Effect of Recombinant a-Interferon on Pharmacokinetics ... · (Rhochek, Rhomed Inc., Albuquerque, NM). The injected ra-diopharmaceutical was always sterile and free of pyrogens as

Vol. 3, 1547-1555, September 1997 Clinical Cancer Research 1547

Effect of Recombinant a-Interferon on Pharmacokinetics,

Biodistribution, Toxicity, and Efficacy of ‘311-Labeled

Monoclonal Antibody CC49 in Breast Cancer:

A Phase II Trial’

Daniel J. Macey, Edward J. Grant, Leela Kasi,

Michael G. Rosenblum, Hua-Zhong Zhang,

Ruth L. Katz, Paula T. Rieger, Donna LeBherz,

Michael South, John W. Greiner, Jeffrey Schiom,

Donald A. Podoloff, and James L. Murray2

National Cancer Institute, Bethesda, Maryland 20815 [J. W. G., J. S.],and the University of Texas M. D. Anderson Cancer Center, Houston,Texas 77030 [D. J. M., E. J. G., L. K., M. G. R., H-Z. Z., R. L. K.,P. T. R., D. L., M. S., D. A. P., J. L. M.]

ABSTRACT

Preclinical studies have demonstrated that recombinantIFN-ot (rIFN.a) can enhance the tumor associated glycopro-

tein 72 (TAG-72) on tumors. To determine whether rIFN-a

could enhance TAG-72 expression in vivo in patients, 15

women with breast cancer were randomized to receive dailyinjections of rIFN-a (3 x 106 units/m2 for 14 days) begin-

ning on day 1 (group 1 7 patients) or on day 6 (group 28 patients). On day 3, all patients received a 10-20-mCi

tracer dose of ‘311-CC49, a high-affinity murine monoclonalantibody reactive against TAG-72, followed by a therapy

dose of 60-75 mCi/m2 of ‘311-CC49 on day 6. Whole body

and single-photon emission computed tomography scansalong with whole blood pharmacokinetics were performed

following tracer and treatment phases. Hematological toxic-ity was considerable; reversible grade 3-4 neutropenia andthrombocytopenia was observed in 12 of 15 patients. Twelve

of 14 patients tested developed human antimouse antibodies3-6 weeks after treatment. For group 1 patients, whole

blood residence time increased significantly between thatpredicted from the tracer doses and therapy doses (42.6 ±

4.7 versus 51.5 ± 4.8 h, respectively; P < 0.01). The calcu-lated radiation absorbed dose to red marrow from therapycompared to tracer activity was also significantly higher forthis group (1.25 ± 0.35 versus 1.07 ± 0.26 cGy/mCi; P <

0.05). Treatment with rIFN-ot was found to enhance TAG-72

expression in tumors from patients receiving rIFN-a (group

1) by 46 ± 19% (P < 0.05) compared to only 1.3 ± 0.95%

in patients not initially receiving IFN (group 2). The uptakeof CC49 in tumors was also significantly increased in rIFN-

a-treated patients. One partial and two minor tumor re-sponses were seen. In summary, rIFN-a treatment alteredthe pharmacokinetics and tumor uptake of ‘311-CC49 in

patients at the expense of increased toxicity.

INTRODUCTION

Radioimmunotherapy of solid tumors with MoAbs3 has

met with limited success due to poor tumor targeting of antibody

(tumor uptakes of 0.001-0.01 %ID/gram; Ref. 1). Hence, meth-

ods to improve tumor uptake of MoAbs and diminish normal

organ toxicity are of paramount importance.

Most solid tumors are heterogeneous with respect to sur-

face antigen expression; in fact, some tumors may express little

or no antigen, thereby preventing MoAbs or immunoconjugates

from binding to a sufficient number of cells to elicit a therapeu-

tic response. Putative methods to overcome antigenic heteroge-

neity might be the use of antibody “cocktails” (2, 3) or methods

to enhance surface antigen expression (4). IFN-a and IFN--y

have been shown to enhance expression of several tumor-spe-

cific antigens both in vitro (5-8) and when administered to mice

bearing human tumors xenografts (9-1 1). Among several breast

cancer-associated antigens, TAG-72, a high molecular weight

glycoprotein, has been shown to be significantly increased on

tumor cells in vitro following exposure to rIFN (12-14). Recent

studies have shown improved tumor targeting, as well as effi-

cacy, of radiolabeled CC49 (a second-generation, high-affinity

murine MoAb against TAG-72) when given in combination

with IFN-�1i in mice bearing TAG-72-positive tumors (15).

On the basis of the above, the main objectives of this study

were to determine the following: (a) whether rIFN-a could

enhance TAG-72 expression and uptake of ‘31I-labeled CC49

(‘31I-CC49) in breast cancer patients; (b) the toxicity of 1311..

CC49 plus rIFN-a; (c) whether rIFN-a could modify the bio-

distribution and pharmacokinetics of CC49; and (6) the response

rate to the combination.

Received 2/3/97; revised 5/19/97; accepted 5/28/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I Supported by United States Public Health Service Contract CM97610

from the National Cancer Institute2 To whom requests for reprints should be addressed, at the Universityof Texas M. D. Anderson Cancer Center, Box 002, 15 15 Holcombe

Boulevard, Houston, Texas 77030.

3 The abbreviations used are: MoAb, monoclonal antibody; TAG, tu-mor-associated glycoprotein; rIFN-a, recombinant IFN alpha; WBTV2;

whole body half-life; BT#{189},blood half-life; WBRT, whole body resi-dence time; BRT, blood residence time; SPECT, single-photon emission

computed tomography; %IA, percentage of injected activity; %ID,percentage of injected dose; ROI, region of interest; HAMA, humanantimouse antibody; MA, mean absorbance; CEA, carcinoembryonic

antigen.

Research. on March 28, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

RANDOMIZE

NO IFN-a

131I-CC49 PUNCH BIOPSY

�2 3 4 5

t t1311-CC49 PUNCH BIOPSY

DELAYED IFN-a : GROUP 2(begin IFN-a) If -�

6 7 8 9 10 11 12 13 14

(etc., off IFN-a Day 20)

Fig. I Schedule of ‘ #{176}1-CC49and rIFN-a administration. Patients began daily rIFN-a injection either on day 1 (group I) or day 6, correspondingwith the therapy dose of ‘311-CC49 (group 2). All patients received 14 days of rIFN-a treatment.

1548 Phase II Trial of CC49 and IFN

Diagnostic Phase (outpatient) Treatment Phase (inpatient)

2�1!.BEGIN IFN-a (3 x 106 units/rn2) -

A GROUP 1 1311-CC497 6 7 8 9 10 11 12 13 141 2 3 4 5

t t

PATIENTS AND METHODS

Patient Eligibility. To be eligible for the study, all pa-

tients had to have a Karnofsky performance status of greater

than 70% and life expectancy of �3 months. Normal hepatic

(bilirubin �2 mg/dl), renal (creatinine �2 mg/dl), and hemato-

logical (absolute granulocytes �2000/mm3; platelets � 100,000/

mm3) function was required for entry into the study. To deter-

mine whether rIFN-a was capable of modulating TAG-72

expression in vivo, all patients were required to have one or

more tumors accessible for punch or excisional biopsy (i.e. , skin

or lymph nodes). This protocol was approved by the National

Cancer Institute and the Surveillance Committee at the Univer-

sity of Texas M. D. Anderson Cancer Center. All patients gave

written informed consent to participate in the study.

Radiolabeled Antibody Preparation. CC49 was pro-

duced using conventional hybridoma techniques and purified

under good manufacturing practice conditions by Dow Chemi-

cal Company (Cincinnati, OH). The MoAb was provided by the

Cancer Treatment Evaluation Program, National Cancer Insti-

tute, in sterile vials containing PBS at a total mass of 15.9

mg/vial ( 10.6 mg/ml). CC49 was radiolabeled with 60-75

mCi/m2 of high specific activity ‘�‘I (500-700 mCi/ml; Du-

pont-NEN, Boston, MA) at a specific activity of 60 p.g of

protein per mCi of 1311 to be used via the Iodogen technique and

purified as described previously ( 16). Purified, radiolabeled

CC49 was collected through a 0.2-p.m sterile filter. Labeling

yield was estimated, and aliquots were taken for quality control

tests. Unlabeled MoAb then was added to the preparation so that

the total mass of protein was 15 mg, and the volume was made

up to 50 ml with 0.05 M phosphate buffer (pH 7.4) containing

1 % human serum albumin for injection.

The labeling yield was 80 ± 5% after purification. The

radiochemical purity and protein-bound iodine were consis-

tently greater than 95%. The immunoreactivity of the labeled

MoAb, as determined by the percentage of radiolabeled CC49

that bound to antigen-coated beads, was greater than 75%

(Rhochek, Rhomed Inc., Albuquerque, NM). The injected ra-

diopharmaceutical was always sterile and free of pyrogens as

assayed using Bactec Sterility Kits (Johnson Laboratories, a

subsidiary of Becton Dickinson, Baltimore, MD) and the

Limulus assay (Whitaker Bioproducts, Walkersville, MD),

respectively.

IFN-ot. Roferon-A (Hoffman-La Roche) was supplied

for this trial by the National Cancer Institute as sterile lyophi-

lized powder for parenteral administration in two concentra-

tions, 3 X 106 and 18 x 106 units. Prior to use, the contents of

each vial were reconstituted with 1 ml of sterile water for

injection. After reconstitution, each via] contained 3 X 106 or

18 X 106 units of rIFN-a along with 9 mg of sodium chloride

and 5 mg of human albumin.

Study Design. Patients were randomized into two groups

using the balanced block method (i.e., patients balanced every

four subjects for a two-armed study), based on the sequence of

rIFN-a administration (Fig. 1 ). For group 1 (7 patients), rIFN-a

was administered s.c. at a dose of 3 X 106 units/m2/day for a

total of 14 days beginning on day 1. Group 2 patients (n = 8)

received the same schedule of injections beginning on day 6. On

day 3, all patients received a tracer amount (10-20 mCi) of

‘31I-CC49 along with 15 mg of unlabeled MoAb iv. over 1 h.

Vital signs (blood pressure, pulse, and respiration) were moni-

tored during and up to 1 h after the infusion. Two days prior to

MoAb infusion, all patients received Lugol’s solution (SSKI-lO

drops p.o. daily for 14 days) to block uptake of free 1311 in the

thyroid. Four total-body Anger camera anterior and posterior

images were acquired at 4, 24, 48, and 72 h after infusion. ROI

counts detected in an Anger camera image from individual

tumors in a patient could be determined with a reproducibility

within ± 10%. All images were acquired using a 20% window

centered over the 364-keV photopeak. Whole blood samples

were collected at various times after the start of the ‘31I-CC49

infusion. Isotope decay-corrected plasma cpm were used to

construct clearance curves for each patient. Whole body reten-

tion of radioactivity was established from the 364-KeV photo-

peak counts recorded with a Nal probe located 1 m from the

patient at various times postinjection as described previously

( 17). The accuracy of whole body retention measurements with

this probe approach was validated with the reciprocal of urine

excretion of Ho-l66 phosphorate DOTMP and was found to

agree within 10% ( I 7). WBT#{189} was determined for each patient

from a monoexponetial fit of the decay-corrected clearance data.

On day 6, the absorbed dose to red marrow was calculated

from the tracer dose of I 3 ‘I-CC49. If this was less than 200 cGy,

patients then went on to receive 60-75 mCi/m2 of ‘31I-CC49

infused over 1 h on a designated high-activity ward in the

hospital. IFN-cs was given 1-2 h prior to ‘31I-CC49 and then

daily for 14 days. The activity used was derived from a previous

Phase I study in breast cancer patients in which the maximum

tolerated dose was 75 mCi/m2 (18). Vital signs were monitored



Clinical Cancer Research 1549

as described. Whole blood samples were collected at various

intervals to measure the clearance of ‘311-CC49 and compared

with the total body retention of radioactivity. Whole BRT and

WBRT, which are the integral of the %IA versus time (area

under the curve) were also calculated for the tracer and therapy

doses. Patients were discharged from the hospital when the total

body activity of ‘�‘I was estimated to be �30 mCi, equivalent

to a dose rate of 5 mRem/h at 1 meter. Following discharge, all

patients had an additional total body image acquired as

described above.

Red Marrow Dose Estimation. The red marrow dose

for each patient was calculated from WBT#{189}and blood retention

data measured for both tracer and therapy activities adminis-

tered to each patient, as described previously (19), using the

Tulane table to estimate blood volume from the height and

weight of each patient (20, 21).

Tumor Dosimetry. Radiation absorbed dose estimates

were calculated for all tumor sites that were visualized in serial

Anger camera images from the number of counts detected after

background substraction. For chest wall tumors, a ROI was

drawn around the perimeter of the uptake site, and a background

region was selected in the area adjacent. For axilla uptake sites,

a contralateral region was selected for background substraction.

The net counts after background substraction were converted to

activity using the sensitivity measured from the image of a

standard lO-ml source of 131J (1% of the administered ‘�‘I) that

was included in every Anger camera image and placed along-

side the boundary of the body. The sensitivity of the Anger

camera whole body image is derived from the number of counts

detected from this source in air and used with no attenuation

correction because lesions with demonstrated uptake were soft

tissue lesions on the chest wall or axillary lymph nodes. The

tumor activity measured on each imaging day was corrected for

physical decay of ‘�‘I to the time of administration of the

radiopharmaceutical and then converted to %IA using the fol-

lowing equation:

[CR01 � Cbgrd],.

%IA,(tumor) = S X A c 100

where [CR01 _Cbgrd] represents the counts detected at time

after administration in a ROI and a background region of the

same size, S is the sensitivity of the camera/collimator system

cps/p.Ci, and A is the administered ‘�‘I in p.Ci.

A trapezoidal method was used to calculate the area under

the curve of %IA versus time for each source in the body.

The area under the curve of %IA versus time (residence

time) was calculated for each tumor site using a trapezoidal

integration scheme that was implemented in a spreadsheet. An

extrapolation scheme was used from the last measured point to

0 assuming that the activity is 0 at 10 times the biological

half-time of the uptake site. The volume of each tumor site was

estimated from a set of contiguous transverse section images

that were reconstructed from a SPECT study that was acquired

at day 4 after the patient received radionuclide therapy.

The SPECT images indicated that the uptake sites were

usually highly irregular and followed the contour of the chest

wall. Because no S values were available for these irregularly

shaped source regions, radiation absorbed dose to each tumor

was calculated by assuming the dose to each site was a combi-

nation of energy deposited by the �3 particles emitted by ‘� 11 and

a photon dose contribution equivalent to the penetrating fraction

from activity in the total body. This was defined as follows:

Dtumor(tumor) = D,�(tumor) + D�(who1e body)

where � (tumor) is the tumor self dose from the �3 particles

emitted by the ‘�‘I in the tumor and D�(total body) is the photon

dose from all of the 131J in the whole body. This equation

applies only to superficial lesions in the chest wall and axilla,

and the sensitivity measured from the image of the l0-ml source

in air was used for all of the activity calculations described in

this study. It is also based on the assumptions that all of the �3

particles from 1311 in the tumors are absorbed locally and the

penetrating fraction of the total ‘�‘I flux in the whole body has

no appreciable gradient across these small tumor volumes. The

reproducibility of this technique for an individual patient is

greater than 90%.

Radiometric and Immunohistochemical Analyses of

TAG-72 and CC49 in Tumor Biopsies. Tumor biopsies were

weighed and analyzed per gram for this %IA, as well as TAG-72

expression and CC49 uptake, in 13 of the 15 patients (2 patients

had insufficient tumor samples for analysis) using quantitative

immunohistochemistry as described previously using a SAMBA

4000 image analyzer (22) as reported previously (23). Data were

expressed as MA index, which is equivalent to a mean labeling

concentration (22).

Measurement of HAMA Response. Blood for HAMA

assay was drawn from each patient 1 week before the study and

at 6-8 weeks. Serum was analyzed for the presence of HAMA

using an Immunostrip#{174} ELISA according to the specification of

the manufacturer (Immunomedics, Warren, NJ; Ref. 16). Values

above 74 ng/ml were considered significant. This value repre-

sents 2 SDs above the mean value of HAMA obtained in sera of

25 healthy individuals.

Response Criteria. Patients were followed at appropri-

ate intervals to assess toxicity and response. Appropriate radi-

ographic and other studies were repeated at 8 weeks after

initiating therapy. Responses were assessed using standard cri-

teria (24).

Statistical Analyses. Comparison of mean ± SD phar-

macokinetic parameters between group I and group 2 patients

was analyzed using the t test for independent means. Differences

in TAG-72 expression as determined by binding of CC49 and

CC49 uptake in tumor by quantitative immunohistochemistry

pre- and post-IFN-a treatment or in patients not receiving IFN-a

were analyzed using the paired t test. This test was also used to

compare differences in BT#{189}and WBT#{189} and residence times

between the tracer and therapy doses of ‘31I-CC49.

RESULTS

Patient Characteristics. Fifteen women with a mean age

of S 1 years were studied. Mean Karnofsky performance status

was 90%. Metastatic sites were primarily skin and soft tissue (14

of 15 patients), lung (6 of 15), liver (3 of 15), and bone (3 of 15).

Fourteen of the 15 patients had received more than one course

of combination chemotherapy, and 10 had received local radi-



0

><

0

7.5

6.0

4.5

3.0

1.5

0.0

I-

>-ImI-m-IC,,

><

0

0 1 2 3 4 5 6 7 8 9 10


Weeks Post Rx

Fig. 2 Hematological toxicity. Significant decreases in WBC and

platelet counts occurred at 4-6 weeks posttreatment; the counts recov-ered by 8-10 weeks.

ation to isolated bone metastases or to chest wall skin recur-

rences.

Toxicity. There were no instances of nonhematological

toxicity from ‘311-CC49. Therapeutic doses of ‘311-CC49 rang-

ing from 60 to 75 mCi/m2 of 1311 caused reversible grade 3-4

hematological toxicity in 12 of the 15 patients treated (Fig. 2).

The initial four patients who received 75 mCi/m2 of ‘31I-CC49

plus rIFN-a developed grade 4 thrombocytopenia with platelets

<25,000 at 4-6 weeks posttreatment; hence, subsequent pa-

tients received ‘� ‘I-CC49 at a reduced activity level of 60

mCi/rn2. Overall, WBC count fell from a mean ± SE of 7400 ±

900 cells/mm3 to 2300 ± 760 cells/mm3 by day 26 of therapy

(range, 14-43) and recovered by 8 weeks. Absolute granulo-

cytes decreased from 53 18 ± 826 to 1083 ± 251 over the same

time interval. Mean ± SE platelet count decreased from

288,000 ± 22,000 to 35,000 ± 9,000. There were no differences

in mean ± SE platelet count between group 1 (34,000 ± 6,800)

and group 2 (37,000 ± 4,500). Similar findings were noted for

WBC count. There was a slight yet significant prolongation of

time before platelets fell below 100,000 in group 2 patients (6 ±

0.2 days) compared to group 1 (4 ± 0. 1 days; P = 0.05). Two

patients had persistent grade 3-4 thrombocytopenia requiring

multiple platelet transfusions. One patient did not recover from

thrombocytopenia despite platelet transfusions and growth fac-

tor support and eventually died of progressive disease.

HAMA Responses. HAMA responses on all 15 patients

are shown in Table 1 . No patient had measurable HAMA prior

to infusion of 131I-CC49. Titers became elevated in 9 of 14

patients studied at 3 weeks post-tracer dose. By 6 weeks, four

patients who were HAMA negative (<74 ng/ml) at 3 weeks had

values greater than 2,000 ng/ml, which is above the limit of

detection by the assay. An additional three patients (patients 3,

5, and 9) also had marked increases in HAMA (>2,000 ng/ml)

at 6 weeks. Interestingly, six patients who were either HAMA

positive or HAMA negative at 3 weeks had no change (patients

8 and 10) or decreases in HAMA by 6 weeks (patients 2, 13, 14,

and 15).

Total Body and Blood Pharmacokinetics of ‘311-CC49.The disappearance curves or biological half-times (tl,2) for

I 3 1 I-CC49 in blood and whole body expressed in h are summa-

Table 1 HAMA re sponse (ng/ml)

Patient Pretreatment 3 wk 6 wk

1

2

3

4

5

67

89

10

11

12

13

1415

<74

<74

<74

<74

<74

<74<74

<74<74<74

<74

<74

<74

<74<74

<74

>2000

201

<74

307

>2000<74

<74

165>2000

NT”

<74

99

>2000380

>2000

460>2000

>2000

>2000

>2000>2000

<74

>2000>2000

NT

405

<74

230<74

a NT, not tested.

Table 2 Total body and blood p harmacokinetics of ‘31I-CC49

Tracer dose Therapy doseParameter (10-20 mCi) (60-75 mCi/m2) P

WBT 1/2 (h)Group 1 82 ± 8” 78 ± 6 NS”Group 2 80 ± 13 63 ± 8 NS

Totalc 81 ± 86 70 ± 5 NS

BT 1/2 (h)Group 1 48 ± 5 56 ± 8 NS

Group 2 43 ± 5 41 ± 6 NSTotal 45±4 48±5 NS

WBRT (h)Group 1 101.0 ± 5 92 ± 5 .07

Group 2 102.6 ± 7 79 ± 67 <0.01

Total 102 ± 6 86 ± 6 NSBRT (h)

Group 1 43 ± 5 51 ± 5 <0.01

Group 2 48.8 46 ± 7 NS

Total 45±7 49±6 NS

a Mean ± SE.b NS, not significant.C Groups 1 and 2 combined.

rized in Table 2. In the first four patients, serial blood samples

were not collected during therapy. Hence, BT#{189}was calculated

for only 1 1 of 15 patients. In short, there were no significant

differences in mean ± SE WBT#{189} between tracer dose (81 ±

8 h) and therapy dose (70 ± 6 h; P > 0.05). Similar findings

were observed for BT#{189}(tracer dose, 45 ± 4 h; therapy dose,

48 ± 5 h; P > 0.05). There were also no significant differences

in WBT#{189} and BT#{189}between group 1 patients (i.e., those

receiving rIFN-a beginning on day I) and group 2 (those

receiving rIFN-a beginning on day 6; Table 2).

In contrast to the above results, in group 1 patients there

was a slight yet significant prolongation in mean ± SE BRT

(i.e., the %ID over time) for the therapy dose of ‘31I-CC49

compared to that predicted from the diagnostic or tracer dose

(43 ± 5 versus 51 ± 5 h; Table 2). A significant decrease in the

WBRT was also noted for the therapy dose compared to tracer

dose for group 2 patients (102 ± 7 versus 79 ± 7 h). A slight

but less significant decrease was seen for group 1 (100 versus



A

Fig. 3 Differences in residence times. #{149},group1; 0, group 2. A, , expected WBRT; area

within the dashed lines, region within 10% of theexpected WBRT. If the tracer dose were identicalto the therapy dose, all points would be expectedto fall in the region defined by the dashed lines.

As shown, the predicted values for 6 of 7 group 2patients and 4 of 8 group 1 patients fell below thedashed line, indicating that WBRT values calcu-

lated from the tracer dose significantly overesti-mated the WBRTs for each therapy dose. B, mdi-

vidual points for 4 of 6 group 1 patients fell abovethe dashed line of identity, indicating that thetracer dose significantly underestimated the BRT

for the therapy dose. Two of 5 group 2 patientsalso fell above the predicted BRT from the diag-nostic dose.

140

120

100

80

60

40

20

0

140

I 20

100

80

60

40

20

0

0 20 40 60 80 100 120 140

B

0 20 40 60 80 100 120 140


I-a:

>�

0.

c�1

0

.�

I-

I-a:

0.Co

a,.�

I-

Diagnostic RI (h)

Diagnostic RI (h)

92.1 h; P = 0.07). The residence time results are presented

graphically in Fig. 3.

Biodistribution of ‘311-CC49. Mean ± SD whole body

%IA as estimated from the tracer study averaged 67.7 ± 12.8%

at 48 h after infusion. Mean ± SD %IA in the liver was 6.8 ±

4.9%, and in the blood, 33.5 ± 15%. The %IA in tumor at 48 h

was 0.57 ± 0.29 a�d remained stable or increased slightly in all

patients.

A comparison of 48 h %IA for total body, blood, liver and

tumor did not differ between groups I and 2 (data not shown).

Red Marrow Activity. Estimates of red marrow dose in

cGy/mCi are shown in Table 3. The cGy/mCi contributed from

activity in the blood increased by 28% between tracer and

therapy studies in group 1 patients (from 0.64 ± 0. 16; mean ±

SD to 0.87 ± 0.28; P < 0.007, paired t test), whereas the

cGy/mCi estimates from whole body activity indicated no sig-

nificant change (tracer dose, 0.43 ± 0.15; therapy dose, 0.38 ±

0. 12; P > 0.05). The total red marrow dose from blood plus total

body activity also increased significantly in this group (from

1 .07 ± 0.26 to I .25 ± 0.34 cGy/mCi; P < 0.05). Similar




Table 3 Predicted absorbed dose to red marrow (cGy/mCi)

Parameter Group 1 Group 2

Tracer doseMean administered activity (mCi) 18 ± 5.7” 15 ± 5.6Blood contribution 0.64 ± 0.16 0.81 ± 0.20Total body contribution 0.43 ± 0.15 0.41 ± 0.18Total I .07 ± 0.26 1.23 ± 0.30

Therapy doseMean administered activity (mCi) 100.9 ± 24 109.9 ± 18

Blood contribution 0.87 ± 0.28” 0.89 ± 0.20

Total body contribution 0.38 ± 0.12 0.32 ± 0.14Total 1.25 ± 0.35’ 1.23 ± 0.27

a Mean ± SD.

/� Significantly increased (P < 0.001) versus tracer dose.C Significantly increased (P < 0.05) versus tracer dose.

findings were not observed for group 2 (Table 3). In summary,

increases in cGy/mCi estimates for the therapy dose paralleled

similar increases in BRT for group 1 patients (i.e., those that had

received more rIFN-ct at the time these parameters were meas-

ured during the treatment phase.)

Tumor Dose Estimates. Four patients had soft tissue

metastatic sites that were visualized in SPECT images following

the tracer dose of ‘ � ‘I-CC49 that were used with computed

tomography images to provide volume estimates. As shown in

Table 4, cGy/mCi estimates in tumors averaged 6.09 ± 1.8, with

total radiation absorbed dose equivalents ranging from 438 to

841 cGy, depending on the total activity of ‘�‘I administered.

There was a direct correlation between radiation dose and the

%IA/g of CC49 in tumor biopsies performed 48 h after admin-

istration of the tracer activity (Table 4). No correlation was

observed between estimated tumor volume and rad dose. Be-

cause only one of the patients with tumors visualized (patient 1)

had begun rIFN-a injections prior to the time of measurement

compared to three that had not yet received treatment (patients

2-4), we could not compare radiation dose estimates between

rIFN-a-treated and untreated patients.

Enhancement of TAG-72 Expression and CC49 Uptake

in Tumor by rIFN-a. Different rIFN-a schedules were de-

signed to compare the %IA/g of CC49 in tumor biopsies from

the two groups of patients who were receiving rIFN-a. Results

of rIFN-a treatment on TAG-72 modulation and CC49 uptake

between pretreatment and 48-h post-tracer dose biopsy speci-

mens from the above patients have been detailed in a separate

report (23) and will only be summarized briefly here. Patients

who begin rIFN-a treatment on day I (group I) had an increase

in TAG-72 expression of46 ± 19% (MA pre-IFN-a, 14.5 ± 4;

post-IFN-a, 20 ± 5; P < 0.05) compared to only I .3 ± 0.95%

(MA pre-IFN-a, 12.5 ± 3.7; post-IFN-a, 13.1 ± 3.9; P > 0.05)

for group 2 patients not receiving IFN-a on day 1. Likewise,

tumor targeting of CC49 in group 1 patients (MA, 8.73 ± I .8)

was 4-fold higher than in group 2 patients (MA, 2.46 ± 0.32;

P < 0.01). In contrast, the %IA in tumor between group 1 and

group 2 patients was only 2-fold higher (mean %ID/g =

0.008 ± 0.002 versus 0.004 ± 0.002; P 0.06).

Antitumor Activity. A partial response was observed in

I of 15 patients studied. A patient with numerous palpable

axillary lymph nodes had a 50% decrease in lymphadenopathy

following treatment (Fig. 4). The same patient also had com-

plete disappearance of numerous pulmonary nodules < 1 cm in

size on chest X-ray along with improvement of bone pain and

partial ossification of lytic metastases. The response lasted 8

weeks following therapy; subsequently, the patient developed

metastases to bone marrow. Minor responses in soft tissue were

observed in two patients, and three other patients had mixed

responses. Ten patients had tumor progression following one

course of treatment. Except for one patient with a persistent MR

in s.c. lesions (all < 1 cm) lasting for 15 months, all patients had

progressive disease by 8 weeks posttreatment.

DISCUSSION

This represents one of the first Phase II radioimmuno-

therapy trials to analyze the biological effects of IFN-a on the

biodistribution pharmacokinetics and tumor uptake of ‘

CC49 in breast cancer patients. A previous study in ovarian

cancer patients demonstrated that the i.p. administration of

IFN-�y (0.1-100 megaunits/week) could enhance the expression

of TAG-72 and CEA in malignant ascites cells (14). In the

above-mentioned trial, IFN--y increased both the percentage of

MoAb B72.3 reactive tumor cells and MoAb staining intensity.

Greiner et a!. (25) have demonstrated elevation of serum CEA

and TAG-72 levels in patients given IFN-�y or IFN-�3.

Significant nonhematological toxicity was not observed.

The majority of patients had significant HAMA responses by 6

weeks. Eight of 15 patients had HAMA greater than 2,000

ng/ml, which is comparable to the results from our previous trial

of murine CC49 (16) and a study of chimeric CC49 (26). The

hematological toxicity observed with the combination differs

substantially from what has been observed with studies of

‘31I-CC49 alone. In our earlier Phase II trial of ‘311-CC49 in

colorectal cancer (16), grade 3-4 thrombocytopenia was only

observed in 7 of 15 patients, compared to 13 of 15 in this study

(P < 0.05, x2). These findings were also validated in a Phase I

trial of ‘31I-CC49 in colon cancer in which the maximum

tolerated dose was 75 mCi/m2 (18). Although these findings

could be due in part to the fact that the breast cancer patients

were more heavily pretreated than the colon cancer patients, it is

also possible that rIFN-a added to the hematological toxicity

because moderate myelosuppression has been shown to occur

with rIFN-a alone. Toxicities at these administered activities

have been observed in patients with prostate cancer receiving

the combination of ‘31I-CC49 and rIFN-a (27). Because radia-

tion can cause stem cell toxicity resulting in irreversible marrow

damage, caution must be exercised in combining radioimmuno-

therapy with drugs/biologicals that might enhance this effect.

An interesting observation that could also explain the en-

hanced hematological toxicity was the significant increase in

whole blood residence time in patients who began rIFN-a

treatment on day 1 . We also observed a significant decrease in

whole body residence time for patients beginning rIFN-a injec-

tions on day 6. A slight decrease was also observed for group 1

(from 101 to 92 h). Although a specific physiological explana-

tion(s) for these findings is lacking, it appears that prolonged

administration of rIFN-a resulted in a shift of ‘31I-CC49 from

the extravascular compartment to the blood resulting in a higher

BRT:WBRT ratio during the therapy phase (i.e., after at least




Fig. 4 Clinical response in left axilla from an individual patient. A, tumor in left axillary lymph nodes prior to therapy; B, posttherapy: note decrease

in size of tumor masses.

Table 4 Predicted absorbe d dose in tumor

TumorDose volume”

Patient (mCi) Tumor site cm3 % IA/g” Total cGy’� cGy/mCi

I 101 Axilla 38 0.014 841 8.32 131 Axilla 61 0.005 438 3.3

3 96 Chestlwall

axilla

71 0.013 632 6.6

4 130 Breast 74 0.011 892 6.2

Mean ± SD 61 ± 16 0.011 ± .003 678 ± 184 6.1 ± 1.807

‘a As estimated from SPECT.b Direct correlation between radiation dose and %IA/g MoAb; r = 0.820; P < 0.01.




7-10 days of rIFN-a treatment). Because rIFN-a has been

shown to alter the metabolism of proteins by affecting cyto-

chrome p450 enzymes in liver (28) it may be feasible that

alterations in whole body and blood distribution of ‘31I-CC49

might reflect changes in antibody metabolism. It is also con-

ceivable that changes in tumor vasculature induced by IFN-a

could be responsible for the findings observed (29). Additional

in-depth studies are required to provide answers to these ques-

tions.

The above findings have important consequences with re-

spect to the predicted radiation absorbed dose to marrow from a

tracer dose of ‘31I-labeled antibody. As detailed above, the

calculated radiation dose to marrow from the tracer dose in

patients already receiving rIFN-a did not predict radiation dose

to marrow for the subsequent treatment dose. In essence, the

“tracer principle,” i.e., the central dogma that biodistribution of

radiolabeled antibodies from the tracer dose should predict

distribution of a subsequent dose of radiolabeled MoAb (30),

did not hold true in this study. Because as much as 20% of the

activity contribution to red marrow may be derived from blood

(31), it is conceivable that the increase in blood residence time

reported above could have resulted in a higher radiation dose to

marrow, with the result of greater toxicity than predicted from

the tracer study.

It is possible that lower doses and/or schedules of rIFN-a

other than those used here might up-regulate TAG-72 to a

similar extent without undue toxicity to marrow. In a previous

study performed in malignant melanoma, we observed an en-

hancement of tumor uptake of the melanoma tumor antigen 96.5

following 3 days of rIFN-a (32). Mahvi et a!. (33) demonstrated

that a single iv. infusion of only 106 units of rIFN-a increased

TAG-72 and CEA expression in 3 of 6 patients with primary

colorectal cancer. In a more recent study, Roselli et a!. (34)

tested four dose schedules of rIFN a (2 doses of 3 X 106 units,

2 doses of 6 X 106 units, 3 doses of 3 X 106 units, and 3 doses

of 6 X 106 units) in colon cancer patients prior to surgical

resection of primary tumor. Significant enhancement of TAG-

72/CEA on resected tumors occurred with either 3 or 6 X 106

units of rIFN-a administered for 3 days, but not 2 days. Biopsies

were obtained 1 day after the final rIFN-a injections; hence, the

duration of increased antigen expression without rIFN-a is not

known. One of the reasons for choosing a 14-day schedule in

our study was the desire to maintain constant expression of

TAG-72 for both diagnostic and treatment phases of the study.

Whether enhanced TAG-72 expression persisted over the dura-

tion of the trial is not known and requires further study.

The mean radiation dose estimate to tumor (cGy/mCi) in

this study was significantly higher (6.09 ± 1.80) than that

calculated for colon cancer patients (2.96 ± 2.30; P < 0.05) in

our previous trial (16). It is possible that several factors (e.g.,

tumor location, tumor site, patient status) might also influence

uptake and contribute to the differences observed. Since the

majority of patients studied had received local radiation to chest

wall tumors, the radiation could have enhanced TAG-72 uptake

and binding (35).

Although modest clinical responses in soft tissue were

observed, it was not possible to determine whether these results

were due to ‘ � ‘I-CC49 or rIFN-a alone or to their combination.

In a previous trial of rIFN-a in breast cancer, modest antitumor

activity was observed (36). Several other studies in the literature

using different MoAbs reactive with breast cancer have demon-

strated clinical response rates of 50% when higher activities are

administered, and peripheral blood stem cell rescue was used to

overcome the increase in marrow ablation (37, 38).

In summary, rIFN-a caused an increase in tumor uptake of

‘31I-CC49 at the expense of increased toxicity. These findings

may have ramifications with respect to the use of radiolabeled

antibodies combined with cytokines or other biologicals. Al-

though the modest antitumor activity observed is of interest, a

0.2-2-fold increase in the %IA and radiation absorbed dose to

tumor induced by rIFN-a may be unlikely to eradicate large

tumor burdens in patients with solid tumors. Future clinical

studies with radiolabeled MoAbs should focus on high-dose

radiotherapy combined with autologous bone marrow or periph-

eral blood stem cell rescue and extending treatment to a minimal

disease or adjuvant seuing, combined with additional strategies

to further enhance the tumor to normal tissue ratio. These

approaches are required to significantly improve the therapeutic

index and subsequent patient benefit from radioimmunotherapy

in patients with solid tumors.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Wei-Ya Xia and Larry Kidd for

expert technical assistance and Madonna M. Yancey for manuscript

preparation.

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1997;3:1547-1555. Clin Cancer Res D J Macey, E J Grant, L Kasi, et al. antibody CC49 in breast cancer: a phase II trial.

monoclonalbiodistribution, toxicity, and efficacy of 131I-labeled Effect of recombinant alpha-interferon on pharmacokinetics,

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