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Clinical Cancer Research 13, 5572s-5576s, September 15, 2007. doi: 10.1158/1078-0432.CCR-07-0891
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Cancer Therapy with Antibodies and Immunoconjugates

Improved Tumor Targeting and Decreased Normal Tissue Accumulation through Extracorporeal Affinity Adsorption in a Two-Step Pretargeting Strategy

Linda Mårtensson1, Rune Nilsson4, Tomas Ohlsson3, Hans-Olov Sjögren2, Sven-Erik Strand3 and Jan Tennvall1

Authors' Affiliations: Departments of 1 Oncology, 2 Immunology, and 3 Medical Radiation Physics, University of Lund; 4 Mitra Medical AB, Lund, Sweden

Requests for reprints: Linda Mårtensson, Department of Oncology, Lund University Hospital, SE-221 85 Lund, Sweden. Phone: 46-46-709-54-2554; Fax: 46-46-286-2461; E-mail: linda.martensson{at}med.lu.se.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Evaluation of the possibilities of reducing the accumulation of radiolabeled streptavidin in radiosensitive organs by extracorporeal affinity adsorption (ECAT).

Experimental Design: Rats were injected with biotinylated antibody and subjected to removal of the antibodies from the circulation by ECAT 24 h after injection (avidin column). Animals were then injected with 111In-1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic acid (DOTA)-streptavidin. In a third step, animals were subjected to a second ECAT 8 h after injection to remove the DOTA-streptavidin from the circulation (biotin column). Biodistribution and tumor targeting of DOTA-streptavidin 24 h after injection was determined.

Results: Elimination of biotinylated antibody by ECAT before injection of DOTA-streptavidin increased the tumor targeting by 50%. In addition, the levels of DOTA-streptavidin in liver and lymph nodes were reduced by 60%, which implied a 4.3- and 3.8-fold increase of tumor-to-liver and tumor-to-lymph node ratios, respectively. By doing a second ECAT to remove DOTA-streptavidin from the circulation, accumulation in normal tissues was reduced. However, this latter ECAT also reduced tumor accumulation by 25% (mostly corresponding to radioactivity in the circulation).

Conclusions: ECAT was efficient as a means of removing biotinylated antibodies and would probably also be efficient for the clearance of streptavidin-conjugated antibodies. Conversely, the use of ECAT for removal of radiolabeled streptavidin seems not to offer any advantage.

The objective of radioimmunotherapy is to obtain an increased concentration of radioactivity in tumor tissue while simultaneously reducing concentrations in healthy tissues to minimize the toxic side effects in normal tissue. Some radiolabeled monoclonal antibodies (mAb) have excellent targeting properties partly due to their persistence in the circulation, and radioimmunotherapy has shown promising results in the treatment of leukemia, lymphoma, and some micrometastatic diseases (1). However, in the treatment of solid tumors, which are less radiosensitive and where tumor cells are less accessible, radioimmunotherapy has met with less success (2). Only a small fraction of the radioactivity, 0.001% to 0.1% injected dose per gram tissue, reaches solid tumors (3). This low accumulation of radioactivity results in a low tumor-to-blood activity ratio and high absorbed doses to radiosensitive organs (e.g., bone marrow, liver, kidneys, and lungs) when attempting to achieve high tumor absorbed doses. To overcome this limitation, the tumor-to-blood activity ratio needs to be increased, which has led to the search for alternative strategies. One such strategy, known as pretargeting, has been shown in several preclinical and clinical studies to improve the tumor-to-blood ratio (4–6). The concept of pretargeting involves the administration of a modified mAb, which, in addition to recognition of and binding to the target antigen, allows a second component to bind to it with high specificity. Uncoupling of the radionuclide from the targeting antibody allows the slow process of antibody localization and clearance to occur before the radioactivity, coupled to a smaller molecule, is administered, allowing faster delivery. Several different pretargeting systems coupled to radionuclides have been reported in the literature: bispecific mAbs containing one antigen-binding site and one hapten-binding site (4), biotinylated mAbs allowing binding to avidin or streptavidin (5), and streptavidin- or avidin-conjugated mAbs enabling binding to biotin (6).

In addition to pretargeting, other methods of improving the tumor-to-blood ratio have been reported. Extracorporeal removal of radiolabeled antibodies in blood offers means of overcoming the problem of low tumor-to-blood ratios and reducing the toxicity in normal organs. The extracorporeal affinity adsorption (ECAT) procedure has been shown to selectively and quickly eliminate radiolabeled antibodies from the blood (7). The selectivity of ECAT 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 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic acid (DOTA) and biotin (8). By passing whole blood extracorporeally through a column coated with avidin, the radioimmunoconjugates are adsorbed in the column and removed from the circulation. Our group has previously shown that ECAT can eliminate 90% to 95% of radioimmunoconjugates from the circulation of rats and can significantly increase the tumor-to-normal tissue ratios of radioactivity in a syngeneic rat tumor model (9). We have also obtained similar results in patients (10).

The aim of this study was to evaluate if ECAT could improve the targeting and tumor-to-blood and tumor-to-normal tissue ratios of radioactivity in a pretargeting system. A two-step targeting system using a biotinylated antibody followed by radiolabeled streptavidin was combined with an ECAT step (avidin column) to eliminate circulating biotinylated antibody and a second ECAT step (biotin column) to eliminate radiolabeled streptavidin. Although the properties of streptavidin, such as its relatively large size (60 kDa), high kidney accumulation, and immunogenicity, are not considered optimal as the labeled molecule in pretargeting, we have chosen this quite simple system for the first evaluation of the effect of ECAT as a clearing step in combination with pretargeting.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
The mAb BR96. BR96 (Seattle Genetics, Inc.) is a chimeric (mouse/human) monoclonal IgG1 antibody binding the tumor-associated glycoprotein Lewis Y (LeY). LeY is expressed on the majority of human epithelial tumors, including cancers of the breast, gastrointestinal tract, lung, cervix, ovaries, and some melanomas (11). As with the majority of tumor-associated mAbs, BR96 also reacts with some normal tissue, primarily human epithelial cells of the gastrointestinal tract (11).

Conjugation of the BR96 mAb with biotin. BR96 was transferred by dialysis into a 0.1 mol/L NaHCO3 buffer at pH 8.4, conjugated with sulfo-NHS-biotin (Calbiochem) by the addition of 100 µg of sulfo-NHS-biotin per mg BR96, and incubated for 2 h at room temperature and then overnight at 4°C. After conjugation, the conjugate was transferred by dialysis into PBS buffer. The number of biotin molecules per BR96 molecule was determined by the 4'-hydroxyazobenzene-2-benzoic acid photometric method (12). This assay is based on the binding of the dye 4'-hydroxyazobenzene-2-benzoic acid to avidin and the ability of biotin to displace the dye in stoichiometric proportions.

Conjugation of streptavidin with DOTA. Before conjugation, streptavidin was transferred by dialysis into a 50 mmol/L HEPES and 1 mmol/L diethylenetriaminepentaacetic acid buffer (pH 8.5). Conjugation was done by adding a molar excess (50:1) of p-SCN-Bz-DOTA (600 µg p-SCN-Bz-DOTA per mg BR96; Macrocyclics) and incubating for 2 h at room temperature and overnight at 4°C. After conjugation, the conjugate was transferred to 0.25 mol/L ammonium acetate storage buffer (pH 5.3).

Radiolabeling and quality control of the streptavidin-DOTA conjugate. Both the streptavidin-DOTA in 0.25 mol/L ammonium acetate buffer and the radionuclide 111InCl3 (MDS Nordion) solutions were preheated at 45°C for 10 min. The DOTA-streptavidin solution was then added to the radionuclide-containing vials and incubated at 45°C for 15 min. The reaction was quenched with an excess of diethylenetriaminepentaacetic acid for 5 min. The radiolabeled conjugate was diluted in 1% human serum albumin. The radiochemical purity of the labeled conjugate was determined by a biotin-binding fraction test. An adsorption column packed with ~0.3 mL avidin-agarose (Mitra Medical AB) was coated with biotin-trimer (Mitra Medical AB) to test the biotin-binding potential of DOTA-streptavidin. A 50 µL sample of radiolabeled DOTA-streptavidin was added to the column and incubated for 10 min at room temperature. The column was washed eight times with 0.5 mL PBST (PBS containing 0.05% Tween 20), and the liquid from each washing was collected separately in tubes. The activity in the column and in each tube was measured in a NaI(TI) scintillator. The biotin-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 Norwegian strain (Harlan) were used. As shown by immunohistochemistry, Brown Norwegian rats express the BR96 epitope in some normal tissues, such as the gastrointestinal epithelium, pancreas, and ventricle, hence mimicking the human situation.5 BN7005 is a single cell clone of a rat colon carcinoma originally induced by 1,2-dimethyl-hydrazine in a Brown Norwegian rat. The BN7005 cells were cultured in RPMI 1640 (Euroclone) supplemented with 10% FCS, 1% sodium pyruvate, 1% 1 mol/L HEPES buffer solution, and 14 mg/L gentamicin at 37°C in a humidified atmosphere containing 5% CO2. Cells were washed in saline, trypsinized, and washed in RPMI 1640 + 10% FCS. Animals were inoculated subperitoneally with 3 x 105 cells (in 50 µL of medium). Experiments to investigate the tumor accretion of mAbs were initiated 12 to 14 days after inoculation (tumor size, 10 x 10 mm). The animals were kept under standard conditions and fed biotin-free food pellets (for 2 weeks before the start of study; Harlan Teklad) and fresh water ad libitum. Studies were conducted in compliance with Swedish legislation on animal rights and protection and approved by the Ethics Committee.

Extracorporeal affinity adsorption. The extracorporeal system (shown in Fig. 1 ) consisted of a 403U/C12 pump (W-Marlow Alitea AB) with 15-cm silicone tubing (1.6 x 6.35 mm inner/outer diameter). The column housing consisted of a modified 2 mL polypropylene syringe (9 x 30 mm) with a 72-µm net at the bottom. The avidin columns for the first ECAT step were packed with 1.5 mL avidin-agarose with ~0.5 mL NaCl above as an extra air trap. For the second ECAT step, the columns were coated with biotin-trimer and are referred to as biotin columns. 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 ~3.5 mL. Before ECAT, the system was flushed with heparin solution (20 IU/mL heparin in 9 mg/mL NaCl) as an anticoagulant.


Figure 1
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  		Figure 1. Experimental setup ECAT. A cannula is inserted into one of the lateral tail veins (1) for the return of blood and is connected to the extracorporeal system [regulated by the pump (2)]. For blood access, another cannula is inserted into the ventral tail artery (3). When the cannulas are inserted, the extracorporeal circulation is started in bypass mode (4) without the column connected. Once the circuit is filled with blood and any air bubbles in the circuit have been collected in the air trap (5), the column (6) is connected to the circuit and the affinity adsorption started.

 
Thirty minutes before anesthesia, a 2% glyceryl nitrate salve (The National Cooperation of Swedish Pharmacies) was applied to the entire tail of each rat to dilate the blood vessels. The animals were anesthetized with isofluran using a U-400 anesthesia unit (AgnTho's). The rats were first anesthetized in a 1.4 L induction chamber (3.3% isofluran, 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 (Neoflon 0.7 x 19 mm; Becton Dickinson) was carefully inserted into one of the lateral tail veins (1-2 cm from the tip of the tail) for the return of blood (Fig. 1). The cannula was secured to the tail with adhesive tape and connected to the extracorporeal system by tubing. 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). Another cannula was inserted into the ventral tail artery ~5 cm from the tip of the tail for blood access. This cannula is correctly inserted when there is a continuous blood flow 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. When the circuit was filled with blood and any air bubbles in the circuit had been collected in the air trap, the affinity column was connected to the circuit and affinity adsorption started. Blood was pumped through the column at a rate of 0.4 mL/min. ECAT was done on six rats in parallel. During ECAT, the rats were anesthetized with a lower level of anesthesia (2.0% isofluran, 575 mL/min air flow) and kept on electrical heating pads (30°C) to keep them warm. After ~2 h (~3 blood volumes; blood volume estimated to be 65 mL/kg body weight) of affinity adsorption, 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 removal of the cannulas. The tail was compressed to stop bleeding.

Study design. Eighteen animals were included in the study and divided into three groups of six (Fig. 2 ). All 18 animals received an i.v. injection of 100 µg biotinylated BR96. Twenty-four hours after injection of biotinylated BR96, the animals in two of the three groups were subjected to extracorporeal adsorption (using the avidin column). Our previous studies have shown that the maximal uptake of BR96 in tumor is attained at this time. Immediately after the adsorption procedure, all animals, including the group not subjected to ECAT, were injected with 100 µg DOTA-streptavidin labeled with 111In. Eight hours after injection of DOTA-streptavidin, one of the two groups previously subjected to ECAT was subjected to additional ECAT (biotin column) to remove DOTA-streptavidin. One of the three groups was thus not subjected to any ECAT. Twenty-four hours after injection of DOTA-streptavidin, the animals were sacrificed and dissected and the following tissues were removed for activity content measurements: tumor, pectoral muscle, kidney, liver, spleen, bowel, mesenteric lymph nodes, lungs, and blood.


Figure 2
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  		Figure 2. Study design. Rats were divided into three groups (n = 6). All animals received an injection of biotinylated BR96. Twenty-four hours after injection of biotinylated BR96, the rats in groups 2 and 3 were subjected to extracorporeal adsorption (avidin column) of the biotinylated BR96 remaining in the circulation. Immediately after the ECAT procedure, all animals, including the group not subjected to adsorption of biotinylated BR96 (group 1), were injected with 111In-DOTA-streptavidin. Eight hours after injection of DOTA-streptavidin, group 3 was subjected to an additional ECAT step (biotin column) for the removal of 111In-DOTA-streptavidin. Group 1 was thus not subjected to any ECAT. Twenty-four hours after injection of 111In-DOTA-streptavidin, the animals were sacrificed and dissected and tissue samples were collected.

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Preparation of biotinylated BR96 and radiolabeling of DOTA-streptavidin. After conjugation, the number of biotin molecules conjugated per BR96 molecule was determined to be, on average, 4.5. After radiolabeling with 111In, the DOTA-streptavidin was purified using size exclusion chromatography (PD10 column; Amersham Pharmacia Biotech) and the specific activity was 77.3 MBq/mg. The biotin-binding fraction of 111In-DOTA-streptavidin exceeded 95% at the time of injection.

Extracorporeal affinity adsorption. Blood samples were collected from the 12 animals subjected to the first ECAT procedure before and after avidin-ECAT to remove biotinylated BR96 (24 h after injection). ELISA results showed that a mean of 90% (range, 89-93%) of the biotinylated BR96 had been eliminated from the circulation. The radioactivity in blood samples taken from the six animals also subjected to the second ECAT step (biotin-ECAT) before and after ECAT for removal 111In-DOTA-streptavidin was measured and the efficacy of removal was found to be 65% (mean value range, 63-68%). This low adsorption can be explained by the fact that 2 weeks on a biotin-free diet is not enough to deplete the endogenous biotin in rats, and some of the 111In-DOTA-streptavidin is blocked and unable to bind to the biotin column. Later studies have shown that, after 3 weeks on a biotin-free diet, 85% of the 111In-DOTA-streptavidin can be eliminated with ECAT 8 h after injection. A biotin-deficient diet is not necessary in patients due to low biotin concentration in human blood compared with rodents (13).

Biodistribution. The results of the biodistribution of 111In-DOTA-streptavidin are shown in Fig. 3 . When 111In-DOTA-streptavidin was injected into animals subjected to ECAT for the removal of biotinylated BR96, the liver and lymph node distribution of 111In-DOTA-streptavidin was reduced by 60% compared with animals not subjected to ECAT (Fig. 3), indicating complex formation of circulating biotinylated BR96 with 111In-DOTA-streptavidin in animals not subjected to ECAT. In addition to a reduction of activity accumulation in these tissues, the tumor accumulation was increased by 52% in animals subjected to ECAT before the injection of 111In-DOTA-streptavidin (Fig. 3). The use of ECAT to remove biotinylated antibodies increased the tumor-to-liver ratio from 0.3 to 1.3 (a 4.3-fold increase) and the tumor-to-lymph node ratio from 0.5 to 1.9 (a 3.8-fold increase). The amount of 111In-DOTA-streptavidin in blood was increased in animals subjected to ECAT to remove biotinylated BR96. This is explained by the fact that, in animals not subjected to ECAT for the removal of biotinylated BR96, complexes are formed between the biotinylated BR96 in the circulation and the injected 111In-DOTA-streptavidin, and these complexes are rapidly deposited in the liver and cleared from the blood. When the second ECAT step to eliminate the 111In-DOTA-streptavidin was used, the activity content in most of the organs was further reduced. However, the activity in tumors was also significantly reduced (by 25%; Fig. 3).


Figure 3
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  		Figure 3. Biodistribution of 111In-DOTA-streptavidin 24 h after injection. The activity contents in tissue samples from the three groups were measured and compared [% injected dose per gram tissue (%ID/g)]. Filled columns, group 1: animals injected with biotinylated mAbs followed by 111In-DOTA-streptavidin (not subjected to ECAT). Striped columns, group 2: animals injected with biotinylated mAbs subjected to ECAT 24 h after injection followed by injection of 111In-DOTA-streptavidin. Empty columns, group 3: animals injected with biotinylated mAbs subjected to ECAT 24 h after injection followed by injection of 111In-DOTA-streptavidin and subjected to additional ECAT 8 h after injection. The effects in the liver, mesenteric lymph nodes, and tumor tissues in animals subjected to avidin-ECAT to remove biotinylated BR96 before the injection of 111In-DOTA-streptavidin are especially noticeable.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study shows that ECAT can be efficiently used as a clearing step in a two-step pretargeting system consisting of biotinylated tumor-targeting mAbs followed by radiolabeled DOTA-streptavidin. By doing ECAT, the liver and mesenteric lymph node accumulation of 111In-DOTA-streptavidin was decreased by 60% and the tumor accumulation increased by 52%. We hypothesized that, because streptavidin clears relatively slowly (compared with biotin) from the circulation, the use of a second ECAT step for the elimination of 111In-DOTA-streptavidin would further reduce the normal tissue activity (Fig. 3). Two variables were considered when choosing the time for doing this second ECAT step. First, sufficient amounts of 111In-DOTA-streptavidin should be allowed to accumulate in the tumor tissue before it is eliminated from the circulation. Second, if the ECAT procedure is to have toxicity-reducing potential, the pharmacokinetics of 111In-DOTA-streptavidin has to be considered and the procedure introduced at a time when 111In-DOTA-streptavidin remains in the circulation. Our results show that the second ECAT step reduced the tumor accumulation of 111In-DOTA-streptavidin to the same extent as in normal tissues (Fig. 3). This was probably due to the fact that the procedure was introduced too early before a maximal tumor penetration and binding of 111In-DOTA-streptavidin had been achieved. A further delay of the ECAT procedure would probably have resulted in higher tumor retention, but reduced depletion, as the blood levels would have been lower. Because only 25% of the 111In-DOTA-streptavidin is left in the circulation 8 h after injection (data not shown), a further delay would minimize the activity reducing potential of ECAT in blood and normal tissues. These results indicate that there is no advantage in eliminating 111In-DOTA-streptavidin using a second ECAT step. Timing, scheduling, and dosing are important factors in pretargeting strategies (14). The ECAT system is probably less dependent on dosing and scheduling of the pretargeting immunoconjugate and radioactivity vector because it is possible to eliminate the excess of immunoconjugate before injecting the radioactivity vector. The fraction of activity targeted to the tumor could probably be increased if the system was optimized.

Most streptavidin-biotin pretargeting methods currently used consist either of a two-step pretargeting method using a mAb-streptavidin conjugate followed by radiolabeled biotin (15) or a three-step pretargeting method consisting of biotinylated mAbs followed by unlabeled streptavidin and finally radiolabeled biotin (5). There are some disadvantages associated with these strategies, such as clinical complexity about optimization of each of the injections, degradation of mAbs due to the long interval between the injection of mAbs and subsequent targeting of molecules, the immunogenicity of streptavidin, and the high absorbed dose in the reticuloendothelial system (14). By using ECAT to remove biotinylated antibodies followed by the injection of radiolabeled DOTA-streptavidin, some of these problems could be circumvented or reduced. The method reduces the interval between the injection of mAbs and subsequent targeting molecules, which reduces the degradation of the mAbs. In addition, as the method reduces exogenous biotinylated mAbs and the endogenous biotin, tumor targeting is increased and there is simultaneously no increase in the absorbed dose in liver or spleen. Reduced uptake of radiolabeled streptavidin in the liver also means less radioactive catabolites excreted in the gastrointestinal tract and less radiation exposure of the intestines. ECAT can probably also be efficiently used as a clearing procedure for pretargeted streptavidin-conjugated antibodies. However, it is uncertain whether the strong immunogenicity of streptavidin (13, 16) in such a pretargeting regimen could be circumvented. Studies have shown that streptavidin may be replaced by avidin conjugated to high-molecular-weight polyethylene glycol polymer chains, which generated bioconjugates with optimal residence in the circulation and reduced immunogenicity (17).

Our two-step pretargeting method, consisting of injecting biotinylated mAbs followed by radiolabeled streptavidin, has been evaluated previously (18–21) but was later abandoned due to the unfavorable properties of streptavidin as a carrier of radionuclides (22). The size of streptavidin (60 kDa) results in relatively slow blood clearance, which makes smaller molecules, such as biotin, more suitable candidates. However, the main disadvantage of this system is the high kidney accumulation of streptavidin. Some studies show kidney accumulation as high as 85% to 100% injected dose per gram (23, 24). The localization of streptavidin in the kidneys has been investigated in detail by Wilbur et al. (25–27), who showed that chemical modification of the streptavidin molecule through succinylation of lysine amines reduced the kidney accumulation of streptavidin. Conjugation of DOTA to lysine amines on streptavidin has the same effect as succinylation [i.e., lower isoelectric point (26)], which explains the low kidney accumulation of radiolabeled DOTA-streptavidin seen in our studies (Fig. 3). The fast clearance of a small molecule, such as biotin, carrying radionuclides is considered largely favorable, but it results in a low percentage of injected activity being delivered to the tumor and high elimination through the kidneys. In the case of very expensive radionuclides, such as some of the {alpha}-emitters, a carrier such as streptavidin would result in a higher fraction of administered radioactivity being delivered to the tumor. In addition, the toxicity of {alpha}-emitters and short-path-length ß-emitters may be reduced if decay occurs in the blood rather than in high concentrations in the kidneys (26). When 90Y-biotin was used in two clinical studies, late renal toxicity appeared (28, 29), illustrating the problem of using a small radiolabeled carrier in the final targeting step.

We conclude that ECAT was efficient as a means of removing biotinylated antibodies and would probably also be efficient for the clearance of streptavidin-conjugated antibodies. It could therefore be used as an alternative to the presently used clearing agents (30) in other pretargeting strategies. Conversely, the use of ECAT as a means of removing radiolabeled streptavidin as the final step of pretargeting procedure seems not to offer any advantage.


   Footnotes
 
Grant support: Swedish Cancer Society, Swedish Medical Society, Mrs. Berta Kamprad's Foundation, Gunnar Nilsson's Foundation, Lund University Medical Faculty Foundation, and Lund University Hospital Fund.

Presented at the Eleventh Conference on Cancer Therapy with Antibodies and Immunoconjugates, Parsippany, New Jersey, USA, October 12-14, 2006.

5 H.O. Sjögren and I. Hellström, personal communication. Back

Received 4/17/07; accepted 5/15/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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