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High-dose radioimmunotherapy combined with extracorporeal depletion in a syngeneic rat tumor model†
Evaluation of toxicity, therapeutic effect, and tumor model
Article first published online: 2 FEB 2010
Copyright © 2010 American Cancer Society
Supplement: Cancer Therapy With Antibodies and Immunoconjugates, Supplement to Cancer
Volume 116, Issue Supplement 4, pages 1043–1052, 15 February 2010
How to Cite
Mårtensson, L., Nilsson, R., Ohlsson, T., Sjögren, H.-O., Strand, S.-E. and Tennvall, J. (2010), High-dose radioimmunotherapy combined with extracorporeal depletion in a syngeneic rat tumor model. Cancer, 116: 1043–1052. doi: 10.1002/cncr.24791
The articles in this supplement were presented at the “12th Conference on Cancer Therapy With Antibodies and Immunoconjugates,” Parsippany, New Jersey, October 16-18, 2008.
- Issue published online: 2 FEB 2010
- Article first published online: 2 FEB 2010
- Manuscript Accepted: 21 OCT 2009
- Manuscript Received: 10 JUL 2009
- radionuclide therapy;
- syngeneic tumor model
The aim of the current study was to investigate the possibility of increasing the maximal tolerated dose (MTD) of a tumor-selective radiolabeled antibody when radioimmunotherapy (RIT) is combined with extracorporeal depletion of radioimmunoconjugates from the circulation. Furthermore, the authors evaluated whether this increase in dose improved the therapeutic effect on solid manifest tumors in an immunocompetent animal model.
Rats were injected with high activities/body weight of lutetium (177Lu)- or yttrium (90Y)-labeled antibody conjugates (monoclonal antibody tetraazacyclododecanetetraacetic acid-biotin) and subjected to removal of the conjugate from the circulation by extracorporeal affinity adsorption treatment 24 hours postinjection. Myelotoxicity was assessed by analysis of blood parameters for 12 weeks. The effect of increased doses in combination with extracorporeal affinity adsorption treatment was evaluated with respect to myelotoxicity and therapeutic effect in a syngeneic rat colon cancer model.
The MTD of 177Lu- or 90Y-labeled immunoconjugates could be increased 2.0× or 1.5×, respectively, when RIT was combined with extracorporeal affinity adsorption treatment. All animals treated with 177Lu- or 90Y-labeled antibodies showed persistent complete response of manifest tumors (approximately 10 × 15 mm) within 16 days postinjection. However, several animals showed disseminated disease 1.5 to 3 months postinjection.
Extracorporeal affinity adsorption treatment is a method that safely and efficiently reduces myelotoxicity associated with RIT. Extracorporeal affinity adsorption treatment allows increased administered activity without increased toxicity, with the aim of increasing the absorbed dose to the tumor. However, because tumor/normal tissue radiosensitivity ratios are more favorable in rodents, it is not possible to draw any conclusions concerning the therapeutic efficacy of increased administered activity in combination with extracorporeal affinity adsorption treatment in this study. Targeted RIT with β-emitting radionuclides seems not to be effective in microscopic disease, because metastases developed at sites without previously known disease. Cancer 2010;116(4 suppl):1043–52. © 2010 American Cancer Society.
The concept of radioimmunotherapy (RIT) is based on the use of tumor-directed antibodies armed with radioisotopes to create a cytotoxic tumor-targeting agent. The objective of RIT is to achieve a toxic concentration of radioactivity in tumor tissue, while simultaneously reducing the concentrations in healthy tissue to minimize toxic side effects. RIT has shown promising results in the treatment of lymphoma.1, 2 RIT of manifest solid tumors has been less successful,3 primarily because of the poor accessibility and low radiosensitivity of solid tumors. Intact monoclonal antibodies (MoAbs) diffuse slowly from the vasculature into the tumor, resulting in slow, limited accretion of antibodies in solid tumors.4 This limited accumulation of radioactivity in solid tumors results in a low tumor-to-blood activity ratio and high absorbed doses to radiosensitive organs, for example, bone marrow, liver, kidneys, and lungs, when attempting to achieve high tumor absorbed doses. The dose-limiting factor is generally myelotoxicity caused by the slow clearance of MoAbs from the circulation. These limitations have led to the search for strategies to improve the low tumor-to-blood activity ratio and to reduce the myelotoxicity associated with RIT.5 Examples of such strategies are bone marrow transplantation6 or pretargeting.7
Extracorporeal depletion of radiolabeled antibodies in blood is a strategy that has been demonstrated to improve low tumor-to-blood activity ratios and reduce the toxicity in radiosensitive organs, that is, the bone marrow, liver, lungs, and kidneys. Extracorporeal affinity adsorption treatment selectively and quickly eliminates radiolabeled antibodies from the blood.8 The selectivity of extracorporeal affinity adsorption treatment is based on the biotin-avidin system using the high-affinity interaction between avidin and biotin. Antibodies are radiolabeled and biotinylated using a trifunctional protein reagent comprising tetraazacyclododecanetetraacetic acid (DOTA) and biotin.9 By passing whole blood extracorporeally through an avidin column, the radioimmunoconjugates are adsorbed in the column and eliminated from the circulation. Extracorporeal affinity adsorption treatment eliminates 90% to 95% of radioimmunoconjugates from the circulation of rats, and significantly increases the tumor-to-normal tissue activity ratio in a syngeneic rat tumor model.8 Similar results have also been obtained in patients.10 In addition, extracorporeal affinity adsorption treatment efficiently reduces myelotoxicity and other organ toxicity associated with RIT, without decreasing the concentration of radioimmunoconjugates in tumors, provided that extracorporeal affinity adsorption treatment is performed at an optimal point in time after antibody administration.11
Several parameters must be considered when combining RIT with extracorporeal affinity adsorption treatment. Sufficient amounts of radioimmunoconjugates should be allowed to accumulate in the tumor tissue before being eliminated from the circulation. Conversely, shortening the time between the injection of radioimmunoconjugates and extracorporeal affinity adsorption treatment is crucial in limiting myelotoxicity. Furthermore, the effective half-life of the radioimmunoconjugate is also important, and must be considered when combining radioimmunoconjugates with extracorporeal affinity adsorption treatment. A previous study conducted in the syngeneic rat tumor model using yttrium (90Y)- and lutetium (177Lu)-BR9611 has shown that conducting extracorporeal affinity adsorption treatment earlier than 24 hours postinjection decreases the tumor accretion of radioimmunoconjugates. Through calculations of the accumulated activities in blood and tumor with simulated extracorporeal affinity adsorption treatment, we have theoretically estimated that it would be possible to increase the maximal tolerated dose (MTD) of an antibody conjugate labeled with 177Lu or 90Y considerably when combining it with extracorporeal affinity adsorption treatment.
The aim of this study was to investigate the possibility of increasing the MTD of a tumor-specific radiolabeled antibody in combination with extracorporeal affinity adsorption treatment, and to evaluate whether this increase has improved therapeutic effects on solid manifest tumors in an immunocompetent animal model.
MATERIALS AND METHODS
Monoclonal Antibody and Immunoconjugates
The chimeric (mouse/human) monoclonal immunoglobulin G1 antibody BR96 (Seattle Genetics, Seattle, Wash), binding the tumor-associated Lewis Y glycoprotein (LeY) was used. LeY is expressed in the majority of human epithelial tumors, including breast, gastrointestinal (GI) tract, nonsmall cell lung, cervix, ovary, and some melanomas.12 As with the majority of tumor-associated MoAbs, BR96 also reacts with some normal tissue, primarily the epithelial cells of the GI tract.12 Trastuzumab, which was used as a control antibody, is an antireceptor antibody with a clinical role in the treatment of breast cancer. It is directed against the extracellular domain of HER–2, a type I tyrosine kinase receptor of the HER family, which is overexpressed and/or amplified in 20% to 30% of human breast carcinomas.
BR96 and trastuzumab were conjugated with the trifunctional chelator 1033, carrying a DOTA moiety and a biotin moiety,9 as described previously.13, 14 Briefly, 80 μg of the 1033 chelator was added per milligram BR96 (in 50 mM N-[2-hydroxyethyl]piperazine N′-[2-ethanesulfonic acid] [HEPES], 1 mM diethylenetriamine pentaacetic acid [DTPA] buffer, pH 8.5) and incubated for 2 hours at room temperature and then overnight at 4°C. After conjugation, the conjugate was transferred to 0.25 M ammonium acetate buffer (pH 5.3) by dialysis (Slide-A-Lyzer Dialysis Cassette, 10K MWCO, Pierce Protein Research Products, Rockford, Ill), eliminating any free 1033. The number of 1033 molecules per BR96 molecule was determined by the 4′-hydroxyazobenzene-2-benzoic acid photometric method.15
The same procedure was used for labeling with 177LuCl3 (MDS Nordion, Vancouver, Canada) and 90YCl3 (MDS Nordion, Fleurus, Belgium). Both the 1033-antibody conjugates in 0.25 M ammonium acetate buffer and the radionuclide solutions were preheated at 45°C for 10 minutes. The 1033-antibody solutions were then added to the radionuclide-containing vials and incubated at 45°C for 15 minutes. The reaction was quenched with an excess of DTPA for 5 minutes. The radiolabeled immunoconjugates were diluted in 1% human serum albumin. The radiochemical purity of the labeled immunoconjugates was determined by instant thin-layer chromatography (ITLC) (1 × 9-cm silica-gel–impregnated fiberglass sheet, eluted in 0.1 M ethylenediaminetetraacetic acid). Separation by size-exclusion chromatography together with high-performance liquid chromatography (HPLC) (7.8 × 300-mm molecular sieving column, Phenomenex SEC S3000, Phenomenex, Torrance, Calif; eluted in 0.05 M sodium phosphate at 1.0 mL/min) was used to control the radiochemical purity and to detect signs of aggregation or fragmentation. To ensure that the labeling had not affected the biotin moiety of the 1033 molecule, the avidin-binding ability of the radioimmunoconjugates was assessed. An adsorption column packed with approximately 0.3 mL avidin-agarose16 was used. A 50-μL sample of radioimmunoconjugates was added to the column and incubated for 10 minutes at room temperature. The column was washed 8× with 0.5 mL PBS containing 0.05% Tween 20, and the liquid from each wash was collected separately in tubes. The activity in the column and in each tube was measured with a NaI(TI) detector. The avidin-binding fraction was expressed as the percentage of radioactivity in the column in relation to the sum of the radioactivity in the tubes and the column.
Syngeneic Rat Tumor Model
Immunocompetent rats of the brown Norway (BN) strain (Harlan, Horst, the Netherlands) were used. As demonstrated by immunohistochemistry, BN rats express the BR96 epitope in some normal tissues, such as the Gl epithelium, hence mimicking the human situation.17
BN7005-H1D2 is a single-cell clone of a rat colon carcinoma originally induced by 1,2-dimethyl-hydrazine in a BN rat. BN7005-H1D2 cells were cultured in RPMI-1640 medium (Euroclone, Devon, UK) supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 10 mM HEPES buffer solution, and 29.3 μM gentamicin at 37°C in a humidified atmosphere with 5% CO2. Cells were washed in saline, trypsinized, and washed in medium + 10% FCS. Animals were inoculated subperitoneally with 3 × 105 cells (in 50 μL medium). Experiments were initiated 12-14 days after inoculation (tumor size, 10 × 15 mm). The animals were kept under standard conditions and fed with standard pellets and fresh water. Studies were conducted in compliance with Swedish legislation on animal rights and protection, and were approved by the ethics committee.
Extracorporeal Affinity Adsorption Treatment
The extracorporeal system included a 403U/C12 pump (Watson-Marlow Alitea AB, Stockholm, Sweden) with a 15-cm silicone tube (1.6/6.35-mm inner/outer diameter). The column housing consisted of a modified 2-mL polypropylene syringe (9 × 30 mm) with a 72-μm filter at the bottom. The column was packed with 1.5 mL avidin-agarose16 with approximately 0.5 mL NaCl above as an extra air trap. Polyvinyl chloride (PVC) tubing (1-mm inner diameter) was used as medical lines. An air trap consisting of PVC tubing (9.5-mm inner diameter) was connected to trap any air bubbles before the blood was returned to the animals. The extracorporeal circuit had a volume of approximately 3.5 mL. Before extracorporeal affinity adsorption treatment, the system was flushed with heparin solution (20 IU/mL heparin in 9 mg/mL NaCl) as an anticoagulant. The system is illustrated in Figure 1.
Thirty minutes before insertion of the cannulae (Neoflon 0.7 × 19 mm, Becton Dickinson, Helsingborg, Sweden), a 2% glyceryl nitrate salve (Apoteket AB, Stockholm, Sweden) was applied to the entire tail of each rat to dilate the blood vessels. The animals were anesthetized with isoflurane using a U-400 anesthesia unit (Agnthos, Stockholm, Sweden). The rats were first anesthetized in a 1.4-L induction chamber (3.3% isoflurane, 575 mL/min air flow) and then placed on a heating pad (30°C). Anesthesia was sustained through anesthesia masks connected to the same anesthesia unit as the induction chamber. A cannula was carefully inserted into 1 of the lateral tail veins (1-2 cm from the tip of the tail) for the return of blood. The cannula was secured to the tail with adhesive tape and connected to the extracorporeal system. To prevent coagulation and to confirm that the cannula was correctly inserted, heparin solution from the extracorporeal circuit was infused for a few seconds and then stopped (regulated by the pump). For blood access, another cannula was inserted into the ventral tail artery, approximately 5 cm from the tip of the tail. This cannula is accurately inserted when there is a continuous flow of blood through the cannula. Before connecting the cannula to the extracorporeal circuit, a blood sample was collected. As soon as the artery cannula was connected to the system, the extracorporeal circulation was started in bypass mode (column not connected). The heparin solution present in the system was infused to prevent clotting, and the whole circuit was filled with blood, bypassing the column. When the circuit was filled with blood and any air bubbles in the circuit had been collected in the air trap, the avidin column was connected to the circuit, and the affinity adsorption started. Blood was pumped through the column at a rate of 0.4 mL/min. Extracorporeal affinity adsorption treatment was performed on 6 rats in parallel. During extracorporeal affinity adsorption treatment, the rats were anesthetized at a lower level of anesthesia (2.0% isoflurane, 575 mL/min air flow) and kept on electric heating pads (30°C) to keep them warm. After approximately 2 hours of affinity adsorption (approximately 3 blood volumes; blood volume estimated to be 65 mL/kg body weight), the procedure was stopped, and the blood in the circuit was returned to the rat. A blood sample was collected from the arterial cannula before withdrawal of the cannulae. The tail was compressed to stop bleeding.
Rats were injected intravenously with 150 μg of radiolabeled 1033-BR96 as specified in Table 1. Two groups (6 rats/group) were injected with 177Lu-1033-BR96: 1 with an activity of 600 MBq/kg body weight, not subjected to extracorporeal affinity adsorption treatment, and 1 with an activity of 1200 MBq/kg body weight in combination with extracorporeal affinity adsorption treatment. Similarly, 2 groups (5 rats/group) were injected with 90Y-1033-BR96, 1 with an activity of 350 MBq/kg body weight, not subjected to extracorporeal affinity adsorption treatment, and the other with 525 MBq/kg body weight in combination with extracorporeal affinity adsorption treatment. Previous studies have shown that the lower activities (600 MBq/kg 177Lu and 350 MBq/kg 90Y) are just below the MTD of these immunoconjugates. The rats injected with the higher activities (1200 MBq/kg 177Lu or 525 MBq/kg 90Y) were subjected to extracorporeal affinity adsorption treatment 24 hours postinjection. Control groups were injected with unlabeled 1033-BR96 (5 rats), or radiolabeled 1033-trastuzumab (5-6 rats) (Table 1). NaCl was also administered as a control substance, but because there was no difference between NaCl and 1033-BR96 in terms of toxicity and therapeutic efficacy, only the 1033-BR96 results are presented. To evaluate the myelotoxicity, blood samples were collected from the tail artery twice a week for 12 weeks postinjection and white blood cell (WBC) and platelet (PLT) counts were analyzed in a Medonic Cell Analyzer-Vet CA530 (Boule Medical, Stockholm, Sweden). At the time of blood sampling, the body weight and physical condition of the animals were recorded.
|Group||No. of Rats Per Group||Treatment||Activity, MBq/kg Body Weight||ECAT|
|6||6||177Lu-1033-BR96||1200||24 hours after injection|
|7||5||90Y-1033-BR96||525||24 hours after injection|
Scintillation camera imaging was performed on all the animals injected with 177Lu-labeled immunoconjugates to assess the tumor accumulation of immunoconjugates. A scintillation camera (SMV DST-Xli, Sopha Medical, Buc, France) equipped with a medium-energy collimator was used to determine whole-body activity and tumor activity. The accumulation of activity in the tumors was investigated by defining a region of interest (ROI) covering the tumor and then comparing the same ROI at different points in time postinjection. Imaging was performed 24 hours postinjection, after extracorporeal affinity adsorption treatment (26 hours postinjection), 48 hours postinjection, and 72 hours postinjection The results are given as counts/pixels not corrected for the decay of 177Lu.
Evaluation of Therapeutic Efficacy
The tumor volume was monitored by measuring the tumors with a caliper. Effects on the primary tumor and outcome were monitored during a total of 6 months postinjection. The animals were sacrificed when the tumor burden became too high (>25 × 25 mm), after dramatic weight loss (>20%), or at the end of the study (180 days postinjection).
Evaluation of Toxicity
To evaluate myelotoxicity, blood samples were collected from the tail artery twice a week during the first 21 days postinjection and then once weekly up to Day 100 postinjection. WBC, red blood cell, and PLT counts were analyzed in a Medonic Cell Analyzer–Vet CA530 (Boule Medical).
In addition, plasma was collected, and the levels of aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase, bilirubin, and creatinine were measured to determine liver and kidney toxicity.
The body weight, tumor size, and general status of the animals were recorded at the time of blood sampling.
The toxicity was graded according to the National Cancer Institute Common Terminology for Adverse Events (version 3.0) to compare the severity of the toxicity between the various regimens used.
Preparation of Radioimmunoconjugates
After conjugation, the average number of 1033 molecules per BR96 molecule was determined to be 2.6. After labeling, the specific activity of 177Lu-1033-BR96 conjugates was 123 MBq/nmol and 53 MBq/nmol. The specific activity of 90Y-1033-BR96 conjugates was 181 MBq/nmol and 113 MBq/nmol. ITLC showed the radiochemical purity to be 94% and 99% for the 2 177Lu-1033-BR96 conjugates and 96% for both 90Y-1033-BR96 conjugates. After conjugation, the average number of 1033 molecules per trastuzumab molecule was determined to 4.3. The specific activity of 177Lu-1033-trastuzumab was 45 MBq/nmol, and that of 90Y-1033-trastuzumab was 133 MBq/nmol. ITLC showed the radiochemical purity to be 99% for 177Lu-1033-trastuzumab and 97% for 90Y-1033-trastuzumab. No aggregation or fragmentation was observed with HPLC. The avidin-binding fraction exceeded 90% for all radioimmunoconjugates at the time of injection.
Extracorporeal Affinity Adsorption Treatment
The extracorporeal affinity adsorption procedure was conducted 24 hours after the injection of 1200 MBq/kg body weight of 177Lu-1033-BR96 or 525 MBq/kg body weight of 90Y-1033-BR96, with 5-6 rats in each group (Table 1). Blood was pumped through the system at a rate of 0.4 mL/min for 2 hours. Blood samples taken before and after extracorporeal affinity adsorption treatment were analyzed regarding activity content. The efficacy of the adsorption was calculated in percentage of activity before extracorporeal affinity adsorption treatment. A mean of 95% (range, 94%-96%) of the activity in the blood was eliminated during extracorporeal affinity adsorption treatment.
Evaluation of Toxicity
Body weight loss
Animals injected with radiolabeled antibodies lost weight (median loss, 5.3%; range, 4%-10%) during the first 7 days postinjection, reaching a nadir on Days 5-7. After Day 7, these animals started to gain weight. The weight progression of control animals injected with 1033-BR96 or NaCl was unaffected by the injections.
Myelotoxicity was monitored by quantification of WBC and PLT counts. All groups injected with radioimmunoconjugates (177Lu-1033-BR96, 177Lu-1033-trastuzumab, 90Y-1033-BR96, or 90Y-1033-trastuzumab) showed a dramatic decrease in WBC count during the first week postinjection (falling to 1%-10% of initial values) (Fig. 2A and C). All of these animals had recovered their initial WBC 2 months postinjection. The PLT count had decreased to approximately 15% of initial values on Day 14 postinjection in animals injected with radioimmunoconjugate (Fig. 2B and D), but the values had recovered in all animals 35 days postinjection (Fig. 2B and D). No bleeding was observed during the study period. No increase in myelotoxicity was seen in animals injected with the higher activity of 177Lu-1033-BR96 or the higher activity of 90Y-1033-BR96 in combination with extracorporeal affinity adsorption treatment, compared with animals injected with lower activities not undergoing extracorporeal affinity adsorption treatment. No myelotoxicity was seen in control animals injected with NaCl (data not shown) or 1033-BR96 (Fig. 2). The control animals were sacrificed 14-21 days postinjection because of large tumor burden.
Neither liver nor kidney toxicity above grade 1 was observed in any group. No clinical symptoms indicating lung, liver, kidney, or other organ toxicity were seen.
Influence of Extracorporeal Affinity Adsorption Treatment on Tumor Concentration of Radioimmunoconjugates
The activity in the tumors of animals injected with 1200 MBq/kg 177Lu-1033-BR96 was twice that in animals injected with 600 MBq/kg 177Lu 24 hours postinjection, but before extracorporeal affinity adsorption treatment (Fig. 3). In the animals subjected to extracorporeal affinity adsorption treatment, the whole-body activity was reduced by approximately 50% (data not shown), and tumor activity was reduced by 17% (Fig. 3). These results correspond well with previous results concerning the effects of extracorporeal affinity adsorption treatment on tumor accretion of antibodies.11 In animals not subjected to extracorporeal affinity adsorption treatment, the tumor activity remained constant for 72 hours, whereas it decreased over time in the animals subjected to extracorporeal affinity adsorption treatment. Control animals injected with the control conjugate 177Lu-trastuzumab showed the same amount of activity in tumors as animals injected with tumor-specific 177Lu-BR96 (data not shown).
Therapeutic Effects on Manifest Tumors
At the time of treatment, the size of the primary tumor was approximately 10 × 15 mm. All animals injected with 177Lu-1033-BR96 or 90Y-1033-BR96 showed persistent complete response within 16 days postinjection (Table 2) until the end of the study (median, 143 days; range, 41-180 days). In the control group injected with 177Lu-1033-trastuzumab, 3 of 5 animals showed complete regression, whereas 2 animals exhibited stable disease, which later progressed (70-80 days postinjection). Of the 3 animals showing complete regression, 1 animal developed disease recurrence with recurrence of the local tumor 97 days postinjection. All animals in the control group injected with 350 MBq/kg 90Y-1033-trastuzumab showed complete regression of tumors. However, 3 of these animals developed disease recurrence, with recurrence of the local tumor 99, 113, and 134 days postinjection. All animals in the control group injected with unlabeled 1033-BR96 displayed progressive disease and were sacrificed after 21 days postinjection because of large tumor burden (>25 × 25 mm). No metastases were detected at dissection.
|Treatment||Activity, MBq/kg Body Weight||ECAT||Local|
|Progressive Disease||Complete Response||Disease Recurrence|
|177Lu-1033-BR96||1200||24 hours after infusion||0/6||6/6||0/6|
|90Y-1033-BR96||525||24 hours after infusion||0/5||5/5||0/5|
Therapeutic Effects on Microscopic Disease
In both groups of animals injected with 177Lu-1033-BR96, 3 of the 6 animals developed disease recurrence with metastases (Table 3). In the group treated with 600 MBq/kg 177Lu-1033-BR96, metastases were detected 41, 48, and 97 days postinjection. In the group treated with 1200 MBq/kg 177Lu-1033-BR96 with subsequent extracorporeal affinity adsorption treatment, metastases were detected 65, 97, and 97 days postinjection. Metastases were observed as ascites, liver, lung, and lymph node metastases in these animals. In the control group injected with 177Lu-1033-trastuzumab, 3 of 5 animals showed complete regression, whereas 2 animals exhibited stable disease, which later progressed. Of the 3 animals showing subsequent complete regression, 1 animal developed disease recurrence with lung metastasis 97 days postinjection. Metastases (liver and lymph) were also detected in 1 of the 2 animals with progressive primary tumors. Thus, 2 animals were disease free at the end of the study.
|Treatment||Activity, MBq/kg Body Weight||ECAT||Distant Metastases||Disease-Free Survivors|
|177Lu-1033-BR96||1200||24 hours after infusion||3/6||3/6|
|90Y-1033-BR96||525||24 hours after infusion||0/5||5/5|
One of the animals in the group treated with 350 MBq/kg 90Y-1033-BR96 developed disease recurrence, showing metastases (systemic lymphatic dissemination) 71 days postinjection, whereas none of the animals treated with 525 MBq/kg 90Y-1033-BR96 with subsequent extracorporeal affinity adsorption treatment displayed any signs of metastasis at dissection at the end of the study (180 days postinjection). In 2 of the animals in the control group treated with 350 MBq/kg 90Y-1033-trastuzumab, metastases were detected 99 and 134 days postinjection (lung and lymph metastases).
We have demonstrated that the maximal tolerable activity of 177Lu- or 90Y-labeled immunoconjugates can be increased 2.0× or 1.5×, respectively, when RIT is combined with extracorporeal affinity adsorption treatment 24 hours postinjection in a rat colon cancer model. We have also shown that all animals treated with 177Lu-1033-BR96 or 90Y-1033-BR96 showed persistent complete response of the manifest tumor (approximately 10 × 10 mm) within 16 days postinjection, which is most likely attributable to the rat tumor model having a much more favorable radiation sensitivity ratio between tumor and bone marrow than that of humans. Because complete response was achieved, we could not determine a dose-response relationship.
When evaluating new therapeutic strategies, it is necessary to use biological models resembling the clinical situation. We used a syngeneic tumor model that has several advantages compared with immunodeficient xenogeneic models. The induction of vascularization and stroma tissue support by syngeneic tumor cells is more adequate than by xenogeneic cells, and the immune response to the tumor is similar to that of the animal in which the original tumor developed. As a consequence, the infiltration of the tumor into surrounding normal tissue and metastasizing at other locations are more similar to the clinical situation than in immunodeficient xenograft tumor models. However, our study demonstrates that rodents are not optimal for RIT studies because of their radioresistance, especially with respect to bone marrow. The maximal tolerable dose per kilogram body weight for the commercially available radioimmunoconjugate Zevalin (a 90Y-labeled murine variant of rituximab) is considerably lower in patients than the lowest activity/body weight (megabecquerels/kilogram) of 90Y-1033-BR96 administered to the rats in this study. The same relationship has been seen for 177Lu-labeled antibodies.18, 19 These findings are also supported by a median lethal dose at 60 days (LD50/60) in rodents estimated at 9 grays (Gy), compared with the LD50/60 of 4-6 Gy in humans.20 Although the bone marrow is considerably more radioresistant in rodents than in humans and other large mammal species,21, 22 no studies have been reported indicating that tumors of rodent origin are more radioresistant than human tumors.
It was thus not possible in the present study to determine whether there is any therapeutic advantage in increasing the administered activity of the tumor-specific antibody in combination with extracorporeal affinity adsorption treatment, because all the animals exhibited persistent complete remission of the primary tumors. However, imaging of the tumors showed that animals treated with higher activities of the tumor-specific antibody in combination with extracorporeal affinity adsorption treatment had a considerably higher amount of activity in their tumors (Fig. 3) during 48 hours postinjection, and the accumulated activity was increased by 60% to 65% between 24 and 48 hours. This results in a higher absorbed dose rate and a higher absorbed dose to the tumor. Before extracorporeal affinity adsorption treatment, there was twice as much activity in tumors treated with the higher activities (megabecquerels) of the tumor-specific antibody. After extracorporeal affinity adsorption treatment, the activity in tumors decreased because of the depletion of radioimmunoconjugates from the blood, and at 48 hours postinjection the same amounts were detected in the 2 groups (Fig. 3). The persistent complete remission of primary tumors in several of the control groups injected with radiolabeled trastuzumab was unexpected, because trastuzumab does not target this tumor cell line in vitro. However, unspecific antibodies do accumulate in tumor tissue in vivo.23 This unspecific accumulation in combination with the high activity injected into these animals might explain the therapeutic effects seen here. In addition, the relatively small body size of rats means that the administration of long-range beta emitters such as 90Y (maximum penetration depth, 12 mm) resembles whole-body radiation, where the cross-dose (ie, not targeted radiation) from surrounding tissues will increase the absorbed radiation dose to the tumor.
Progressive local disease was seen in all control animals treated with unlabeled tumor-specific antibodies, and these animals were sacrificed because of large tumor burden 2 weeks postinjection. This indicates that the antibody per se has no significant therapeutic efficacy. There was no evidence of metastases at dissection, which supports our assumption that dissemination was microscopic at the time of treatment.
Our results underline the problem of choosing relevant animal tumor models for the prediction of the therapeutic efficacy and toxicity of RIT in a clinical situation. The use of a tumor model in a small animal is also compromised by geometry. It would most likely be possible to increase the administered activity of 90Y-labeled MoAb beyond 1.5× in humans because of the decrease in cross-dose to the bone marrow from adjacent organs (because of the larger body size of humans). Nemecek et al are presently evaluating extracorporeal affinity adsorption treatment in a nonhuman primate model without tumors.24, 25 The results of their study will hopefully provide important information in radiosensitivity in normal tissues, but no information will be obtained on the ratio of radiosensitivity between tumor and bone marrow. A tumor model in a large syngeneic mammal would be optimal for predicting the therapeutic effects and toxicity of radiolabeled MoAbs regarding both tissue sensitivity and size.
Several animals in the study exhibited disseminated disease after local complete and persistent responses to radiolabeled BR96 (mean, 83 days postinjection; range, 41-134 days postinjection). No difference was detected between animals treated with different activities. However, there was a trend toward fewer metastases in animals treated with 90Y-labeled MoAb. This could be explained by the long range of the β-emission of 90Y, resembling whole-body radiation, as discussed above. The development of disseminated disease at sites without previously known disease has also been seen in a clinical study of patients treated with RIT.26 This indicates that targeted RIT with high-energy β-emitting radio nuclides may not be effective in cases of microscopic disease. This is most likely because of the radiation energy being deposited outside the tumor cell cluster. Auger-emitting radioisotopes such as 111In, and α-particle emitting radioisotopes such as 213Bi, may be better alternatives in the treatment of disseminated, single-cell, or oligocellular disease.27 Another possible explanation could be selection of disseminated cells after therapy not expressing the target antigen.
Extracorporeal affinity adsorption treatment can safely and efficiently reduce myelotoxicity associated with RIT.10 Extracorporeal affinity adsorption treatment allows increased administered activity without increased toxicity, with the aim of increasing the absorbed dose to the tumor. The present study demonstrates that the administered activity of 177Lu- or 90Y-labeled immunoconjugates can be increased 2.0× or 1.5×, respectively, without increased toxicity when RIT is combined with extracorporeal affinity adsorption treatment in a rat tumor model. However, because tumor-to-normal tissue radiosensitivity ratios are much more favorable in rodents, it was not possible to draw any conclusions concerning the therapeutic efficacy of increased administered activity in combination with extracorporeal affinity adsorption treatment.
Targeted RIT with beta-emitting radionuclides seems not to be effective in cases of microscopic disease, because metastases develop at sites without previously known disease.
CONFLICT OF INTEREST DISCLOSURES
The papers in this supplement represent proceedings of the “12th Conference on Cancer Therapy With Antibodies and Immunoconjugates,” in Parsippany, New Jersey, October 16-18, 2008. Unrestricted grant support for the conference was provided by Actinium Pharmaceuticals, Inc.; Bayer Schering Pharma; Center for Molecular Medicine and Immunology; ImClone Systems Corporation; MDS Nordion; National Cancer Institute; National Institutes of Health; New Jersey Commission on Cancer Research; and PerkinElmer Life & Analytical Sciences. The supplement was supported by an unrestricted educational grant from ImClone Systems Corporation, a wholly-owned subsidiary of Eli Lilly and Company, and by page charges to the authors. Supported by grants from The Swedish Research Council, The Swedish Cancer Society, Mrs. Berta Kamprad's Foundation, Gunnar Nilsson's Foundation, Lund University Medical Faculty Foundation, and the Lund University Hospital Fund.
- 14Blood pharmacokinetics of various monoclonal antibodies labeled with a new trifunctional chelating reagent for simultaneous conjugation with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid and biotin before radiolabeling. Clin Cancer Res. 2005; 11( 19 pt 2): 7171s-7177s., , , et al.
- 20Physics and Radiobiology of Nuclear Medicine. New York, NY: Springer-Verlag; 2003: 196..