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Clinical Trials |
Stanford University School of Medicine, Department of Radiation Oncology [S. J. K.], and Department of Diagnostic Radiology, Division of Nuclear Medicine [M. L. G.], Stanford, California 94305; University of Nebraska Medical Center, UNMC/Eppley Cancer Center [M. T., D. C.], Omaha, Nebraska 68198; Virginia Mason Medical Center, Medical Oncology, Seattle, Washington 98101 [P. L. W., H. B.]; Janssen Research Foundation, Titusville, New Jersey 08560 [L. G., I. D. H.]; Fox Chase Cancer Center, Department of Medical Oncology, Philadelphia, Pennsylvania 19111 [G. P. A., L. M. W.]; NeoRx Corporation, Seattle, Washington 98119 [D. A., S. G., K. B.]; and Pacific Northwest National Laboratory, Richland, Washington 99352 [D. R. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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48 h after
the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA.
Lastly, 24 h after administration of clearing agent, patients
received biotin-DOTA-labeled with 110 mCi/m2
90Y. All three components of the therapy were administered
i.v. Both hematological and nonhematological toxicities were observed.
Diarrhea was the most frequent grade 4 nonhematological toxicity (16%;
with 16% grade 3 diarrhea). Hematological toxicity was less severe
with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16%
grade 4 thrombocytopenia. The overall response rate was 8%. Two
partial responders had freedom from progression of 16 weeks. Four
patients (16%) had stable disease with freedom from progression of
10–20 weeks. Despite the relatively disappointing results of this
study in terms of therapeutic efficacy and toxicity, proof of principle
was obtained for the pretargeting approach. In addition, valuable new
information was obtained about normal tissue tolerance to low-dose-rate
irradiation that will help to provide useful guidelines for future
study designs. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pretargeting of RIT is a promising approach that has the potential to increase achievable tumor doses, improve tumor:normal tissue ratios, and therefore to increase the therapeutic index of pretargeted RIT compared with the use of directly labeled MABs. Pretargeting approaches dissociate the delivery of unlabeled antibody from the delivery of the radionuclide. This is potentially advantageous because the antibody that is administered first is not radiolabeled and therefore does not expose normal organs to radiation. The unlabeled antibody localizes in tumor and is then cleared from the circulation with a clearing agent. The radionuclide-hapten complex is then administered that reacts with the pretargeted antibody, thereby concentrating the radionuclide in the tumor. Pretargeting approaches use either bifunctional antibodies (3, 4, 5) , a biotin-streptavidin approach, or similar high-affinity ligand systems (6, 7) .
The optimal timing of administration of the radionuclide is achieved when the tumor:background ratio of unlabeled antibody is maximum. This is optimized by accelerated clearance of unlabeled antibody from circulation using a clearing agent based on the biotin-avidin or streptavidin system (8) . Advantages of this system include the small molecular weight of biotin, which can quickly circulate throughout the body, and the high-binding affinity between biotin and avidin or streptavidin (1015 M-1). Because the hapten-radionuclide complex is relatively small, it is cleared by the kidneys, and unbound radionuclide is cleared rapidly. Because biotin is a tetravalent molecule, the availability of four binding sites per streptavidin molecule also multiplies radionuclide deposition in tumor. Studies using both two- and three-step approaches have demonstrated that these pretargeting approaches permit the administration of much higher doses of radionuclide with acceptable toxicity than is possible with RIT using directly labeled antibodies (2 , 9) .
In the Phase II study reported here, a three-step pretargeting approach was used in which streptavidin-conjugated NR-LU-10 MAB was administered and allowed to localize in tumor. Next, a biotin-containing clearing agent was administered, followed by administration of yttrium-90-biotin. The murine MAB NR-LU-10 recognizes a noninternalizing Mr 40,000 glycoprotein antigen (Ep-CAM) expressed on several epithelial tumors, such as carcinomas of the lung, colon, breast, prostate, and ovary (10) , as well as on some normal tissues including gastrointestinal epithelium. NR-LU-10 possesses two desirable characteristics for pretargeting: reactivity with a high percentage of tumor cells in a broad range of adenocarcinomas (11) , and efficient in vivo tumor cell localization in both animal models and in humans (12) .
NR-LU-10 linked to SA was administered as a first step and allowed to
localize in tumor. Next, biotin-galactose-human serum albumin was
administered as the clearing agent. This biotinylated protein,
administered when the peak MAB uptake in the tumor had occurred,
rapidly and quantitatively complexes circulating NR-LU-10/SA, which is
then removed from the circulation by hepatocytes in the liver. Removal
of circulating antibody conjugate prior to administration of the
radionuclide reduces the amount of circulating antibody by 
95%
(13)
, which if radiolabeled would be a source of
nonspecific dose deposition that would result in additional toxicity.
In addition, because the clearing agent is cleared rapidly, it has
little opportunity to bind to the tumor-localized NR-LU-10/SA and
thereby compromise the binding of the radiolabeled biotin to tumor.
Lastly, the therapeutic radionuclide (third component of therapy) was
administered after confirmation of NR-LU-10/SA clearance from the
blood. 90Y was selected for therapy because it is
a pure ß emitter with a half life of 64 h, a maximum energy of
2.28 MeV, an average energy of 0.935 MeV, and a mean range in tissue of
2.5 mm. The pathlength over which 90% of the emitted energy is
absorbed is 5.3 mm (14, 15, 16)
. 90Y was
linked to biotin by the linker known as DOTA. Previously, studies have
shown that DOTA binds to 90Y with a favorably
high level of stability so that leaching (in vitro) is
minimized (17)
.
The three steps of this pretargeting regimen (including doses of the components and the timing of their administration) were optimized in patients to maximize tumor:normal tissue ratios (13) . A Phase I dose escalation trial was performed using this optimized regimen. The MTD was determined to be 110 mCi/m2. The dose-limiting toxicity at 140 mCi/m2 was GI toxicity. Two of three patients experienced grade 4 diarrhea requiring hospitalization and i.v. hydration. Two of four patients at the 120-mCi/m2 dose level also experienced grade 3/4 GI toxicity. One patient experienced grade 4 diarrhea requiring hospitalization, and another patient experienced grade 3 diarrhea requiring i.v. hydration. The onset of diarrhea occurred between 5 and 14 days after treatment with 90Y. Six subjects were treated at the 110-mCi/m2 cohort, with one grade 3 diarrhea unrelated to study medication. For this reason, the dose of 110 mCi/m2 was chosen to be the dose used in the Phase II study (18) .
In the Phase II study reported here, patients with metastatic colorectal cancer were treated with 110 mCi/m2 of 90Y-DOTA-biotin pretargeted by NR-LU-10/SA. The primary objective of the study was to evaluate the efficacy and safety of this therapy in this patient population. Secondary objectives included: evaluation of the duration of tumor responses; time to tumor progression; quality of life; and the incidence and titer of HAMAs, HASAs, and HACAs after a single dose of murine NR-LU-10/SA.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Before entry in the study, patients
met the following eligibility criteria: (a) pathologically
confirmed colon cancer with metastases; (b) age, 18 years;
(c) bidimensionally measurable disease; (d) no
prior cancer therapy for 4 weeks; (e) WBCs
3000/mm3, platelets
100,000/mm3, absolute neutrophil count
1500/mm3; (f) creatinine 
1.5
mg/dl, serum aspartate aminotransferase and serum alanine
aminotransferase <3 x upper limit of normal and total bilirubin
<1.5 x upper limit of normal; (g) baseline Eastern
Cooperative Oncology Group performance status 0 or 1; (h) no
prior high-dose chemotherapy requiring stem cell support or radiation
to >25% of the bone marrow; (i) no known HIV positivity;
(j) no concurrent malignancy (except basal cell or
T0–2
N0M0 squamous cell
carcinoma of the skin); (k) no history of previous
malignancy or treatment for any cancer in the last 5 years, except for
colon cancer and skin carcinoma; (l) HAMA/HASA/HACA results
<2 SDs above the geometric mean of a control population with no prior
history of MAB administration for imaging or therapy; (m) no
current treatment with another investigational drug; (n) no
prior mitomycin C or nitrosoureas; (o) at least one prior
standard therapy; (p) life expectancy >3 months;
(q) no active and untreated brain metastases; (r)
no serious illnesses that would, in the opinion of the investigator,
preclude the patient from participation or study completion; and
(s) no administration of Metastron (strontium-90) or
Quadramet (sumarium-153) within 12 weeks of trial entry. All patients
had prior abdominal/pelvic surgery, and all had failed at least one
course of conventional chemotherapy, with most having had two courses
of prior therapy. Five patients had received prior radiation therapy.
Additional eligibility criteria added after study initiation for safety
reasons included: (a) no medical history of irritable bowel
syndrome; (b) no grade 1 diarrhea at baseline;
(c) no prior external beam radiotherapy to the pelvis; and
(d) no uncorrected hypokalemia. In addition, all patients
had to agree to comply with the protocol stipulations and sign the
informed consent form prior to any pretrial testing. Patients were
instructed not to take vitamins containing biotin during the study, and
females of child-bearing potential had to use an accepted method of
birth control for the duration of the study.
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48 h
after the NR-LU-10/SA administration. The dose of the clearing agent
was calculated to be 1.04 times the dose of NR-LU-10/SA. The clearing
agent was given i.v. in up to 150 ml of saline as a single-bolus
injection over a period of 5 min. Patients were observed for acute
toxicities and had vital signs monitored immediately prior to and at
10, 30, and 60 min and 2 and 4 h after administration. Patients
were hydrated from 18 h prior to DOTA-biotin administration to 6
h post-DOTA-Biotin with 1 liter of 5% dextrose in water with one-half
normal saline plus 20 mEq KCl plus 10 mg of Furosemide every 6 h
as tolerated.
Seventy-two h after administration of NR-LU-10/SA and 24 h after
administration of clearing agent, patients received 0.5 mg of
90Y-DOTA-biotin labeled with 110 mCi/m2
90Y. The 90Y-DOTA-biotin was
given in up to 60 ml of saline as a single, rapid i.v. bolus injection
(15–20 s). Patients were again observed for acute toxicities and had
vital signs monitored immediately prior to and 10, 30, and 60 min after
administration of 90Y-DOTA-biotin and then as
clinically indicated. Stools were tested for occult blood when
clinically indicated. Patients were discharged when stable and when
their level of emitted radiation met revised Nuclear Regulatory
Commission release guidelines. They were discharged with Imodium and
antiemetics to use at the first sign of any GI toxicity. Any patient
experiencing grade 2 diarrhea was seen by a physician, and
patients with grade 3 or 4 GI toxicity were referred to a
gastroenterologist for evaluation.
Antibody and Radioimmunoconjugate Preparation.
90Y was obtained from the United States
Department of Energy’s isotope production program at the Pacific
Northwest National Laboratory (Richland, Washington). Patients were
treated with 90Y-DOTA-Biotin (110
mCi/m2) pretargeted by NR-LU-10/SA under
BB-IND-5247. The NR-LU-10 was conjugated to SA and tested for general
safety, sterility, pyrogenicity, polynucleotides,
Mycoplasma, and adventitious virus contamination. The
90Y-DOTA-biotin was prepared as follows. The
90Y (1.3 x patient dose in mCi) was
buffered with ammonium acetate and mixed. Ascorbic acid (0.05 ml) was
then added to the reaction vial and mixed. Ammonium acetate buffer (0.8
ml) was subsequently added to the DOTA-biotin vial and mixed. Next,
0.25 ml of the diluted DOTA-biotin vial was added to the vial and
mixed. The shielded 90Y reaction vial was
incubated in a water bath at 80°C for 60 min. After removal from the
water bath, 0.06 ml from the diethylene triamine-pentaacetic acid vial
was added to the reaction vial as a precautionary measure to scavenge
any unchelated 90Y. The final dilution for
patient administration was prepared by transferring the contents of the
90Y reaction vial into a 30-ml syringe containing
8 ml of PBS and 1.0 ml of ascorbic acid. Flushing of the vial with 15
ml of PBS ensured that the transfer was complete. The entire
preparation was filtered through a 0.2 µm filter.
Quality control assays were then performed on a 0.3-ml aliquot from the
30-ml syringe as follows. Three release assays (LAL testing,
determination of percentage of binding, and radiochemical purity) were
performed on site prior to the release of the
90Y-DOTA-biotin. One safety assay (Relative
Biotin Binding) was performed before the patient was injected with
90Y-DOTA-biotin. The LAL gel-clot method is a
qualitative test for Gram-negative endotoxin. Gram-negative bacterial
endotoxin catalyzes the activation of a proenzyme in the LAL. The
initial rate of activation is determined by the concentration of
endotoxin present. The activated enzyme (coagulase) hydrolyzes specific
bonds within a clotting protein (coagulation) also present in LAL. Once
hydrolyzed, the resultant coagulin self-associates and forms a
gelatinous clot. This assay was performed using a positive product
control for each sample tested. Typically, the assay sensitivity of the
LAL kit (BioWhittaker, Inc., Walkersville, MD) ranged between 0.25 and
0.125 endotoxin units/ml, and the 90Y-DOTA-biotin
was always less than this level of endotoxin. Determination of the
percentage of binding was accomplished by measuring the percentage of
binding of SA to biotin in a mixture of sample to its binding partner
coated on agarose beads. Using radiolabeled samples, the percentage of
the activity associated with the beads compared with the total activity
was determined by a gamma counter. Briefly, 0.2 ml of avidin-coated
beads were washed twice with 500 µl of PBS and reconstituted with 500
µl of PBS. The patient preparation (10 µl of a 1:1000 dilution) was
added to each of two Microfilterfuge tubes (one with the beads and one
with 500 µl of PBS only), incubated for 10 min at room temperature,
and centrifuged in a microcentrifuge for 20–30 s. The beads were
washed twice with PBS. Then 900 µl of each filtrate were removed into
separate test tubes and counted in the gamma counter. The percentage of
binding was determined by comparing the cpm of the Microfilterfuge tube
section associated with the beads to the total cpm of the sample:
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90% for patient administration, and the mean purity of the
90Y-DOTA-biotin administered to patients was
98.4 ± 2.6%. A relative biotin binding assay was performed to
determine the biotin binding capacity of SA-conjugated antibodies
remaining in circulation after in vivo administration of the
clearing agent. This assay is used as a safety measure, by determining
the amount of biotin capable of binding to SA conjugate in patient
serum prior to the administration of radiolabeled biotin derivatives.
The relative biotin binding (percentage bound) was calculated as [1
-(mean unbound/mean total counts)] x 100%. For patient
administration, it was required that binding for 40 µg/ml control be
>80% and that the patient serum contain <5 µg/ml of biotin binding
conjugate. For the patients treated in this study, their serum
contained a mean level (±SD) of biotin binding conjugate of 1.4 ± 1.6 µg/ml, with a range of 0–4.5 µg/ml, and no detectable
biotin binding conjugate in the serum of 10 patients after
administration of the clearing agent, prior to administration of the
90Y-DOTA-biotin. This represented clearance of
95% of the circulating antibody after clearing agent administration.
Dosimetry.
Dosimetric studies were not required by the protocol and were not
routinely performed in this Phase II study. However, in three patients
treated at Virginia Mason Medical Center, dosimetry studies were
performed using 111In-DOTA-biotin, as described
previously (20)
, in which absorbed radiation doses were
estimated for normal organs and tissues, the whole body, and for tumor
masses using methods that are consistent with those recommended by the
MIRD Committee of The Society of Nuclear Medicine
(21, 22, 23)
. These methods account for both the penetrating
gamma and the nonpenetrating ß radiation emitted by radioactivity
distributed throughout the body. Dosimetry calculations were based on
gamma-camera measurements of 111In-labeled-biotin
in the major source organs, tumors, blood serum, and in the total body
at various times after administration using methodology described
previously (20
, 24, 25, 26)
. These calculations were used to
estimate the range of doses to normal organs and tissues delivered by
90Y-DOTA-biotin at the MTD in these patients to
try to better understand the observed toxicity.
The S values (the absorbed dose per unit cumulated activity in cGy per µCi-h, or Gy per Becquerel-s) used for these calculations were the same as those that were used previously in the International Commission on Radiological Protection Publication 30 (27) and implemented in MIRDOSE2 computer software (Oak Ridge Associated Universities, Oak Ridge, TN). S values for tumors were estimated by extrapolation using normal organs of similar size and location in the body. Published S values are not available for mucosal tissue of the small and large intestines. Therefore, we calculated the S values from first principles using a mathematical model of the intestinal wall, mucosa of the wall, and lumen (bowel contents). These S values were calculated (28) for 90Y activity deposited in the mucosa, the wall, or lumen using a Monte Carlo code (EGS4, Stanford Linear Accelerator, Palo Alto, CA). The small and large intestines were modeled as parallel-packed cylinders (>30 cm length) for these calculations. We assumed a wall thickness for small intestine of 0.35 cm, which includes a mucosa of 0.06-cm thickness. We assumed a small intestine luminal diameter of 0.57 mm. Radiation absorbed doses to mucosa were then obtained by multiplying the calculated S values by the cumulated activities, Ã, that were obtained from 111In gamma camera measurements for 90Y in the small or large intestines.
Clinical Parameters Monitored.
After treatment, a number of parameters were followed. These included
blood counts, chemistry panels, thyroid function tests, pancreatic
enzyme levels, urine analyses, carcinoembryonic antigen levels, and
HAMA/HASA/HACA tests. Patients were seen for follow-up examinations at
least every 2 weeks for the first 3 months and then monthly until
disease progression was documented. The exact timing of these visits
was determined in part by the patient’s clinical status. A toxicity
assessment, with special emphasis on gastrointestinal toxicity, was
performed three times/week for 3 weeks after 90Y
administration. An electrocardiogram was performed at baseline and at
the time patients went off study. The European Organization for
Research and Treatment of Cancer Quality of Life assessment was
performed on day 1, weeks 4, 8, and 12, and then every 3 months as long
as the patients remained on study. Restaging was performed at weeks
4–6 and 8–10 after treatment and then every 2 months until
progression and was based on physical examination and a variety of
radiographic studies including chest, abdominal, and pelvic computed
tomography scans. Standard response criteria were used and defined as
follows: a complete response was defined as disappearance of all
clinical evidence of tumor by physical examination,
roentgenography, and computed tomography scans for a minimum of
4 weeks. A PR was a 50% or greater decrease in the sum of the product
of the diameters of the measurable sentinel lesions for a minimum of 4
weeks without any increase in size of other lesions and the appearance
of no new lesions. Stable disease was any change in the size of the
sentinel lesions not meeting the criteria of a complete response or PR
or progression. Progressive disease was a 25% or greater increase in
the sum of the product of the diameters of the measurable sentinel
lesions and/or the appearance of a new lesion. The duration of response
was the number of days between the first documentation of a PR or
complete response and the first documentation of progression of
disease.
HAMA/HASA/HACA Response.
Patients were monitored for the production of HAMA, HASA, and HACA.
Antiglobulin levels were measured in patient sera using an ELISA as
described previously (29)
. Briefly, streptavidin,
NR-LU-10, or NR-LU-10/SA was used as a capture antigen for HASA, HAMA,
and HACA, respectively. In each case, antigen was coated on 96-well
polyvinyl microtiter plates (Falcon Plastics, Oxnard, CA) in PBS (Sigma
Chemical, St. Louis, MO). Patient sera was added in 4-fold dilutions to
wells in PBS containing 0.5% Tween and 4% chicken serum (PCT buffer).
After washing unbound sera components, peroxidase-labeled goat
antihuman (heavy and light chain) antibody was added in PCT for each of
the three assays. After additional washes, the chromogen substrate,
2,2' azino-bis-3-ethylbenzothiozoline-6-sulfonic acid, was added, and
color development was monitored spectrophotometrically. Relative
reactivity was determined by measuring the HASA, HAMA, and HACA immune
response relative to a pooled serum source of untreated normal
individuals. To be considered a positive response, posttreatment levels
needed to be at least 2-fold higher than pretreatment levels.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The actual dose administered is shown
for each patient, as well as the subsequent best response and FFP in
months. The response rate was 8% with two PRs in patients 20 and 21
with FFP of 16 weeks. Four patients (16%) had stable disease with FFP
of 10–20 weeks, as determined by tumor measurements made at the study
sites.
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. Diarrhea was the most frequent grade 4
toxicity. Twenty-two of the 25 patients (88%) had diarrhea of grade 1 toxicity, 4 patients (16%) had grade 3, and 4 patients
(16%) had grade 4 toxicity. Of the patients with grade 3 or 4
diarrhea, 50% had received prior radiation therapy to a field
including bowel. Of the two patients with grade 4 diarrhea that had not
received prior radiation therapy, one had a history of diverticulitis
and one had an underlying partial small bowel obstruction. One patient
with severe diarrhea, dehydration, and hypokalemia died of a cardiac
arrest after refusing hospitalization for 2 consecutive days
immediately prior to her death. Although autopsy revealed severe
underlying cardiac disease, it is possible that diarrhea with
hypokalemia contributed to this death. Nausea and vomiting occurred in
76% and 60% of patients, respectively, with all but one patient
experiencing only grade 1 or 2 toxicity. Fatigue (grades 1–3) occurred
in 84% of patients, with 68% of patients with grade 1 or 2 toxicity.
Anorexia also occurred in 68% of patients, with a 56% incidence of
grade 1 or 2 toxicity. Other nonhematological toxicities occurred less
frequently. The other grade 4 toxicities observed included: two
patients with dehydration secondary to diarrhea, nausea, vomiting, and
anorexia; one patient with jaundice with elevated creatinine with
end-stage hepatorenal syndrome thought to be unrelated to the RIT
(patient had progressive disease and post-operative complications after
surgery for a bowel obstruction); one patient with stomatitis; and one
patient with both abdominal cramping and abdominal pain secondary to
small bowel obstruction from progressive disease. The number of
patients with elevated liver function tests is shown in Table 3
. Grade
3 or 4 abnormalities in alkaline phosphatase, transaminases, and total
bilirubin were observed in 32% of patients. In 50% of the patients
with grade 3 or 4 elevated liver function tests, these elevations were
transient and reversible, and in 50% of patients, these abnormalities
persisted and were attributable to progressive disease. Grade 1 or 2
only liver function test toxicities were observed in 60% of patients.
Again, these abnormalities were either transient and reversible in 27%
of patients or persistent and apparently secondary to progressive
disease in 73% of patients. Of concern, two patients (patients 2 and
3) developed significant elevations in serum creatinine levels 7–8
months after treatment to 3.1 and 6.8, respectively, that cannot be
explained by progressive disease.
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. The numbers of patient with grades
1–4 leukopenia, granulocytopenia, thrombocytopenia, and anemia are
shown. There were more patients with grade 4 thrombocytopenia than
grade 4 neutropenia. Mean nadir counts are based on absolute nadir
counts or the lowest count that occurred prior to the initiation of
granulocyte-colony stimulating factor in three patients or platelet
transfusions in three patients. Nadirs generally occurred between 5 and
6 weeks after treatment and usually resolved by approximately 6–8
weeks after treatment (Table 5)
.
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2 SD
above the mean of a control population in measured normal human serum
units. Positive antibody responses were observed in 70–80% of
patients by 2 weeks after treatment and in all patients by 4–5 weeks
after treatment.
Dosimetry.
Three patients treated at Virginia Mason Medical Center on the Phase II
study underwent dosimetry studies with estimated doses to the small
intestine (standard MIRD calculation), kidney, and bone marrow of
2102 ± 591 cGy, 2864 ± 840 cGy, and 33 ± 8 cGy,
respectively. Tumor doses were estimated for two of these patients at
479 cGy (patient 6, lung mass) and 2885 cGy (patient 8, liver lesion).
A tumor dose was not calculated for the third patient (patient 7)
because of poor imaging of disease in that patient.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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95% of circulating antibody to serum
levels <5 µg/ml of biotin bonding conjugate, and useful information
was obtained that will allow for improved RIT using similar approaches
in the future.
The response rate in the Phase II study was similar to that observed in
the Phase I study for patients treated at 80
mCi/m2, with PR rates of 8% (current study) and
9% (Phase I study), respectively. However, only 16% of patients in
the Phase II study had stable disease compared with 54% of patients in
the Phase I study treated with 80 mCi/m2 90
Y-DOTA-biotin, who achieved either a minor response (2 patients) or
stable disease (16 patients). Furthermore, the mean FFP for responses
was shorter in the current study. The discrepancy in responses between
the two studies may be attributable in part to the histology and
associated natural history of the disease types studied, because only
22% of the patients in the Phase I study had colorectal cancer (29%
had prostate cancer and 22% had ovarian cancer, with the remaining
27% comprised of breast, kidney, lung, cervix, and endometrial
cancers).
The incidence and severity of GI and hematological toxicity at the MTD, determined by the previous Phase I trial (18) , were surprising and not predicted by the results of that study. In the Phase I trial, 40 patients with advanced adenocarcinoma of a variety of sites were treated in a dose escalation study (dose increments 5–20 mCi/m2), with total doses ranging from 25 to 140 mCi/m2 90Y-DOTA-biotin. In an interim analysis, grade 3/4 diarrhea, nausea/vomiting, thrombocytopenia, and neutropenia occurred in 4, 3, 7, and 4 of 40 patients, respectively. The dose-limiting toxicity in this Phase I trial was diarrhea at a dose level of 140 mCi/m2 90Y. The reason for the discrepancy in the toxicity results of the Phases I and II studies is unclear but could be attributable in part to small numbers of patients at each dose level in the Phase I trial as well as to the heterogenicity of tumor types in the Phase I trial as compared with the Phase II trial. The patients in the Phase II study were not more unfavorable than those treated in the Phase I study in terms of the extent of prior myelosuppressive therapy or the presence of risk factors for GI toxicity (prior abdominal or pelvic radiation therapy and/or laparotomy). The observed renal toxicity was also not predicted by the prior Phase I study, perhaps because there were only small numbers of patients treated at high doses, with many not surviving long enough to develop late toxicities of treatment. Of note, one of the patients treated previously in the Phase I study with 140 mCi/m2 now has a diminished creatinine clearance. In the Phase II study, the timing of onset of elevated serum creatinine levels is consistent with radiation-induced nephritis (30) .
Estimated doses to bowel, kidney, and bone marrow from the Phase I study were 10.6 ± 3.9 cGy/mCi, 11.5 ± 4.2 cGy/mCi, and 0.15 ± 0.06 cGy/mCi, respectively. Similarly, doses estimated for small intestine (standard MIRD calculation), kidney, and bone marrow for three patients in the Phase II study were 2102 ± 591 cGy, 2864 ± 840 cGy, and 33 ± 8 cGy, respectively. Because dosimetry studies were not performed routinely as part of this Phase II study, doses to these normal tissues were also estimated by extrapolation from the Phase I experience. For patients receiving 110 mCi/m2, assuming a body surface area of 2.0 m2, the average estimated dose to the kidney and bone marrow could have ranged between 1606 and 3454 (mean, 2530) cGy and 11–55 (mean, 33) cGy, respectively. Using a new model for calculating radiation absorbed dose to intestinal tissues for the three Phase II study patients above (28) , Fisher et al. (28) have estimated the dose to the wall of the small intestine in the GI tract to be 59 ± 2.0 cGy/mCi (compared with 9.4 ± 2.5 cGy/mCi as predicted by standard MIRD calculations for the small intestine, which assumes that the activity resides in the bowel contents as compared with the tissue itself), with doses for the large intestine slightly >50% of the dose to the small intestine (because of the greater mass of the large intestinal wall). Therefore, these patients in the Phase II study received on average a small intestinal wall dose of 13,334 cGy. This is because of cross-reactivity of the NR-LU-10 antibody with the bowel epithelium and not because of GI excretion of the 90Y-DOTA-biotin. NR-LU-10 also cross-reacted with kidney tubules; therefore kidney doses were secondary to both renal excretion of 90Y as well as to the targeting of bound NR-LU-10/SA by 90Y-DOTA-biotin. Given that conservative estimates of tolerable whole-organ doses from conventional (high-dose rate) radiation therapy are 1500–1700 cGy for kidneys and 4000–4500 cGy for the small bowel (31) , the doses to these organs in this study may have greatly exceeded these "tolerable" ranges in many patients. These dose estimates are more than sufficient to explain the observed toxicity. If these estimates are accurate, it is in fact surprising that more toxicity was not observed and demonstrates the impact of dose rate effects on toxicity. RIT results in continuous exponentially decreasing low-dose-rate radiation. Little has been known about normal organ tolerance to low-dose-rate radiation, and these observations provide new insight into the radiobiology and toxicity of this form of therapy. It is important to emphasize, however, that dose estimates for radioimmunotherapy lack the precision of the dosimetric methodology used to calculate tumor and normal tissue doses from conventional external beam radiation therapy. It is possible that the imprecision associated with estimating doses in this study could have resulted in overestimation of doses to normal tissue, which would affect our interpretation of the findings with regard to expected toxicity as a function of dose rate.
In this study, proof of principle was obtained for the pretargeting approach used with documentation of excellent clearance of circulating antibody. New information about normal tissue tolerance to low-dose-rate irradiation was obtained that will help to provide useful guidelines for future study designs. Clearly, future studies should use antibodies directed to a different antigenic target because reactivity of the NR-LU-10 MAB with normal GI epithelium and collecting tubules in the kidney clearly contributed to toxicity. Ideally, the targeted tumor antigen should have highly restricted expression in normal tissues. Efficacy may be further improved by using less immunogenic agents (e.g., chimeric or humanized MABs) that may allow for multidose fractionated RIT. The tumor-targeting vehicle should be multivalent and highly tumor avid, yet small enough to penetrate into tumors from the vasculature and rapidly clear from normal organs. If this is possible, a formal clearance step may not be necessary. The high-affinity interactions between the tumor-targeting vehicle and the radionuclide should be mediated by nonimmunogenic proteins, again ideally of human origin, and the high-affinity interactions should not involve potential cross-reactivity with host elements, such as endogenous biotin. Pretargeted RIT remains a promising area of clinical investigation that merits further study. Modifications of the pretargeting strategy, such as those described above, will enable pretargeted RIT to achieve its full potential.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Human Health Service Grant
M01-RR00070, General Clinical Research Centers, National Center for
Research Resources, NIH, and a grant from the Janssen Research
Foundation. 
2 To whom requests for reprints should be
addressed, at Stanford University Medical Center, Department of
Radiation Oncology (A-093), Stanford, CA 94305. Phone: (650) 723-5832;
Fax: (650) 725-8231.
3 The abbreviations used are: MAB,
monoclonal antibody; RIT, radioimmunotherapy; SA streptavidin; DOTA,
tetra-azacyclododecanetetra-acetic acid; MTD, maximum tolerated dose;
GI, gastrointestinal; HAMA, human antimouse antibody; HASA, human
antistreptavidin antibody; HACA, human anticonjugate antibody; LAL,
Limulus amebocyte lysate; MIRD, Medical Internal
Radiation Dose; FFP, freedom from progression; PR, partial response.
Received 7/26/99; revised 10/27/99; accepted 11/ 5/99.
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