeoRx Corporation.410 W. Harrison. Seattle. WA 98119.

2The abbreviations used are: r„>,radius of a sphere in mm within which 90ccof the energy of a point source is absorbed; MAb. monoclonal antibody; RIT.radioimmunotherapy; SCLC, small cell lung carcinoma: N3S, triamidothiolate:MAGG. mercaptoacetylglycylglycyl--y-aminobutyrate; TLC, thin-layer chroma-tography; HPLC, high-performance liquid chromatography; II >,,„,.50Cf lethaldose within 30 days.

186Reis intermediate in fi particle energy and physical half-life between '"I and 90Y. '""Re offers the advantages of an

appropriate /rfparticle energy and path length (rw = 1.80 mm)(5), a sufficiently long physical half-life, an ideal y emission,and good postmetabolic distribution kinetics. Continued progress in the pursuit of improved chelates for 99l"Tc-antibodylabeling has produced in parallel good chelates for its "matchedpair" rhenium (9).

Building on experience with Wn'Tc-labeled antibodies forimaging, the ligand technology was extended to """Re to eval

uate its therapeutic potential as a MAb conjugate. A transitionalSCLC xenograft was selected as a model target since this typeof cancer is known to be relatively radiosensitive and thereforeappropriate to evaluate efficacy ( 10). A multiple dosing regimenwas chosen to take advantage of antibody delivery kinetics andreduced toxicity. It was postulated that repeated doses at sufficiently short intervals would maintain continuous low dose rateexposure to tumor, while clearance from the whole body wouldpermit intermittent recovery of normal tissues during the latephase of each dose cycle.

Considering the moderate uptake of radiolabeled MAb, theinitiation of treatment against well-established tumors, and theadministration of doses well below the tolerable limit, a non-optimal experimental regimen was tailored to reflect betterradioimmunotherapy in human clinical trials. We describe forthe first time experimental evaluation of a l86Re-radioimmu-

noconjugate in a nude mouse human xenograft model.

MATERIALS AND METHODS

Monoclonal Antibodies. NR-LU-IO. a murine monoclonal antibody(IgG2b), recognizes a M, 40,000 glycoprotcin antigen associated withepithelial tumors (11, 12). NR-ML-05, a murine monoclonal antibody(IgG2b), recognizes a high-molecular-weight proteoglycan antigen ofmelanoma ( 13) and was used as an irrelevant isotyped matched control,since it is not cross-reactive with the NR-LU-10 epitope. NR-LU-10Fab affinity was measured at 4.4 x 10gM~' and recognizes an estimated3.3 x 10' sites on cultured SCLC cells as assayed by Scatchard analysis

using a published technique (14). The monoclonal antibodies wereproduced commercially by in vitro fermentation (Invitron Corp.. St.Louis, MO) and purified to >98cc using aniónexchange chromatog

raphy.SHT-1 SCLC Cell Line. The SHT-1 cell line was originally derived

from malignant pleural effusion taken from a patient with primary,untreated small cell lung carcinoma and maintained continuously intissue culture (15). The SHT-1 line is anchorage independent and growsin culture as loose clumps characteristic of transitional small cellcarcinoma. Cell cultures were periodically checked and shown to beMycoplasma free (GenProbe, San Diego. CA). Cells were expanded ina cell factory (Nunc, Roskilde, Denmark) and maintained in Dulbecco'smodified Eagle's medium supplemented with 5% fetal calf scrum, 5%

Serum Plus (Hazelton Biologies, Lenexa, KS), a 4 HIMi.-glutamine,and 2 niM sodium pyruvate (Whitaker Bioproducts. Walkersville, MD).Flow cytometric analysis of cultured SHT-1 cells using direct conjugates of NR-LU-10 MAb and standard techniques was used to confirmstable expression of the target antigen (16).

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RHENIUM-186 RADIOLABELING OF ANTIBODIES

,0

HN NM

Fig. 1. "'Re antibody labeling scheme, including perrhenate reduction andcomplex formation and coupling of "'Re-MAGG preformed chelate via amide

linkage to antibody.

SHT-1 SCLC Xenograft. Eight-wk-old female National Cancer Institute nu/nu mice (Simonsen, Gilroy, CA) used in these studies wereidentified individually with ear tags, housed 5/cage in microisolators ina temperature- and humidity-controlled environment, and maintainedon sterilized Lab Blox chow and water ad libitum. Treated mice wereisolated in a shielded area with bedding changed daily during theexcretion of radioactivity.

Growth characteristics for the SHT-1 xenograft were established inpreliminary experiments in which a series of cell concentrations wereimplanted s.c. in the left side midline of nude mice. Injection of 5 xIO6 cells resulted in >60% implantation and consistent growth rates(tumor volume reached roughly 100 mm3 in 14 days). SHT-1 xenograftmorphology was examined by hematoxylin-eosin staining of flash-frozen cryostat sections using standard techniques (16). Antigen density-was evaluated using biotinylated NR-LU-10 applied at 10 ¿jg/mlto 5-fim-thick frozen sections and detected with streptavidin-horseradishperoxidase at 1 Mg/ml, followed by 0.5 mg/ml of 3,3'-diaminobenzidine

(16). Autoradiography was done on flash-frozen sections using IlfordK5D emulsion prepared in 2% glycerol (Ilford, Ltd., Basildon, Essex,United Kingdom) (16). Slides were viewed and photographed under adark field to enhance contrast.

""Re Radiolabeling Procedure. 186Re (tv, = 89.2 h,3 1.07-MeV ßenergy maximum, 137-keV y (9.2% abundance), 0.73-g*cGy/MCi*haverage equilibrium dose constant (17), was obtained from the MissouriUniversity Research Reactor. As outlined in the labeling scheme shownin Fig. 1, perrhenate was purified following a published procedure (18),reduced with stannous citrate, and chelated to a tetrafluorophenyl-activated ester derivative of a N3S ligand system, MAGG (19). Thepreformed IS6Recomplex was coupled to 15 to 20 mg of intact MAb

by the reaction of the active ester with lysine amino groups resulting ina covalent amide linkage. Labeled antibody was purified by gel filtrationand/or microcentrifugation to >95% as assayed by thin-layer chroma-tography. Protein concentration was determined spectrophotometri-cally measuring absorbance at 280 nm. The injected dose was standardized to 75 ^g/mouse by adding unlabeled MAb as needed. Immuno-reactivity was assessed by a rad iolabeled cell-binding assay (14). Labelstability was confirmed by TLC and HPLC under multiple challengeconditions including incubation at 37°Cin serum and in 40 m,viDTPA,

7 HIMphosphate-buffered isotonic saline over 24 h. For multiple dosestudies fresh preparations were prepared for administration on Days 0and 7, and portions of these same preparations were repurified, assayed,and used for administration on Days 3 and 10, respectively.

Biodistribution of Labeled Antibodies. Biodistribution studies wereconducted to quantify immunospecific localization and to provide abasis for developing dosimetry estimates. The biodistribution of NR-LU-10-MAGG-'86Re and NR-ML-05-MAGG-'86Re was performed

separately in tumored nude mice at 1, 2, 5, and 7 days. The injecteddose (percentage) was calculated from dilution standard and theweighed volume of injectate. At selected time postinjection, groups of

3 D. D. Hoppes. National Institute of Standards and Technology, personal

communication.

4 mice/time point were weighed, bled via the retroorbital plexus, andsacrificed by cervical dislocation. Thirteen organs and tissues werecollected: blood; tail: tumor; skin; muscle: bone; lung: liver: spleen;stomach; neck; kidneys; and intestine. Tissues were blotted when appropriate, weighed, and counted along with standards of the injecteddose in a 7 scintillation counter (Packard Instrument Co., LagunaHills, CA) setting the energy window at 40 to 1000 keV to bracket the137-keV photopeak and other lower abundance photons of '86Re. Blood

was taken as 8.0% of the body weight (20). Other tissues (skin, muscle,and bone) were taken as 18.4%, 44.5%, and 4.7% of the body weight,respectively, based on biodistribution analysis of 50 normal nude mice.Taken with the other whole-organ values listed above, these percentagesallowed estimation of the percentage of injected dose retained in thewhole body. Limited biodistribution studies were also carried out fordosimetry estimation. Radioactivity remaining in the whole body wasdetermined directly by dose calibrator (Capintec, Inc., Ramsey, NJ)ignoring tumor which accounted for <3% of the injected dose. Micro-curies in the tumor were determined by calibration of count rates withradioactivity standards measured by dose calibration.

Radioimmunotherapy Toxicity Screening. To determine the toxicityof 186ReRIT, single- and multiple-dose screening studies were under

taken. To estimate the maximum tolerable dose, each of four groups of7 to 8 mice received an escalating, single i.v. injection of '86Re-NR-LU-

10 MAb, averaging 294, 430, 579, and 721 pd at a specific activity of5.8 /jCi/Mg. Body weight and survival were followed as indicators oftoxicity. Linear regression of the percentage of survival versus meanpCi administered and whole-body absorbed dose allowed the approxi

mation of an LDso/jo.A comparison was made between a group of mice receiving a single

bolus dose of 430 MCiof 186Re-labeled NR-LU-10 MAb and anothergroup of mice which received radiolabeled MAb in a multiple-doseregimen (177,88, 168, and 57 /¿Cion Days 0, 3. 7, and 10, respectively,totalling 490 pCi). The toxicities of the single- and multiple-doseregimens were compared by monitoring WBC counts twice/wk. Twenty^1 of whole blood were sampled from the retroorbital plexus, diluted inphysiological phosphate-buffered saline, 0.5 mivi EDTA, and countedin triplicate using a Sysmex blood cell analyzer (Toa Medical Electronics, Ltd., Kobe, Japan). The two closest determinations were averaged,and the outlier was dropped.

Radioimmunotherapy Study Design. A multiple-dose protocol con

sisting of single doses of 75 /ig of protein and up to 300 ^Ci/injectionto each of 8 mice/group was initiated. Doses of 186Re-labeledNR-LU-

10 were administered on Days 0 and 7 or on Days 0, 3, 7, and 10.Control groups received two or four doses of labeled irrelevant NR-ML-05 at matched specific activity, and two additional control groupsreceived matched doses of unlabeled NR-LU-10 or saline. Treatmentregimen parameters are detailed in Table 1.

Dosimetry Estimation Method. Dosimetry estimates for tumor andwhole body were calculated from decay-corrected biodistribution dataand tumor and whole-body counting. Only the nonpenetrating radiationcomponent was considered, and homogeneous distribution throughouttumor or whole body was assumed. Complete radiation absorption wasassumed in the whole body. Dosimetry to the tumor was adjusted usingan infinite volume boundary' correction factor of 0.75 to account for

the size of the tumors used in these studies (21). These approximationsare reasonable, given the low abundance of the 186Rey emission and

the mean range of its ßparticle (90% deposition within 2 mm). Theabsorbed dose was calculated by integration of the time activity usingnonlinear curve-fitting software (Rstrip; MicroMath Scientific Software, Salt Lake City, UT) or the trapezoidal method.

Table 1 Dose regimens and dosimetry estimates

Mean ^Ci administered onMAb treatment Tumor Whole

(frequency) Day 0 Day 3 Day 7 Day 10 (cGy) body (cGy)

NR-LU-10 (2 doses) 213+ 279 =492 2012 828NR-ML-05 (2 doses) 124+ 282 =406 468 752NR-LU-10 (4 doses) 218+ 60+280+ 45 =602 2671 1099NR-ML-05 (4 doses) 135+197+271+20 =623 727 1167

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EXPERIMENTAL "*Re RADIOIMMUNOTHERAPY

Therapeutic Efficacy Evaluation. Mice bearing uniformly sized tumors were selected from a larger group of implanted animals. Tumordimensions were followed until a diameter of >20 mm was attained,after which the animals were sacrificed. Digital calipers (Mitutoyo, Inc.,Tokyo, Japan) were used to measure tumor length, width, and heighttwice/wk. Tumor progression was followed by monitoring volume,calculated using the formula for the volume of an ellipsoid

Tumor mass could be linearly correlated with volume (assuming I mg= 1 mm3) in agreement with observations made by Tomayko and

Reynolds (22) relating tumor mass to volume. Tumor volume doublingtime was determined by linear regression of the log of tumor volumeversus time. Mean growth delay was measured based on the averagenumber of days required for tumor volume to reach 900% of the initialvolume (taken as 100% at the start of treatment) relative to control.This criterion was selected since xenograft growth reaches midpoint atthis stage in its exponential growth pattern. The statistical significanceof tumor growth delay was assessed using ranked analysis of variance(Stata; Computing Research Center, Santa Monica, CA). Tumor remissions were defined as the absence of detectable tumor beyond 140days posttreatment.

RESULTS

In Vitro Assessment of Labeled Antibody. Radiolabeled antibody was obtained at an overall yield of 40%, the metalion:MAb mohmol substitution ratio ranging from 0.4 to 1.6:1,with a specific activity of 2 to 6 fiCi/ng. Label stability studieswith either 186Re-labeled NR-LU-10 or NR-ML-05, relative toradioiodinated control, demonstrated a loss of <2% of protein-bound activity/day. The cell-binding assay of freshly radiola-beled NR-LU-10 gave an absolute immunoreactivity of 64 ±10% (n = 5) (>95% of trace-radioiodinated control) usingfreshly harvested, cultured SHT-1 cells. The cell-bound percentage for radiolabeled irrelevant NR-ML-05 MAb was <3%.Stability and immunoreactivity measurements for repurifiedpreparations at 3 days postlabeling were equivalent to freshlylabeled preparations.

Innnunohistology of SHT-1 Xenografts. Immunohistologicalanalysis of SHT-1 xenograft sections revealed an antigenexpression summary reactivity of 60%. (Summary reactivityvalues, 0 to 100%, are calculated from a weighted combinationof antibody reactivity values measuring intensity, uniformity,and percentage of cells antigen positive.) Structurally, SHT-1consists of poorly differentiated, neuroendocrine-like carcinoma cells distributed throughout a delicate murine fibrovas-

cular stroma. Tumors are uniformly well vascularized withminimal presence of ischemie necrosis. Xenografts variedgreatly in tumor/stromal tissue composition. The tumor accounted for 35 to 95% and stromal matrix for 5 to 65% of thexenograft cross-sectional area. Dark field-enhanced autoradi-ography revealed homogeneously distributed radiolabel scattered throughout the xenograft (Fig. 2). Flow cytometric analysis of the viable cell fraction obtained from mechanically dissociated SHT-1 xenografts confirmed stable antigen expressionon the viable high forward scatter cells as indicated by maintenance of a consistently high fluorescein equivalents ratio whichaveraged 28.7 ±3.1 (n = 6). Parallel evaluation of control MAbNR-ML-05 indicated background expression as confirmed bya fluorescein equivalents ratio of <2.0.

Biodistribution and Dosimetry Estimates. Biodistribution datafor immunospecific NR-LU-10 and control NR-ML-05 MAbs

are summarized in Table 2. As expected, both specific andcontrol antibody characteristically cleared slowly from bloodand whole body. Moderate uptake of specific MAb in tumorreached 8.3% of injected dose/g at 1 to 2 days compared withcontrol MAb which reached only 3.1% of injected dose/g in thesame interval (Table 2). Tumor:whole-body absorbed dose ratios based on decay-corrected data were estimated at 2.52:1 forNR-LU-10 and 0.80:1 for NR-ML-05.

The time activity profiles for a 200-¿iCidose of '86Re-labeledNR-LU-10 and NR-ML-05 in tumor and whole body are shownin Fig. 3. The area under the curve predicted radiation absorbeddoses of 1211 ¿¿Ci»h/gto tumor and 499 /¿Ci*h/gto wholebody for NR-LU-10, compared with 290 ^CUh/g and 469/iCi*h/g for NR-ML-05. These results point out the approximate 4-fold delivery advantage attained with tumor-specificMAb.

Toxicology Screen of l86Re RIT. The single-dose toxicity

screening study estimated that doses averaging 294, 430, 579,and 721 ^Ci deliveredy 442, 644, 867, and 1033 cGy to thewhole body with 8 of 8, 6 of 8, 5 of 8, and 2 of 8 mice,respectively, surviving more than 30 days. From linear regression analysis based on the fraction surviving versus the whole-body absorbed dose [X= -0.0011*X(cGy) + 1.532, r2 = 0.94]and the fraction surviving versus fiC\ administered [Y =-0.0011*X (cGy) + 1.496, r2 = 0.96], an approximate LD50/3oof 600 pC\ (880 cGy of whole-body radiation) was calculated.A mean body weight depression of >10% was noted in the twohighest dose treatment groups and was accompanied by a dropin WBC count to a nadir of 20% of initial on Day 15 withprolonged and incomplete recovery. Control WBC countsshowed a wide fluctuation from 9,000 to 13,000 cells/mm' as

reported by others (2) as well myeloproliferation apparentlyinduced by blood sampling (Fig. 4).

A single dose of 430 /¿Ci(see above) resulted in delayedrecovery of WBC counts which remained depressed in 3 of 6surviving mice at 2 mo posttreatment (Fig. 4). In contrast, 490¿iCiadministered in multiple doses delivered 700 cGy to thewhole body and were considerably less toxic. All mice treatedunder the multiple-dose regimen survived, and WBC countsreturned to initial levels in 6 of 6 mice within 24 days.

Whole-body clearance based on dose calibrator measurements from 0 to 211 h estimated a monoexponential half-lifeof 44.4 h with a 5% SD.

Response of SHT-1 Xenografts to ""Re RIT. SHT-1 s.c. tu

mors progressed with a doubling time of 4.54 days | Y [log(vol,mm')] = 0.066** (days) + 1.60, r1 = 0.99. Xenograft volume

could be linearly correlated to measured weight by linear regression relating xenograft volume to weight: Y (mm3) = 0.74*^(mg) + 12.21 (r2 = 0.97, n = 19).

Dosimetry estimates, summarized in Table 1, indicate thecomparable whole-body dose delivered by l86Re-labeled relevant

and control MAb (within 10%). A comparison of treatmentregimen efficacy based on tumor growth delay is compiled inTable 3. Since values for the relevant NR-LU-10 (2 doses, 70.8±17.5 days, n = 8; 4 doses, 68.7 ±9.1 days, n = 7) regimenswere equivalent, and values for the unlabeled MAb (12.6 ±2.0days, n = 8) and saline (14.3 ±2.9, n = 8) regimens were alsoequivalent, these results were condensed into a NR-LU-10group and a control group. Data for the NR-ML-05 (2 dosesand 4 doses) regimens, however, were statistically distinct andare presented separately.

Tumor volume progression in response to the above-described treatment regimens is illustrated in Fig. 5. Plotted on a

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EXPERIMENTAL IMRe RADIOIMMUNOTHERAPY

Fig. 2. Dark field enhanced autoradiography of a s.c. SHT-1 SCLC xenograft, illustrating the uniform distribution of grains (""Re radiolabel) throughout thetumor cross-section.

Table 2 Biodislribution ofNR-LU-IO MAb-MAGG-'^Re and NR-ML-05 MAb-MAGG-'^Re in nude mice grafted with SHT-I SCLC s.c. tumors

Tissue 1 day 2 days 5 days 7 days

Specific NR-LU-10 (% of injected dose/ g oftissue)BloodTailTumorSkinMuscleBoneLungLiverSpleenStomachNeckKidneyIntestineWhole

body12.852.238.253.101.081.214.032.912.550.492.992.140.82*2.56±

0.45"±0.48±

0.82±0.22±0.07±0.18±0.24±0.78±

0.46±0.11±

0.34±0.55±0.12±0.1411.471.508.292.550.870.873.141.701.640.472.331.710.572.10Control

NR-ML-05 (% ofinBloodTailTumorSkinMuscleBoneLungLiverSpleenStomachNeckKidneyIntestineWhole

body10.342.353.032.751.101.103.862.272.330.862.652.301.11*2.43±

1.95±0.42±0.76±0.43±0.25±0.26±

1.02±0.56±

0.72±0.10±0.76±0.61±

0.25±0.418.521.363.062.440.790.782.581.881.730.521.620.271.080.430.150.060.520.280.200.080.210.140.150.304.76:0.86

dt0.75t

0.196.13±1.291.08

±0.170.38dt0.040.47±0.052.01

d0.91:t0.35t

0.150.96±0.140.26dbO.081.29±0.030.74:0.22:t0.17bO.040.91

±0.113.250.514.970.92±

0.44±0.06±0.16±0.150.40

+0.120.281.050.670.540.270.730.510.210.72±0.04±0.08+

0.09±0.09±0.07±0.04±0.04±0.02±0.11ected

dose/ g oftissue)0.920.130.780.380.110.060.100.110.240.181.92

±0.101.45±0.160.68±0.111.94

±0.184.80:b

0.460.78±0.071.301.310.430.411.801.030.930.251.060.890.36:1.04

:0.610.050.020.030.290.090.080.060.150.11bO.05bO.042.630.470.830.760.240.310.960.570.520.240.710.500.220.62±

0.44+0.07+0.38±0.17+

0.02±0.03±0.10±0.12±0.09±0.03±0.13±0.04±0.02±0.59

°Mean ±SD; 4 animals per time point.6 Whole-body percentage of injected dose/g excludes tumor and was deter

mined from the percentage of injected dose remaining in all other organs,including scaled estimates for blood, bone, muscle, and skin taken as the percentage of body weight.

semilog scale, the different curves are nearly linear and parallelin the 500 to 2000% of initial volume range, indicating thatonce escaping therapeutic control, tumor progression reverts to

75 100 125TIME (HR)

150 175 200

Fig. 3. NR-LU-10-""Re in tumor (O) and whole body (A) (absorbed dose ratio2.43:1); NR-ML-05-"6Re control in tumor (•)and whole body (A) (absorbeddose ratio, 0.62:1). Mice received 189 pd i.v. SHT-1 xenograft volume over alltime points averaged 123 ±59 mm3 in mice weighing 24.2 ±4.0 g (n = 32) withn = 4 mice/data point.

its characteristic initial exponential growth rate. Treatmentwith saline or unlabeled NR-LU-10 had no measurable effecton tumor progression, and the volume doubling time was equivalent to that of untreated controls.

Even though control NR-ML-05 does not target tumor specifically, '*6Re-NR-ML-05 (2 doses and 4 doses) treatment didproduce significant growth delays. NR-ML-05 (2 doses) delivered 468 cGy to tumor and produced a 10-day mean growthdelay statistically different from control (P < 0.001) or NR-ML-05 (4 doses) (P < 0.001) regimens (Table 3). The NR-ML-05 (4 doses) regimen delivered >700 cGy to tumor and produced a 20-day mean growth delay. This treatment regimenwas lethal to one of 8 mice (1200 cGy of whole-body radiation),presumably due to hematopoietic toxicity. Additionally, NR-ML-05 (4 doses) produced one remission (>140 days).

The I86Re-NR-LU-10 regimen delivered -2000 to 2700 cGyto tumor. This dose produced a 53-day mean growth delay that

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EXPERIMENTAL ""Re RADIOIMMUNOTHERAPY

' .

. O

-•i

:,

2020 25 30

TIME (DAYS)

Fig. 4. O, mean percentage of initial WBC count in untreated control mice. D,mean percentage of initial WBC count after a single i.v. injection of 430 fid ofNR-LU-10-MAGG-ls6Re. Six of 8 mice survived >30 days. •.mean percentageof initial WBC after multiple injections on Days 0, 3. 7, and 10 totaling 490 /jCiof NR-LU-IO-MAGG-'"Re. All treated mice survived. Bars, SD.

Table 3 Therapeutic responses

Days for tumortoreach900% ofini-MAb

treatment (frequency)0 tialvolumeNR-LU-10

(2 doses, 4 doses) 66.4 ±12.6 (n =13)NR-ML-05(2 doses) 23.5 ±7.3 (n =8)NR-ML-05(4 doses) 33.3 ±7.2 (n =6)'Controls

(4 doses, 4 doses/ 13.4 ±2.8 (n = 16)Tumor

remission*3010Statisticalsignificance'<O.OOI1/<0.001<0.001

* Treatment groups consisted of 8 mice (combined groups. 16).* No visible nodule present at 140 days posttreatment.' Level of significance relative to controls using Scheffé'smultiple comparison

test in analysis of variance of ranks.' NR-LU-10 growth delay was also statistically different (<0.01) from NR-

ML-05 (2 doses and 4 doses).' One mouse died.'Saline or unlabeled NR-LU-10 MAb.

1200 r

0t l

20 30 40 50DAYS POST-TREATMENT

60

Fig. 5. SHT-1 s.c. xenograft progression after various treatment regimensbegun 16 days postimplantation. O, four doses of saline or cold specific MAb (n= 16). A, two doses of "*Re-NR-ML-05 irrelevant MAb administered on Days 0and 7 (total 418 fiCi; n = 8). A, four doses of "'Re-NR-ML-05 irrelevant MAbadministered on Days 0, 3, 7, and 10 (total 797 pCi; n = 8). D, two (506-^Citotal) or four doses (735-jiCi total) of specific '"Re-NR-LU-10 MAb administeredon Days 0 and 7 or on Days 0, 3, 7. and 10. Xenograft volume at the start oftreatment averaged 65.8 ±50.4 mm3 in mice weighing 23.8 ±1.8 g (n = 48). The

time to 900% of starting xenograft volume was arbitrarily chosen as the standardfor comparison of tumor growth delay.

was statistically different from any of the ""'Re-NR-ML-05 (2doses or 4 doses) regimens (P < 0.01). The 186Re-NR-LU-10

regimen produced complete but transient tumor regression in6 of 16 mice which persisted an average of 39 days prior torelapse. There were three remissions that appeared durable andshowed no evidence of recurrence at 140 days. Mean tumor

volume decreased to a nadir of 27% of initial volume 21 daysfrom the start of treatment.

DISCUSSION

We present in detail for the first time an experimental evaluation of 186Re-labeled MAb for RIT or SCLC. 186ReNxSychelated antibody was developed as the therapeutic "matchedpair" analogue of 99mTcNxSy chelated antibody (23), because

the two radionuclides have remarkably similar coordinationchemistry. The in vitro integrity of the immunoconjugate post-labeling was confirmed by the demonstration of complete retention of immunoreactivity and negligible label loss followingchelate or serum challenge. In vivo studies demonstrated tumortargeting and retention of radiolabel in tumor, substantiatingthe metabolic durability of the 186Re(V)-MAGG "covalent"

chelation and amide linkage chemistries.Among human solid tumors, SCLC is considered to be one

of the most sensitive to radiation; thus, it may be especiallyamenable to RIT (3, 24, 25). Accordingly, the SCLC xenograft,SHT-1, was selected as a relevant experimental model. Histológica!examination revealed uniform morphology, rich vascu-larization, and moderate antigen expression. Autoradiographyconfirmed the uniform distribution of radiolabel throughoutthe tumor cross-section. Tumor-specific MAb uptake (8.3% ofinjected dose/g) was modest and consistent with data reportedby others using an SCLC model (25). In spite of moderateimmunospecific tumor uptake, a 4-fold tumonwhole-body absorbed dose advantage was achieved with """Re-NR-LU-lO ascompared with irrelevant """Re-NR-ML-05 (Fig. 3).

Toxicity screening measured by WBC recovery and survivalindicated that 490 pCi of '"'Re-labeled MAb administered in

multiple doses were less toxic than a single dose of 430 ¿/Ci(Fig. 4). We estimated an LD50/.„,of 600 ¿(Ci(880 cGy of whole-body radiation) for a single bolus dose of "IARe-NR-LU-10 in

nude mice. This is less than the LDsn/.mof 900 cGy reportedfor immunocompetent mice exposed to acute total-body irradiation (26). We have also found (data not shown) that a totaldose of 1200 ¿¿Ci(3020 cGy of whole-body radiation) administered in weekly fractions of 300 //Ci permitted the survival of5 of 16 mice for more than 30 days. By interpolation based onthe single dose toxicity screening study, this is equivalent to713 /uCi (1039 cGy of whole-body radiation). These findingssuggest the clinical advantage attainable with MAb-deliveredradiation using a multidose regimen, assuming human anti-mouse antiglobulin response can be avoided by, for example,the use of chimeras.

A significant antitumor response, including 3 remissions of>140 days in duration and a prolonged mean tumor growthdelay of 53 days, was demonstrated by using specifically targeted radiation. There was no difference in therapeutic responsebetween the NR-LU-10 (2 doses and 4 doses) regimens, probably because the midweek doses (see Table 1) were small. In 13of the 16 mice treated with labeled NR-LU-10, tumors recurred.

Recurrences arose from inadequately targeted or relativelyinsensitive cells' growth limited by oxygen or nutrient depriva

tion. Such cells would cycle through radiosensitive mitosis lessfrequently during peak radioimmunotherapeutic intensity (26).This hypothesis is compatible with (a) the interpretation offeredby Duchesne and Peacock (27) regarding radiation sensitivityof SCLC spheroids ¡nvitro; (b) the temporal pattern of extendedgrowth delay observed (Fig. 5), since outgrowth could onlyoccur after reestablishment of good vascular supply; and (c) our

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EXPERIMENTAL ""Re RADIOIMML'NOTHERAPY

observation (data not shown) that durable compléteremissions(>150 days) could be achieved in 10 of 10 mice bearing rapidlyproliferating tumors (treating at 7 days postimplantation, 17-mm' tumor volume). Since a single dose of 240 fid of 186Re-

NR-LU-10 (2175 cGy to tumor) was apparently tumoricidal, aradioresistant cell population had not developed in these tumors. This finding was dependent on immunospecific targeting,since 8 of 10 tumors recurred in mice treated with a matcheddose (210 fid, 308 cGy to tumor) of irrelevant MAb. Insummary, these animal model data suggest that IS6Re-labeledM Ab can reduce but not cure 60- to 100-mm' tumor masses,but may be able to cure 20-mm3 tumors.

It is interesting to consider the implications of 186Reenergydeposition (r90 = 1.80 mm), given the 2- to 5-mm diameter ofthese xenografts. At these low /uCi and tumor-absorbed doseestimates, little energy wastage could occur without nullifyingthe response. Energy deposition therefore must be relatively"local," restricted to zones well accessed by antibody carrier

distributed via vascular supply. These results support the efficacy, and even advantage, of the moderately energetic ßof l86Re

that deposits most of its released energy throughout and withintumor.

Clinical trials are currently under way with 186Re-immuno-

conjugates (28) and have to date demonstrated instances ofgood tumor targeting and a general lack of retention in normalorgans such as liver, spleen, and bone. Dosimetry estimates areencouraging, and an objective response has been confirmed atthe 150-mCi dose level in an escalating single-dose Phase Istudy. Future animal studies will focus on evaluating RIT in amultiple low-dose regimen and in combined modality therapy.

ACKNOWLEDGMENTS

We wish to thank Jeff Parkins for the Scatchard analysis, LauraMorgan for immunoreactivity assessment, and Rich Klein for flowcytometric analysis. We thank Becky Bottino who supplied antibodyand Catherine Jackson and Denise Du Pont for providing excellenttechnical assistance in therapy and biodistribution studies. Joe Bugajassisted in '8<1Relabeling, and Nick Ranieri and Lolan Chang contrib

uted histopathology support. We gratefully acknowledge Gary Ehrhardtof the Missouri University Research Reactor for supplying ""Re and

Debra Leith for reviewing the manuscript and providing helpful editorial comments. We thank Susan Duke for performing the rankedanalysis of variance statistical evaluation of the therapy data. We thankBarry Wessels and Ellen Yorke for assistance in tumor dosimetryestimate calculation.

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1991;51:676-681. Cancer Res   Paul L. Beaumier, Prasanna Venkatesan, Jean-Luc Vanderheyden, et al.   Xenografts in Nude Mice

Re Radioimmunotherapy of Small Cell Lung Carcinoma186

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