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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0107.
NEOPLASIA
From the Metabolism Branch, Center for Cancer Research,
National Cancer Institute; Nuclear Medicine Department, Clinical
Center; Radiation Oncology Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD; and NeoRx Corporation, Seattle, WA.
We used a pretargeting technique to treat a nonobese
diabetic/severe combined immunodeficient murine model of human adult T-cell leukemia with an anti-Tac antibody-streptavidin (HAT-SA) conjugate, which recognizes CD25, followed by bismuth 213 (213Bi)-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
acid (DOTA)- biotin. In the 3-step pretargeting
radioimmunotherapy protocol, HAT-SA (140 or 400 µg) was administered
intravenously (i.v.) to bind to the interleukin 2 receptor Adult T-cell leukemia (ATL) develops in a small
proportion of individuals infected with human T-cell lymphotrophic
virus-I (HTLV-I).1 The leukemia consists of an
overabundance of malignant activated T cells, which are characterized
by the expression of CD25 (interleukin 2 receptor The observation that IL-2R Leukemia generally provides better access than solid tumor to target
antigens. This quality, along with the inherent radiosensitivity of
hematologic malignancies, has made clinical radioimmunotherapy (RIT) of
lymphomas one of the few applications with established clinical
efficacy.5,7-9 However, the long serum half-lives of
antibodies prolong radiation exposure to normal organs and to
radiosensitive bone marrow, which limits the radiation dose that can be
safely administered.10,11 Furthermore, the large size of
antibodies yields only slow access to malignant cells in large tumors,
precluding the use of short-lived radionuclides, including most
available To overcome some of the obstacles encountered by conventional RIT, a
technique involving a pretargeting system was introduced for tumor
targeting to take advantage of the extremely high affinity of
avidin-biotin binding and rapid pharmacokinetics of the small molecule
biotin.12-22 In this system, antibody and radionuclides are administered separately, and radioactivity is rapidly and selectively accumulated in tumors with a parallel reduction of radioactivity in normal tissues.
A promising approach, reported by the NeoRx Corporation (Seattle,
WA),19 consists of 3 steps. In step 1, the
antibody-streptavidin (SA) conjugate is administered intravenously
(i.v.) and allowed to target and accumulate in the tumor. In step 2, the unbound antibody-SA is cleared from the circulation by in vivo
complexation with a synthetic biotinylated poly(GalNAc)-clearing agent
(sCA) to prevent it from binding the biotin-radionuclide used in the following step. The resultant complexes are rapidly cleared into the
liver by the asialogalactose receptor and metabolized.23 The clearing step is essential to maintain low absolute blood concentration of antibody-SA that would bind to the radiolabeled reagent administered in the next step. In step 3, radiation is delivered to the antibody-SA on the tumor by the administration of
radiolabeled biotinidase-resistant
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA)-biotin. This low molecular weight cytotoxic molecule readily
reaches the tumor where it is captured by the prelocalized antibody-SA.
Unbound radiolabeled DOTA-biotin is rapidly eliminated from the body by
the urine. Rapid uptake of the therapeutic nuclide at the tumor and
efficient elimination of excess radioactivity represent the fundamental
advantages of the pretargeting approach as compared with conventional
RIT, in which the radionuclide is directly conjugated to the antibody.
Animal studies have characterized the pharmacokinetics and optimized
the 3 reagents used in this approach in solid tumor
models.19 The results have shown favorable specific and
rapid targeting of radionuclide that has resulted in good tumor
responses. Clinical trials have also been performed, and results showed
the feasibility of the technique.20-22
For select situations, especially with isolated malignant cells as in
leukemia, In this study, we investigated the use of the pretargeting technique
with the Tumor cell lines
Monoclonal antibody
Preparation of antibody-SA conjugate HAT or B3 was conjugated to SA by use of succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate described previously.19Synthetic clearing agent The sCA, provided by NeoRx, consists of a bifunctional moiety with multiple N-acetyl-galactosamine residues linked to biotin (molecular weight = 8651).23 The sCA binds rapidly to the circulating antibody-SA conjugate and clears rapidly from the circulation into the liver by the asialogalactose receptor present on hepatocytes. In this process, it carries with it any SA-bound antibody that had attached to it.23Radiolabeling Bismuth 213 was eluted from an actinium 225 (225Ac) generator.33 The 225Ac was supplied by Oak Ridge National Laboratory (Oak Ridge, TN), and 90Y was obtained from NEN (Boston, MA) as 90YCl3. For pretargeting therapy, biotinidase-resistant DOTA-biotin (molecular weight = ~900) (NeoRx) was labeled with 213Bi or 90Y at specific activities of 1 mCi/µg (37 MBq/µg), as previously described,19 and these labeled DOTA-biotin reagents consistently bound more than 95% to avidin gel.For directly labeled antibody therapy, HAT-CHX-A" was labeled with 213Bi at a specific activity of 16.2 µCi/µg (0.6 MBq/µg) as previously described.34 Unmodified HAT and HAT-SA conjugate were labeled with iodine 125 (125I) for immunoreactivity assay and internalization studies at a specific activity of 3 µCi/µg (111 kBq/µg) by using the chloramine-T method. Immunoreactivity assay and internalization study Immunoreactivity of the conjugate was evaluated by using Kit225-IG3 cells compared with the unmodified HAT.35 The internalization of 125I-labeled HAT-SA conjugate was evaluated by using an in vitro method described previously.36Tumor model and tumor burden evaluation The leukemia model was established by intraperitoneal injection of 1.5 to 2.0 × 107 MET-1 cells into SCID/NOD mice. MET-1 cells were separated from enlarged spleens or from subcutaneous tumors of MET-1 tumor-bearing mice with a high (> 400 000 pg/mL) serum soluble IL-2R (sIL-2R) level.6 The therapy
experiment was performed on these mice when their sIL-2R levels were
more than 1000 pg/mL serum, which occurs approximately 10 to 14 days
after tumor inoculation.
Pretargeting technique Tumor-bearing mice were injected i.v. with 140 or 400 µg (0.67 or 1.91 nmol) of HAT-SA conjugate for pretargeting. After 24 hours were allowed for distribution and tumor localization, 100 µg (11.56 nmol) sCA was injected i.v. to clear circulating HAT-SA conjugate from the blood. Four hours after injection of the sCA, 0.3 or 1 µg (0.33 or 1.11 nmol) 213Bi-DOTA-biotin was injected i.v.Seven days before pretargeting, the mice were fed with a biotin-free diet to reduce the endogenous biotin level (Biotin Deficient Rodent Diet 5836C-I; Purina Mills, Richmond, IN). One day after the administration of 213Bi, the normal diet was restored. Therapy study The therapeutic protocol is shown in Table 1. In the dose-escalating therapeutic trial, different doses (0, 50, 150, 250, or 350 µCi [0, 1.85, 5.55, 9.25, or 12.95 MBq]) of 213Bi-DOTA-biotin were used. Tumor-bearing mice with sIL-2R from 1000 to 10 000 pg/mL were
selected and divided into 6 groups of 5 mice. The mice were injected
intravenously with 140 µg HAT-SA conjugate for pretargeting for 24 hours. Then, 100 µg sCA was injected intravenously 4 hours later.
213Bi-DOTA-biotin (0.3 µg) was administered
intravenously. One group without treatment served as a
control.
In the small-tumor-burden therapeutic trial, there were 4 groups, 10 mice each, with the same sIL-2R In the large-tumor-burden therapeutic trial, mice with serum sIL-2R To compare the therapeutic effect of a Monitoring of tumor growth Measurements of the serum concentrations of the sIL-2R and/or
soluble  -2-microglobulin (2µ) were performed by using
enzyme-linked immunosorbent assay at 2-week intervals after therapy to
monitor the growth of the leukemia. The assay kits were purchased from R&D System (soluble Tac-Cat. No. DR2A00; soluble 2µ Cat. No. DBM200; Minneapolis, MN).
Toxicity study Six groups of 10 healthy SCID/NOD mice each were used to define the toxicity of 213Bi. For groups 1 to 3, increasing doses of 213Bi-DOTA-biotin (100, 250, and 500 µCi [3.7, 9.25, and 18.5 MBq]) were administered following the pretargeting approach with larger doses of HAT-SA and DOTA-biotin as used in the large-tumor-burden therapeutic trial. Group 4 also received 250 µCi (9.25 MBq) 213Bi-DOTA-biotin but without pretargeting and sCA. Group 5 received pretargeting, sCA, and 250 µCi (9.25 MBq) 213Bi-DOTA-biotin followed by 100 µg unmodified HAT at days 1, 7, 14, and 21, respectively. Group 6 did not receive any treatment.The body weight and the complete blood count were measured before and
after treatment (initially at weekly and subsequently at monthly
intervals). The serum levels of creatinine, blood urea nitrogen (BUN),
alanine aminotransferase, aspartate aminotransferase, creatine kinase,
and Definition of the maximum-tolerated dose Prior to initiation of PRIT, the maximum-tolerated dose of 213Bi-DOTA-biotin was determined in both healthy SCID/NOD and MET-1 tumor-bearing SCID/NOD mice. Doses of 0, 50, 150, 250, and 350 µCi (0, 1.85, 5.55, 9.25, and 12.95 MBq) 213Bi-DOTA-biotin for tumor-bearing mice and doses of 100, 250, and 500 µCi (3.7, 9.25, and 18.5 MBq) 213Bi-DOTA-biotin for healthy mice were evaluated. Renal functional abnormality developed in the mice receiving 500 µCi (18.5 MBq). Therefore, doses of 250 µCi (9.25 MBq) 213Bi-DOTA-biotin were used in the PRIT studies.Statistical analysis The serum levels of sIL-2R, 2M, and BUN, as well as body
weight, at different time points for the different treatment groups and
the data of internalization studies were analyzed statistically using
the t test for unpaired data. In terms of the mouse survival plots, StatView was used to generate Kaplan-Meier cumulative
survival plots.
Immunoreactivity The 125I labeled HAT-SA conjugate and HAT antibody bound to Kit225-IG3 cells comparably. They showed similar maximal bindings of 83% to 87% and 86% to 89%, respectively, suggesting that SA conjugation did not affect the bindability of the antibody.Internalization of HAT-SA conjugate It is important for the pretargeting technique that antibody-SA conjugate stay on the tumor cell surface until the radiolabeled biotin is administered. Therefore, the rate of internalization of HAT-SA by leukemic cell lines (MET-1, Kit225-IG3, HUT102, and MT1), as well as nonlymphoid cell lines (SP2/Tac and ATAC4), was investigated. All 6 cell lines had more than 80% of the cell-associated radioactivity on the cell surface initially (Figure 1). Acid soluble activity decreased rapidly in nonlymphoid SP2/Tac and ATAC4 cells. For the SP2/Tac cell line, only 10% and 3% were left on the cell surface after incubation for 6 and 24 hours, respectively. In contrast, approximately one half and one third were present on the cell surface after 6 and 24 hours' incubation, respectively, with the 4 leukemic cell lines, values significantly higher than those with SP2/Tac and ATAC4 cell lines (Figure 1A, P < .001).
As the radioactivity on the cell surface decreased, that of the supernatant increased accordingly. In particular, the amount in the supernatant with SP2/Tac cells (Figures 1B) was significantly higher than that with Kit225-IG3 cells (Figures 1C). At 6 hours after incubation, more than 70% was found in the supernatant with SP2/Tac cells, whereas less than 30% was present with Kit225-IG3 cells. The radioactivity in the supernatant of SP2/Tac cells was mainly free iodine (more than 40% at 6 hours), which reflects the release of iodine from the cell after internalization and processing of the labeled conjugate. In contrast, the amount of free iodine in the supernatant with Kit225-IG3 cells was less than 10% at 6 hours of incubation, significantly lower than that of SP2/Tac cells (P < .001). The pattern of catabolism with the ATAC4 cells was similar to that of the SP2/Tac cells, and the patterns with MET-1, HUT102, and MT1 cell lines were similar to that of the Kit225-IG3 cells. The internalization of HAT-SA by the 4 leukemic cell lines studied was relatively slow, which provides binding sites for the subsequent radiolabeled biotin for tumor targeting. Therapeutic study PRIT with the
The effective results with PRIT were repeated in mice with both small- and large-tumor burdens (Tables 2,3 and Figures 3,4). The tumor growth was inhibited with PRIT in both therapeutic studies. On day 28 after therapy in the small-tumor-burden therapeutic trial, the serum concentration of 2M was 0.44 µg/mL in the PRIT group as
compared with 7.74 µg/mL in the control group (Table 2,
P < .0001). At 14 days after therapy in the
large-tumor-burden therapeutic trial, 2M was 0.38 µg/mL in the
PRIT group as compared with 7.17 µg/mL in the control group (Table 3,
P < .0001). Furthermore, the survival of the mice in the
PRIT groups was significantly prolonged as compared with the control
groups (Figures 3,4; P < .0005). The median survival
durations of the control groups were 42.3 and 23.8 days in the small-
and large-tumor-burden therapeutic trials, respectively, whereas
they were prolonged to 89.8 and 53.7 days, respectively, in the
PRIT groups.
Compared with HAT immunotherapy, PRIT showed more therapeutic efficacy (Tables 2,3 and Figures 3,4). The survival of the mice in the PRIT groups was prolonged significantly as compared with HAT therapy groups (Figures 3,4; P < .05). In the therapy with large-tumor burden, 2M was also significantly lower with PRIT than with HAT
treatment on day 14 after therapy (Table 3, P < .0001).
The specificity of therapeutic effect with PRIT was confirmed by
comparing specific HAT-SA conjugate PRIT with nonspecific PRIT using an
irrelevant control antibody-SA conjugate, B3-SA. The levels of 2M
were significantly lower with the specific PRIT than with the
nonspecific PRIT (Tables 2,3; P < .001), and there were
significant prolongations of survival of the mice in the specific PRIT
groups compared with the nonspecific PRIT groups (Figures 3,4;
P < .0001).
Immunotherapy (HAT).
Treatment of MET-1 tumor-bearing mice with the unmodified HAT antibody
showed effective therapeutic results with partial remissions of the
leukemia and a prolongation of the life of the leukemia-bearing mice,
similar to those we previously reported.6 In the
therapeutic study with small-tumor burden, initial sIL-2R RIT with 213Bi-labeled intact HAT.
To compare the pretargeting approach with RIT with directly labeled
intact antibody, 213Bi-CHX-A"-HAT was administered to
mice bearing large-tumor burdens. The tolerated dose of 50 µCi (1.85 MBq) 213Bi-HAT was used. The PRIT with the
Combination therapy involving PRIT with 213Bi and
immunotherapy with unmodified HAT.
Combination therapy, involving pretargeting 250 µCi (9.25 MBq)
213Bi in the protocol used above followed by weekly doses
of HAT at 100 µg on day 1, 7, 14, and 21 showed improved therapeutic results when compared with either PRIT or HAT alone. In the therapeutic study involving mice with large-tumor burdens (sIL-2R Toxicity In the 3-step pretargeting regimen in SCID/NOD mice, the animal body weight did not show significant changes with 250 µCi (9.25 MBq) or less 213Bi-DOTA-biotin with or without HAT-SA pretargeting, whereas mice that received 500 µCi (18.5 MBq) 213Bi-DOTA-biotin showed weakness and significant loss of body weight (P < .01).The platelet count was reduced with PRIT in a dose-related manner
(Figure 6). The nadir occurred 1 week
after radiation therapy and recovered 2 to 3 weeks later. Minor changes
of the platelet count were shown with 250 µCi (9.25 MBq)
213Bi-DOTA-biotin without pretargeting. The serum values
of BUN in the mice receiving 500 µCi (18.5 MBq)
213Bi-DOTA-biotin were significantly higher than those of
the mice in the control group at 2 and 5 weeks and at 2 months after
treatment (P < .05). However, significant elevations in
BUN were not observed in the groups receiving 250 µCi (9.25 MBq)
213Bi-DOTA-biotin. Histopathologic examination showed that
the mice treated with 250 µCi (9.25 MBq)
213Bi-DOTA-biotin without pretargeting or 500 µCi (18.5 MBq) 213Bi-DOTA-biotin manifested hydronephrosis 4 months
after treatment. In contrast, the mice receiving 100 to 250 µCi
(3.7-9.25 MBq) 213Bi-DOTA-biotin with the pretargeting did
not show the pathologic changes.
ATL is a malignancy of T lymphocytes with a median survival duration of 9 months in the acute form of the disease.1 Various combination chemotherapies have not significantly increased the survival of patients with ATL.1 In light of the disappointing results using conventional combination chemotherapy, IL-2R-directed therapy was developed that included the use of unmodified murine and humanized antibodies (eg, anti-Tac) directed toward IL-2R. Although such therapy yielded partial or complete remissions in one third to one half of patients, most suffered a disease relapse.5,37 The use of monoclonal antibodies armed with toxins or radionuclides to specifically target these cytotoxic agents to the leukemic cells provides a valuable augmentation of therapy. There are a number of components that must be considered in designing an optimal RIT agent, including (1) the selection of the monoclonal antibody and thus the antigenic target, (2) the choice of the delivery system used to target the radionuclide to the tumor cell, and (3) the choice of the radionuclide. As just noted, a pivotal issue to be addressed is the selection of the
monoclonal antibody that targets the tumor and thereby the type of
malignancy chosen as the target for RIT. In the present study we have
chosen the human IL-2R A second issue in designing an optimal RIT reagent is the choice of the
method used to deliver the radionuclide to the tumor cell. In our
clinical trials, we have used intact monoclonal antibodies to deliver
the radionuclide 90Y. There are a number of limitations in
this approach. First, there are physiologic and structural barriers
that limit rapid delivery of high molecular weight molecules such as
intact antibodies to the tumor cells. Second, meaningful tumor uptake
of antibody may not occur until 24 to 48 hours after injection.
Unfortunately, the long serum half-lives of the monoclonal antibody
prolong radiation exposure to normal organs, including radiosensitive
bone marrow which limits the radiation dose that can be safely
administered.10,11 Finally, because of the slow
equilibration of intact monoclonal antibodies with the tumor cell, it
is limited to relatively long-lived To obviate some of the obstacles encountered by conventional RIT with intact radiolabeled monoclonal antibodies, a series of multistep strategies has been described to de-couple the pharmacokinetics of the radionuclide delivery from that of the antibody.12-17,19-22,39,40 The approach used in the present study involves a pretargeting approach that includes 3 steps. This approach delivered large quantities of radioactivity to the tumor with the remaining radionuclide rapidly cleared by the kidney. A pivotal requirement for the success of this technique is that the conjugated antibody stays on the tumor cell surface until the radiolabeled biotin is administered. In this study, the internalization of HAT-SA conjugate into the leukemic cells was investigated with 6 cell lines. Although a similar percentage of the cell-associated radioactivity was present on the cell surface at the beginning, HAT-SA was internalized rapidly in the nonlymphoid SP2/Tac and ATAC4 cells with 60% internalized by 6 hours. In contrast, approximately 50% was present on the cell surface with the 4 leukemic cell lines examined. Thus, internalization of HAT-SA by the 4 leukemic cell lines studied was relatively slow, which made the pretargeting technique promising for the therapy of ATL leukemia with radiolabeled biotin. The third component of an optimal RIT regiment to consider is the
nature of the radionuclide used. There are various We have observed such an advantage of the One potential radionuclide that is studied in the present report,
213Bi, decays with a physical half-life of 46 minutes and
with an The results of the pretargeting trial in the MET-1 model of ATL were encouraging; however, this aggressive T-cell leukemia was not completely eliminated by a single course of therapy with 213Bi. A paradigm is emerging which suggests that, for cancer therapy, the addition of 2 therapeutic agents that interrupt the cell cycle at 2 distinct points may be more than additive in their cytotoxic action leading to malignant cell death. This paradigm has been shown for monoclonal antibodies added at therapeutic doses with chemotherapeutic agents as has been reported for the combination of Herceptin and Paclitaxel.42 In our combination trial, we wanted to obtain the complementary actions of receptor-saturating doses of the anti-Tac monoclonal antibody to yield antibody-dependent cellular cytotoxicity (ADCC) and cytokine deprivation-mediated leukemic cell death with the tumor cytoreduction provided by irradiation mediated by the radionuclide 213Bi delivered to the leukemic cell surfaces. Indeed, whereas neither HAT alone nor 213Bi used in a pretargeting regime yielded complete long-lasting remissions, such remissions were observed in most of the mice receiving both agents in conjunction (Figure 4). In conclusion, our emerging understanding of the IL-2/IL-2R system in normal and leukemic cells opens the possibility for novel IL-2R-directed therapeutic approaches. In particular, pretargeting of ATL cells | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-emitting radionuclide, bismuth 213
Top
Abstract
Introduction
, and CD25+.
Kit225-IG3, HUT102, and MT1 are leukemic T-cell lines, which also
express CD25 on their cell surfaces. SP2/Tac and ATAC4 are nonlymphoid
cells transfected with the IL-2R
-glutamyl transpeptidase were also measured at 2 and 5 weeks and
at 2 and 4 months after treatment. Two to 3 animals in each group were
killed at 5 weeks and at 2 and 4 months after treatment, and the
tissues (liver, kidneys, lung, spleen, intestine, and femur) were
evaluated histopathologically (Pathology Laboratory, National Cancer
Institute, Frederick Cancer Research and Development Center, Frederick, MD).
