Extracorporeal immunoadsorption (ECAT) is a method of reducing activity in radiosensitive organs by removing excess monoclonal antibodies (MAbs) from the blood. Previously, the authors experimentally evaluated ECAT based on the avidin-biotin concept after intravenous administration of radioimmunoconjugates. The aim of the current study was to determine whether ECAT could be used to reduce activity after intraperitoneal (i.p.) administration of indium-111(111In)–HMFG1–biotin in rats, and to compare the pharmacokinetics of 111In-HMFG1 with or without attached biotin after i.p. injection.
HMFG1, a murine immunoglobulin G1 MAb that recognizes an epitope on the polymorphic epithelial mucin (PEM) antigen, was labeled with 111In and then biotinylated. ECAT was explored from unseparated blood using an avidin-agarose adsorption column. Thirty rats were used as controls and 13 underwent ECAT. The whole-body (WB), blood, and organ activity were monitored.
The binding capacity of 111In-HMFG1-biotin to avidin was high. Biotinylation did not enhance the excretion of HMFG1. When ECAT was employed, the WB and blood radioactivity were reduced by 35–40% (P < 0.05) and 75–86% (P < 0.01), respectively. After the completion of ECAT, the activity uptake in organs was significantly decreased.
Extracorporeal immunoadsorption (ECAT) is a new method of enhancing radioimmunotargeting of tumors by removing excess radiolabeled monoclonal antibodies (MAbs) from the blood by affinity adsorption, thus reducing the activity in radiosensitive organs and improving the tumor–to–normal tissue uptake ratio (Fig. 1).1 Since the concept of the current model is based on the high binding affinity of avidin to biotin (pK1015 M−1), a variety of selected radiolabeled MAbs can be removed from the circulation at a determined optimal interval after injection, provided that these MAbs are biotinylated.
We have previously developed and experimentally evaluated ECAT after intravenous administration of various biotinylated MAbs, namely, 96.5, L6, and BR96, labeled with different radionuclides. These studies have shown that ECAT applied to a rat tumor model can manifestly reduce whole-body (WB), blood, and organ activity without significantly affecting tumor uptake.2–5 ECAT can also be repeated, leading to a further reduction of WB activity.5 In contrast to avidin chase, the ECAT approach might improve radioimmunotargeting without overloading the liver with radioactivity.6 The ex vivo biocompatibility of the avidin immobilized on the agarose matrix, which is a key component of the adsorption column, was proven to be good when fresh blood from eight healthy donors was used.7
There are several proponents of intraperitoneal (i.p.) administration of radiolabeled antibodies in the treatment of ovarian cancer. Their argument is that the i.p. route exhibits better pharmacokinetics, resulting in a better tumor–to–normal tissue ratio and allowing the administration of higher levels of activity with relatively low toxicity.8–12 However, other investigators found no essential differences in tumor uptake in a study comparing i.p. and intravenous (i.v.) delivery of radiolabeled c-MOv18 in ovarian cancer patients.13 The arguments against i.p. administration in radioimmunotherapy of ovarian cancer are preexisting adhesions and peritoneal carcinomatosis, which might impair locoregional delivery or make this approach hazardous.13 It has also been reported that retroperitoneal, lymph node, and hematogenous metastases are not successfully targeted with i.p. administration.11 Furthermore, the i.v. route might be superior for bulky stage disease, such as tumor nodules larger than 2 cm in diameter.14
The primary aims of the current study were to determine whether ECAT could be used to reduce WB and blood activity after i.p. administration of biotinylated and radiolabeled MAb (indium-111[111In]–HMFG1–biotin) and to determine the optimal time for initiation of the adsorption. Since ECAT requires biotinylation of MAb, a comparative biodistribution study of nonbiotinylated and biotinylated 111In-HMFG1, following intraperitoneal administration in rats, was also performed.
Monoclonal Antibodies, Radiolabeling, and Biotinylation
HMFG1-CITC-DTPA (Theragyn, Antisoma Ltd, UK) is an antipancarcinoma murine immunoglobulin G1 MAb that recognizes an epitope on the polymorphic epithelial mucin (PEM) antigen. Ten μL of 1 M sodium acetate was added to a vial containing 22 MBq 111In chloride. Nine hundred μL of HMFG1-CITC-DTPA was added to the 111In solution and incubated for 30 minutes at room temperature. The quality of the radioconjugate was determined by both thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC).
Subsequent to radiolabeling, biotinylation of 111In HMFG1 was performed by adding 4 μL biotin solution to a vial containing 500 μL 111In-HMFG-1 conjugate (2.25 mg/mL). After the mixture had been incubated for 4 hours at room temperature, low-molecular-weight components were removed by gel filtration. Immunoreactivity in vitro was determined using enzyme-linked immunoadsorbent assay (Bioinvent Ltd., Lund, Sweden). The biotinylated HMFG1 was tested for binding capacity to avidin. Fifty μL of biotinylated and radiolabeled HMFG1 was applied on avidin-agarose microcolumns and incubated for 5 minutes at room temperature. The columns were then washed with phosphate-buffered saline and measured for radioactivity in a gamma counter.
All studies were performed in accordance with Swedish legislation on animal rights and protection. Forty-three rats of the F1 breed of Brown Norway (BN) and Wistar Furth (WF) were used in the experiments. Thirty of these rats served as controls in the biodistribution study and were injected i.p. with approximately 150 μg of nonbiotinylated (n = 12) or biotinylated (n = 18) 111In-HMFG1 (4.5–5.5 MBq per rat). Thirteen rats underwent ECAT starting 12 hours (n = 11) or 18 hours (n = 2) after i.p. injection of 111In-HMFG1-biotin.
One to 2 days before the ECAT, the rats underwent surgery under i.v. anesthesia (0.25% Nembutal solution) so that catheters could be inserted into the arteria carotis and vena jugularis to gain blood access. The method by which ECAT removes exogenous targeting molecules has been described previously.15 Before initiating ECAT, the system was flushed with buffer A, containing 20 IU/mL heparin, in order to prevent thrombotic complications and activation of the complement cascade. ECAT was explored from unseparated blood using an adsorption column containing Mitra Avidin Agarose (Mitra Medical Technology Ltd., Lund, Sweden). Before connection to the tubing, the column was carefully washed with 0.9% NaCl solution to eliminate free avidin from the adsorbent. Blood was pumped from the arterial catheter through an adsorption column at a flow rate close to theoretical clearance for 21/2 hours, i.e., approximately three blood volumes were passed through the column and then returned to the rat via the venous catheter.
WB imaging was performed using a General Electric 400T scintillation camera (GE, Milwaukee, WI) equipped with a medium-energy collimator. Images were stored and analyzed with Nuclear MAC 2.7 software (Littleton, CO) to obtain the total number of counts in the entire body. After correction for radioactive decay and background subtraction, the number of counts was used to calculate the activity retention in the body.
Blood samples were drawn from the periorbital venous plexus at 5 minutes and 8, 16, 24, 48, 72, and 96 hours after i.p. injection in order to determine the blood activity. In six rats, the blood activity was analyzed every 20 minutes during the ECAT procedure.
Control rats were dissected at 8, 24, 72, and 96 hours postinjection (n = 3 at each time point). The rats subjected to ECAT were sacrificed directly after completion of the procedure at 16 hours p.i. (n = 10) or were monitored and killed at 24 hours p.i. (n = 3). At dissection, organs and tissues of interest were removed and weighed and the activity content measured.
The radioactivity in blood and in the dissected organs was measured in an automatic NaI(Tl) scintillation well counter (LKB, Turku, Finland). The number of counts was corrected for the decay of 111In. The activity uptake was expressed as a percent of the injected activity per gram of tissue (%/g).
All the results are reported as mean values ± 1 standard deviation. The program Excel 2000 SR1 (Microsoft Corporation, Bellevue, WA) was used to estimate mean differences by calculating sample statistics and a Student t test, assuming equal and unequal variances. Confidence levels greater than 95% (P < 0.05) were considered to be significant.
Both the in vitro and in vivo binding capacities of 111In-HMFG1-biotin to avidin-gel were high (97% and 86%, respectively). The radiochemical yield after 111In labeling was 97.2% without signs of aggregation. The immunoreactivity of the MAb was retained. All the animals tolerated surgery and the ECAT procedure well. The WB retention, blood activity, and uptake in normal organs of 111In-HMFG1 were similar to those of biotinylated MAb. The uptake in the liver was insignificantly (P = 0.07) higher at 24 hours postinjection in the biotin group. Peak radioactivity transferred to blood after i.p. injection of the MAb (Fig. 2) seemed to be between 8 and 16 hours (about 3%/g of the injected activity, or about 65% of the administered activity). Hence, the optimal time for initiation of ECAT assumed to be 8–12 hours after i.p. administration of 111In-HMFG1-biotin.
By employing ECAT, the WB radioactivity was reduced by 35–40% immediately after ECAT and continued to decrease slightly during the next 9 hours after completion of ECAT (Fig. 3A).
The blood radioactivity during the ECAT procedure was reduced by 75–86%, rising slightly from 0.42%/g directly after completion of the procedure to 0.7%/g during the following 9 hours (Fig. 3B). The major activity reduction in the blood (approximately 80%) occurred during the first 60 minutes of ECAT. Both immediately and 9 hours after the completion of ECAT, the activity uptake in the organs showed a significant decrease when compared with the controls (Fig. 4).
Extracorporeal immunoadsorption is a strategy that can be used to accelerate MAb clearance by removing excess antibodies from the blood. It is based on the avidin-biotin concept and has shown a reproducible and good capability to enhance the relative tumor uptake in different rat tumor models after intravenous injection of 96.5, L6, and BR96 MAbs labeled with iodine-125, indium-111, rhenium-188, or yttrium-90.16, 17 As there are several advocates of the i.p. administration of radiolabeled antibodies in radioimmunotherapy of ovarian cancer patients,8–10 we have experimentally studied the ECAT procedure after intraperitoneal administration of radiolabeled and biotinylated MAb.
The current study demonstrates a reduction in radioactivity in the blood of 75–86% and a reduction in WB radioactivity of 35–40% in non-tumor-bearing rats, when ECAT is started 12 hours after i.p. administration of the antibody. Thus, the ECAT procedure seems to be applicable after i.p. administration of radiolabeled MAb, provided that the optimal time for initiation of the adsorption has been predetermined from the pharmacokinetic profile of the radiolabeled and biotinylated MAb.
The biodistribution of MAb after i.p. injection is thought to be more complex than that after i.v. injection regarding the redistribution of activity between the abdominal cavity and other compartments (adjacent lymph and blood vessels, visceral organs, and other tissues). Peak radioactivity in the blood after i.p. injection of 111In -HMFG1, specifically, the resorption rate from the abdominal cavity in rats, was observed much earlier than that in patients (10–12 hours in rats vs. 50–54 hours in patients).10 ECAT starting 12 or 18 hours after i.p. administration of MAb manifestly reduced the retention of activity in the WB, blood, and radiosensitive organs. The reduction of activity in radiosensitive organs persisted at least 9 hours after completion of ECAT. The optimal starting time for ECAT, when applied only once, is at the time of peak activity, or just before, as the area under the time-activity curve (AUC) in blood is generally considered to be directly correlated to the myelotoxicity.10 Repeated ECAT may also be performed,5 but this has not yet been studied following i.p. injection.
As biotinylation of the antibody is required for the avidin-biotin concept, comparative biokinetic study of biotinylated versus nonbiotinylated 111In-HMFG1 preceded the adsorption studies. The study showed that HMFG-1 could be biotinylated with retained pharmacokinetics and high binding capacity to the avidin gel. Previous studies of biotinylation of MAb in vivo have demonstrated that biotinylation does not significantly change the biodistribution of the antibody.18, 19 Alternative pretargeting strategies for blood clearance, such as secondary antibodies or binding protein (avidin, streptavidin), to some extent redistribute the circulating blood activity to the excretory organs in the reticuloendothelial system, implying the risk of further accumulation of 111In/90Y-labeled conjugates in these organs.6 The ECAT approach may completely circumvent further “overloading” of the reticuloendothelial system.
Recently, Bosch et al.7 tested the biocompatibility of our avidin-agarose columns using ex vivo on-line perfusion of minicolumns with fresh blood from healthy donors in single-pass mode. The minicolumns showed good biocompatibility upon contact with human blood, without signs of hemolysis, thrombogenicity, or low activation of blood cells and the complement system. As these safety studies on human blood gave good results, preparations are underway for clinical Phase I–II studies of ECAT.
In conclusion, ECAT proved to be successful in reducing radioactivity in the WB, blood, and radiosensitive organs when employed after i.p. injection of the 111In-HMFG1-biotin MAb. Due to redistribution, which results in slowly increasing blood activity during the 9 hours after completion of ECAT, it may be advisable to extend the adsorption time or repeat ECAT. Biotinylation of the 111In-HMFG1 MAb did not significantly change the WB, blood, or organ biodistribution after intraperitoneal injection.