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Clinical Cancer Research Vol. 9, 3873S-3879S, September 1, 2003
© 2003 American Association for Cancer Research

Session II: ISOTOPIC CONJUGATES, PRECLINICAL AND PRETARGETING

211At-labeled and Biotinylated Effector Molecules for Pretargeted Radioimmunotherapy Using Poly-L- and Poly-D-Lysine as Multicarriers1

Sture Lindegren2, Börje Karlsson, Lars Jacobsson, Håkan Andersson, Ragnar Hultborn and Gunnar Skarnemark

Departments of Radiation Physics [S. L., B. K., L. J.] and Oncology [H. A., R. H.], Göteborg University, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden, and Department of Nuclear Chemistry, Chalmers University of Technology, SE-412 96 Göteborg Sweden [G. S.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Poly-L- and poly-D-lysine were evaluated as carriers of astatine and biotin for prospective use as effector molecules in pretargeted radioimmunotherapy of micrometastases. The precursor polylysine was derivatized in a three-step, single-pot procedure, including biotinylation with biotin amidocaproic N-hydroxysuccinimide, astatination via the intermediate reagent N-succinimidyl 3-(trimethylstannyl)benzoate, and, finally, charge modification using succinic anhydride. The chemistry was shown to be very facile, with a biotinylation efficiency of 75 ± 5%, and overall radiochemical yields in the range of 50–70%. After charge modification, no amines could be detected in the final product. The biotin function was unaffected by the chemistry and the radiation, as confirmed by almost complete binding of the effector molecule to avidin beads using a convenient filter tube assay. The effector molecules were evaluated in tumor-free female nude mice with regard to whole-body retention and tissue distribution after i.p. administration. The distribution of the L-isomer effector molecule showed rapid whole-body clearance with low uptake in all tissues, whereas the D-isoform showed whole-body clearance related to uptake in the kidneys. Both D-isomer and L-isomer showed faster blood clearance and generally lower tissue uptakes than labeled antibodies. The normal tissue distribution after the peritoneal administration implies that pretargeting using L-structure polylysine as the effector molecule may give a higher therapeutic index than that achieved in conventional radioimmunotherapy.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Endoradiotherapy of malignant diseases using radiolabeled tumor-specific monoclonal antibodies is a treatment modality that has been investigated for several decades. Despite some promising clinical reports (1, 2, 3) , the application of radiolabeled antibodies has thus far been limited in cancer therapy. The main problem in using antibodies for RIT3 applications is the nonideal pharmacokinetics, generally resulting in slow tumor uptake, where only a small proportion of injected antibodies bind to the tumor cells. In addition, the major nonbound fraction is slowly cleared from normal tissues, which results in low tumor:normal tissue ratios, especially if nuclides with short half-lives (e.g., 211At and 213Bi) are used.

To circumvent the problem of distribution of the radioactivity, PRIT approaches have been proposed by several investigators. The most common technique for pretargeting is based on various chemical constructs of antibody and radionuclide, using biotin and streptavidin as intermediates (4, 5, 6) . Protocols for PRIT using this system can be varied in a number of different ways, but the prevailing strategy for systemic treatment is pretargeting with streptavidin-conjugated antibodies followed by radiolabeled biotin [effector molecule] (7, 8, 9) . An intermediate step using a clearing agent, before administration of the effector molecule, is introduced to facilitate clearance of the nonbound streptavidin-conjugated antibody from the circulatory system. In this way, tumor uptake and clearance of nonlocalized radioactivity can be improved. Indeed, using this pretargeting protocol, it has been shown that significant improvement in the pharmacokinetics can be achieved both preclinically (10 , 11) and clinically (12 , 13) .

Pretargeting can also be applied for intracavitary treatment, such as ovarian carcinoma, using i.p. administration. Due to late symptoms of patients with ovarian cancer, the disease is often at a late stage at the time of diagnosis (FIGO stage IIB–IV), with occult metastases accompanying the macroscopic tumor. Despite standard treatment of patients with malignant ovarian disease using surgery and chemotherapy, the 5-year survival is no higher than 30%. The dissemination is, as a rule, restricted to the peritoneal surface and is primarily in the form of micrometastasis. As the tumor disseminates in this way, a high-LET, short-range {alpha}-emitting nuclide such as 211At may be a suitable choice of nuclide for the adjuvant treatment. An appropriate pretargeting system for ovarian carcinoma may be a two-step protocol with avidin-conjugated antibody followed by the effector molecule. A clearing agent is not necessary because the avidin-antibody conjugate will eventually enter the circulation and be cleared rapidly via the liver.

To further improve the pharmacokinetics of the effector molecule for i.p. treatment, we have previously proposed poly-L-lysine as a multicarrier for biotin and radionuclide (14) . Poly-L-lysine can be obtained in different molecular weights and may therefore allow improved control of the distribution within the peritoneal compartment compared with the monoderivative of biotin and radionuclide. Furthermore, this chemistry allows multi-N-acylation modifications, thus enabling increased avidity for avidin and increased specific radioactivity.

In this study, effector molecules based on biotinylated, astatinated, and charge-modified poly-L-lysine and poly-D-lysine for prospective use in pretargeted i.p. microtumor therapy are investigated. The effector molecules were evaluated with regard to efficacy in chemical modification of the precursor polylysine, avidin binding capacity, and whole-body retention and biodistribution in tumor-free nude mice


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Poly-L-lysine and poly-D-lysine (molecular weights, 7,000 and 10,000), biotinamidocaproate N-hydroxysuccinimide ester, N-bromsuccinimide, and N-iodosuccinimide were obtained from Sigma Chemical Co. Avidin beads (ImmunoPure Immobilized Avidin Gel), TNBSA, and HABA were purchased from Pierce Chemical Co. All additional nonradioactive commercial chemicals and solvents used were of analytical grade or better.

211At was produced by the 209Bi({alpha},2n)211At reaction using internal target irradiation on the Scanditronic MC32-NI cyclotron at the Cyclotron and PET Unit, Rigshospitalet [Copenhagen, Denmark (15) ]. Sodium radioiodine, Na125I and Na131I, was obtained from NEN Life Science Products Inc.

General
After irradiation, the target was immediately shipped to the Department of Radiation Physics (Göteborg University, Göteborg, Sweden), and the astatine was isolated from the target by dry distillation, according to the method described previously (15) . All high-activity targets and samples (>100 kBq) were measured in an ionization chamber (Capintech CRC-15). Low-activity samples (<100 kBq) were measured using a NaI(Tl) gamma-well counter (Wizard Wallac). The two devices were cross-calibrated for the 70–90 keV 211Po X-rays after the decay of 211At. Whole-body radioactivity of the astatinated polymers in athymic mice was determined using a NaI(Tl) scintillation detector (Harshaw Chemie B.V.).

High performance liquid chromatography was conducted on a Waters 600/486 system with Waters Baseline 820 analysis software. Reversed-phase chromatography was performed using a Kromasil C-18 column (4.6 x 250 mm; EKA-Nobel AB), and size-exclusion analysis was carried out on a Superdex-200 FPLC column (Amersham Pharmacia Biotech). Preparative gel permeability chromatography was conducted on disposable Sephadex G-25 PD-10 columns (Amersham Pharmacia Biotech). Mass spectral data were obtained using inductively coupled plasma ion source mass spectroscopy.

Labeling of m-MeATE
Astatination of the polylysine was achieved via the labeling of m-MeATE, according to the previously reported procedure (16) . Briefly, an aliquot (20–30 MBq) from the distilled 211At activity in chloroform was transferred to a microvial, and the solvent was evaporated. To the dry residue were added N-iodosuccinimide (0.5 nmol) and m-MeATE reagent (1.0 nmole in 1% acetic acid/methanol). The 211At was allowed to react with the m-MeATE reagent for 15 min during gentle agitation at room temperature. Finally, the reaction was stopped by the addition of 50 nmol of sodium metabisulfite. The labeling efficiency was determined by running an aliquot of the reaction mixture on a reversed-phase C-18 column, isocratically eluted with acetonitrile/2 mM phosphoric acid (60:40) as the mobile phase.

Iodine labeling with 125I or 131I of the m-MeATE precursor was performed according to the same method as the astatination, except that N-bromosuccinimide was used as the oxidizing agent. The product, N-succinimidyl-3-[211At]astatobenzoate, N-succinimidyl-3-[125I]iodobenzoate, or N-succinimidyl-3-[131I]iodobenzoate, was used immediately, unpurified, for conjugation to the biotinylated polymer.

Modification of Polylysine
Biotinylation.
Before conjugate labeling, the polylysine was biotinylated using the biotinamidocaproate N-hydroxysuccinimide ester. The biotin reagent was dissolved in dimethylformamide to a concentration of 5–10 mg/ml. To poly-L-lysine or poly-D-lysine in 0.2 M carbonate buffer (pH 8.5), a 10-fold molar excess of the biotinylation reagent was then added during vigorous agitation, and the reaction was allowed to proceed for 1 h with gentle agitation at room temperature. Further chemistry was conducted on the crude biotinylated product.

Conjugate Labeling.
To the crude labeling mixture, 100 µg of the biotinylated poly-L-lysine were added to give a final volume of 50 µl. The reaction was allowed to proceed for 10 min with gentle agitation at room temperature.

Succinylation.
The biotinylated and astatinated polymer was finally subjected to charge modification using succinic anhydride as reagent. The anhydride was added in large excess over available amines to convert the remaining unsubstituted amino groups to carboxylic residues. Solid succinic anhydride, in the form of flakes, was added to the reaction mixture. The pH was adjusted with 1 M carbonate buffer (pH 8.5) during the reaction to maintain the amino residues unprotonated. After 10 min of reaction time, the polymer fraction was isolated by passage over a G-25 PD-10 column (Pharmacia).

The final product was analyzed with regard to unbound radioactivity gel filtration chromatography. The efficiency of the biotinylation was calculated using the HABA dye method (17) , and the degree of substitution of the amines was determined using the TNBSA spectrophotometric method (18) . The average molecular weights of the modified polylysines were estimated to be 18,000.

In Vitro Binding of Modified Polylysine
The avidin binding capacity of the effector molecules was determined using a convenient fast filter tube assay. To Costar spin-x, 45-µm filter tubes (Corning Inc.), 50 µl of the avidin bead slurry were added. Effector molecule (25 ng) was added, and the volume was adjusted to 150 µl with PBS. The binding reaction was allowed to proceed for 30 min at room temperature with agitation. The beads were then washed three times with PBS, and bound radioactivity was measured in a gamma-well counter.

Nonspecific binding to the filter tubes was determined by incubation with the modified polymer in tubes not containing avidin beads, and nonspecific binding to avidin was determined by incubating nonbiotinylated, labeled polylysine with the beads.

Animal Experiments
Whole-body retention and tissue distribution of the poly-D- and poly-L-effector molecules were evaluated in tumor-free, female athymic mice (Balb/C nu/nu), 6–8 weeks of age. The effector molecules were administered i.p., corresponding to activities in the range of 550–800 kBq/animal for the astatinated products and 150 kBq/animal of iodinated products. The mice were sacrificed by cervical dislocation at 1, 6, and 24 h postinjection, in groups of 4 animals/time point. Whole blood was collected by cardiac puncture immediately after the animals had been killed, and tissues including neck, lungs, stomach, liver, small intestine, large intestine, kidneys, spleen, i.p. fat, and muscle were excised and washed in saline. The tissues were blotted dry and weighed, and the radioactivity measured in a gamma-well counter. After measurement, all tissue data were corrected for radioactive decay and expressed as the percentage of injected radioactivity per gram of organ, except for the neck, in which it was expressed as the percentage of injected activity. The clearance rate of the modified polymers was determined by following whole-body radioactive retention in groups of four mice for 24 h, measured with a sodium iodide detector.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Modification of Polylysine.
The modification of poly-L- and poly-D-lysine, via N-acylation of the {epsilon}-amino groups, by biotinylation; labeling with 211At-, 125I-, or 131I-reagent; and charge modification with succinic anhydride all showed high efficiency.

Biotinylation was performed before conjugate labeling and succinylation, and the efficiency was in the range of 70–80% as determined by the HABA dye method. After the succinylation step, which is very important for minimizing nonspecific bindings in vitro and in vivo, no amines were detected using a TNBSA assay. The substitution of the amines of the precursor polylysine was therefore considered to be complete. Because the succinic acid residues are in large excess, the average increase in molecular weight was approximated from complete succinylation, i.e., an increase of approximately 80% of the final product.

Radioconjugation.
All three steps were performed in a single-pot procedure to make optimum use of input radioactivity, therefore resulting in high conjugation efficiencies. Overall radiochemical yields were in the order 125I > 131I > 211At, corresponding to 85%, 70%, and 60% efficiency, respectively. The differences in the conjugation efficiencies are probably related to radiation quality after the different decays.

Because the reactions were performed without purification of the labeling mixture, conjugation of the m-MeATE precursor may occur. The tin residue of the precursor may be toxic if released in vivo. However, <0.0004% of input m-MeATE was detected in the final products, as determined by inductively coupled plasma ion source mass spectroscopy analysis.

In Vitro Binding of Modified Polylysine.
The only biologically active part of the effector molecule is the biotin moiety, which was proven to be unaffected by the chemistry and the radiation, as determined by reaction with avidin beads, using a rapid filter tube assay. To examine nonspecific binding in this system, two additional control assays were run simultaneously. These were (a) nonbiotinylated labeled and succinylated polylysine together with the avidin beads to determine nonspecific binding to the avidin beads and (b) measurement of nonspecific filter binding in tubes not containing avidin beads. Typical results from the biotin assay, showing the binding properties of the astatinated effector molecule, are shown in Fig. 1Citation . As can be seen, almost complete specific binding of the effector molecule was achieved.



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  		Fig. 1. Avidin binding capacity of biotinylated, astatinated, and charge-modified polylysine (211At-PLsuc-B) using a filter tube assay. Results are expressed as the percentage of applied activity ± SD for four experiments. At-211-PLsuc-B refers to the D- or L-product of modified polylysine.

 
Animal Experiments.
Three separate experiments were conducted to determine clearance rates and biodistribution of the effector molecule in tumor-free nude mice, after i.p. administration. In the first study, the astatinated L-isomer effector molecule was examined, followed by a study on the corresponding astatinated D-isomer. To compare and confirm the results from the astatinated effector molecules, a paired labeled experiment was performed using 125I-labeled D-isomer and 131I-labeled L-isomer effector molecules. Results from determinations of the clearance are shown in Fig. 2Citation . As can be seen, there is an obvious difference in whole-body retention between the different isoforms, with longer retention for the D-structure. The L-structure shows very rapid whole-body clearance rates with a biological half-life of approximately 2 h, whereas the D-structure shows biological half-lives of about 14 and 24 h for the iodinated and astatinated molecules, respectively. The clearance of an 125I-labeled IgG antibody (16) is also shown in Fig. 2Citation , which illustrates the considerable difference in retention, especially compared with the L-structure effector molecule. The discrepancy in retention between the two isomers can be explained by the biodistribution data, which are summarized in Tables 1Citation and 2Citation . High uptake and retention of the D-isomer in the kidneys is clearly shown at all time points, which is almost completely related to the observed prolonged whole-body retention.



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  		Fig. 2. The whole-body retention of astatine and iodine effector molecules, based on poly-D- and poly-L-lysine compared with whole-body retention of an IgG antibody, in tumor-free nude mice after i.p. administration. Results are given as mean ± SE for four animals per time point. I-125-MAb refers to iodinated antibody. I-125-PLsuc-B, I-131-PLsuc-B, and At-211-PLsuc-B refer to radiohalogenated (125I, 131I, or 211At), biotinylated, and charge-modified polylysine.

 

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  		Table 1 Distribution of biotinylated, 211At-labeled, and charge-modified poly-L- and poly-D-lysine in nude mice after i.p. administration

Results are presented as mean ± SD of the percentage of injected dose per gram of tissue; n = 4 mice/time point.

 

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  		Table 2 Distribution of biotinylated, radioiodinated, and charge-modified poly-L- and poly-D-lysine in nude mice after i.p. administration

Results are presented as mean ± SD of the percentage of injected dose per gram tissue; n = 4 mice/time point.

 
Comparison of the different labels, iodine and astatine, shows somewhat higher uptakes in most tissues for the astatinated molecules. It is interesting to note that the astatinated D-isomer shows less uptake in tissues known to accumulate astatine (i.e., the thyroid, lung, spleen, and stomach) as compared with the L-isomer. This indicates that structure (e.g., nonmetabolizing configurations such as in D-peptides) is of importance for the susceptibility to deastatination in vivo. This effect was not seen with iodinated effector molecules, where the L-structure shows less uptake in all tissue at all time points as compared with the D-structure.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Despite recent advances in the use of radiolabeled monoclonal antibodies for adjuvant treatment of certain forms of disseminated cancer, general applications are hampered by nonideal pharmacokinetics. In other words, IgG-sized antibodies suffer from low targeting efficiency in vivo and low whole-body clearance rates, which lead to high radiation burdens on normal tissues from nonlocalized radioactivity. In particular, the long antibody half-life in blood results in a high absorbed dose to bone marrow, limiting the activity that can be administered in RIT.

To overcome this inherent problem in the distribution of radiolabeled antibodies, here we present a strategy to improve the pharmacokinetics, using biotinylated, astatinated, and charge-modified polylysine for prospective use as effector molecule in pretargeted microtumor RIT. The system is based on the high-affinity interaction between avidin/streptavidin and biotin, which can be used as intermediates in the construction of radionuclide and antibody conjugates for a multiple administration protocol (4 , 5) . In such a system, the targeting agent can be either a biotin- or avidin/streptavidin-conjugated antibody. Likewise, the corresponding effector molecule is based on either radiolabeled biotin derivatives or labeled avidin/streptavidin. Most pretargeting systems use radiolabeled biotin, which, due to its small size and low molecular weight, exhibits rapid clearance via the kidneys (19, 20, 21, 22) . A possible protocol for general systemic treatment of micrometastasis, using the effector molecule presented in this work, may be a three-step procedure including (a) pretargeting with streptavidin-conjugated antibody for binding to the tumor antigens, (b) rapid clearance of nonlocalized conjugate using a chase molecule, and (c) tumor cell killing by irradiation after the binding of the effector molecule. To achieve rapid diffusion into tumor tissues and fast clearance via the kidneys, a low molecular weight polymer precursor (i.e., molecular weight < 10,000) would probably be the most appropriate choice for derivatization. Note that the chemical modification of the polylysine, using the conditions described, results in an increase in molecular weight of the final product of approximately 80%. For i.p. treatment of ovarian cancer, a two-step protocol would be sufficient, including avidin-conjugated antibody for pretargeting followed by the effector molecule. The reason for using avidin instead of streptavidin is that the avidin-conjugated antibody, when entering the circulation from the peritoneal cavity, will be rapidly cleared by the liver. This means that no clearing agent is required. Furthermore, in the case of i.p. treatment, a high molecular weight effector molecule (i.e., molecular weight > 60,000) may increase retention within the peritoneal compartment, thus affecting the tumor uptake and toxicity in a positive way.

Because the tumor dissemination of ovarian cancer is generally restricted to the peritoneal surface, primarily in the form of micrometastasis, the choice of nuclide is crucial for effective treatment. For microtumor therapy, increasing interest has been focused on {alpha}-emitting radionuclides, due to their short range and high LET. Among the {alpha}-particle-emitting nuclides, only a few fulfill most of the criteria for endoradiotherapeutic applications including 211At, 212Bi, 213Bi, and 225Ac (23, 24, 25) , with the most studied being 211At and 213Bi (11 , 26, 27, 28) . Comparison of these two nuclides suggests that 211At is perhaps the more versatile, due to its longer half-life, which permits time for chemical synthesis and time in vivo to be distributed to the tumor tissue (28) . Indeed, our research group has previously demonstrated the great potential of 211At in the successful treatment of ovarian tumors in nude mice using the 211At-labeled monoclonal antibody MOv18 (29) . On the basis of these encouraging results, a second-generation form of treatment was suggested using a pretargeting strategy to minimize radiation to normal tissues, especially the bone marrow.

The reason for using polylysine as an effector carrier was to further improve the pharmacokinetics, especially with regard to i.p. treatment, compared with that of labeled biotin. Polylysine is essentially a number of repeating units of the amino acid lysine, determined by its molecular weight. Each subunit contains one {epsilon}-amino group, which is the functional group on which N-acylation reactions can be performed. Because polylysine is a multi-amino compound, multi-N-acylation modifications are feasible, meaning that a number of different molecules can be attached to it. For example, multi-biotin and multi-radionuclide substitutions are possible, which implies higher avidity for avidin and increased specific radioactivity of the final product.

The concept of using polylysine as a carrier molecule has been examined previously in a few investigations. del Rosario and Wahl described a method for the synthesis and distribution of biotinylated and iodinated polylysine for prospective use in pretargeted radioimmunotargeting (30) . To increase the specific radioactivity of radiometal-labeled effector molecules, Torchilin studied the conjugation of biotin and chelating agents of polymers (31) .

The polylysine precursor is positively charged and is a highly basic molecule that, when unmodified, exhibits a high degree of nonspecific binding in vitro and in vivo. In the present study, only a few of the total amount of the amino groups are actually substituted with biotin and astatine at the reaction conditions used, and this biotinylated and astatinated molecule cannot be used without further modification. Therefore, a final step, using succinic anhydride, was introduced to react with and convert the remaining amino groups to carboxylic residues (i.e., succinic acid residues). In this way, nonspecific binding can be minimized.

The chemical modification of polylysine has been shown to be extremely facile, with high yields in all three chemical steps. Besides the facile chemistry, predetermination of the distribution is possible, as shown previously by our research group (10) . This work is an extended evaluation based on that study, with the focus on poly-L-lysine and poly-D-lysine. The precursor polymers were of low molecular weight (10,000), resulting in a final weight of the product of approximately Mr 13,000–18,000, and were expected to be rapidly cleared via the kidneys. Surprisingly, the effector molecule based on the D-isomer showed a much longer whole-body retention time than the L-isomer. The difference was almost completely due to uptake in the kidneys. Some kidney uptake of the L-effector molecule is also seen early on, but this declines rapidly with time. This may indicate metabolic decomposition in the kidney. The fact that the D-isomer is retained despite its high negative charge seems to exclude renal filtration of the intact L-isomer. The mechanism for the renal clearance of these molecules has not yet been completely elucidated. However, in renal clearance of radiometal-labeled antibody fragments, it has previously been shown that the main metabolite is a lysine-{epsilon}-amino-chelate-metal product (32) . A similar kind of mechanism of decomposition is also likely in the clearance process of the L-isomer effector molecule with a lysine-{epsilon}-amino-astatine derivative as the major metabolite.

A paired labeled study was also conducted using 125I-labeled poly-D-lysine and 131I-labeled poly-L-lysine of the same molecular weight as the astatinated products. The results of the astatine and the iodine studies are well correlated. Somewhat increased tissue uptake was seen with the astatinated polylysine, which may be related to the lower strength of the astatine-carbon bond than the iodine-carbon bond (33) . However, the organ uptake for astatinated L-effector molecules was generally lower than that observed for antibodies. A major difference between antibodies and the effector molecules is the blood content, affecting bone marrow dose. Compared with antibodies, the blood retention is much lower, and the content in blood is at least a factor of 30 lower already at 6 h postadministration for the polymers. This implies that a higher therapeutic index may be achieved using a pretargeting strategy with an effector molecule based on these effector molecule constructs, because higher radioactivity levels can be administered before severe bone marrow toxicity is reached.

In summary, the synthesis and biodistribution of biotinylated, astatinated, and charge-modified poly-L- and poly-D-lysine for prospective use in PRIT are described. After i.p. administration, a marked difference in whole-body retention was seen between the two isoforms with a cumulative uptake in the kidneys for the D-effector molecule. This indicates a clearance route determined by renal decomposition of the L-isomer.

Tissue uptake was generally lower than that for antibodies, particularly with regard to the blood content. This is important for a prospective endoradiotherapeutic treatment using the {alpha}-emitter 211At. Because nonlocalized radioactivity (i.e., L-structure effector molecule) will be rapidly excreted, and blood clearance rates are fast, increased radioactivity can be administered in a complete pretargeting protocol for i.p. therapies. This will most likely result in higher tumor doses compared with conventional RIT using radiolabeled antibodies.


   ACKNOWLEDGMENTS
 
We thank Elizabeth Warnhammar (Department Oncology, Göteborg University, Göteborg, Sweden) and Tom Bäck (Department of Radiation Physics, Göteborg University) for excellent technical assistance with the animal study.


   FOOTNOTES
 
1 Presented at the "Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates," October 24–26, 2002, Princeton, NJ. This work was supported by grants from the Swedish Cancer Foundation (Grant 3548) and the King Gustaf V Jubilee Clinic Cancer Research Foundation in Göteborg, Sweden. Back

2 To whom requests for reprints should be addressed. Fax: 46-31-822-493; E-mail: sture.lindegren{at}radfys.gu.se Back

3 The abbreviations used are: RIT, radioimmunotherapy; FIGO, International Federation of Gynecology and Obstetrics; LET, linear energy transfer; PRIT, pretargeted radioimmunotherapy; m-MeATE, N-succinimidyl-3-(trimethylstannyl)benzoate; TNBSA, 2,4,6-trinitrobenzene sulfonic acid; HABA, 2-(-4-hydroxyazobenzene) benzoic acid. Back


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Crippa F., Bolis G., Seregni E., Gavoni N., Scarfone G., Ferraris C., Buraggi G. L., Bombardieri E. Single-dose intraperitoneal radioimmunotherapy with the murine monoclonal antibody I-131 MOv18: clinical results in patients with minimal residual disease of ovarian cancer. Eur. J. Cancer, 31A: 686-690, 1995.[CrossRef]
  2. Goldenberg D. M. The role of radiolabelled antibodies in the treatment of non-Hodgkin's lymphoma: the coming of age of radioimmunotherapy. Crit. Rev. Oncol. Hematol., 39: 191-201, 2001.
  3. White C. A., Weaver R. L., Grillo-López A. J. Antibody-targeted immunotherapy for treatment of malignancy. Annu. Rev. Med., 52: 125-145, 2001.[CrossRef][Medline]
  4. Stoldt H. S., Aftab F., Chinol M., Paganelli G., Luca F., Testori A., Geragherty J. G. Pretargeting strategies for radio-immunoguided tumour localisation and therapy. Eur. J. Cancer, 33: 186-192, 1997.
  5. Sakahara H., Saga T. Avidin-biotin for delivery of diagnostic agents. Adv. Drug Deliv. Rev., 37: 89-101, 1999.[CrossRef][Medline]
  6. Wilbur D. S., Pathare P. M., Hamlin D. K., Stayton P. S., To R., Klumb L. A., Buhler K. R., Vesella R. L. Development of new biotin/streptavidin reagents for pretargeting. Biomol. Eng., 16: 113-118, 1999.[CrossRef][Medline]
  7. Breitz H. B., Weiden P. L., Beaumier P. L., Axworthy D. B., Seiler S., Su F-M., Graves S., Bryan K., Reno J. M. Clinical optimization of pretargeted radioimmunotherapy with antibody-streptavidin conjugated and 90Y-DOTA-biotin. J. Nucl. Med., 41: 131-140, 1999.
  8. Weiden P. L., Breitz H. Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin‘s lymphoma (NHL). Crit. Rev. Oncol. Hematol., 40: 37-51, 2001.[Medline]
  9. Grana C., Chinol M., Robertson C., Mazzetta C., Bartolomei M., De Cicco C., Fiorenza M., Gatti M., Calicceti P., Paganelli G. Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: a pilot study. Br. J. Cancer, 86: 207-212, 2002.[CrossRef][Medline]
  10. Sharkey R. M., Karacay H., Griffiths G. L., Behr T. M., Blumenthal R. D., Mattes M. J., Hansen H. J., Goldenberg D. M. Development of streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Studies in a human colon cancer xenograft model. Bioconjugate Chem., 8: 595-604, 1997.[CrossRef][Medline]
  11. Zhang M., Yao Z., Garmestani K., Axworthy D. B., Zhang Z., Mallett R. W., Theodore L. J., Goldman C. K., Brechbiel M. W., Carrasquillo J. A., Waldman T. A. Pretargeting radioimmunotherapy of a murine model of adult T-cell leukaemia with the {alpha}-emitting radionuclide, bismuth 213. Blood, 100: 693-713, 2002.
  12. Press O. W., Corcoran M., Subbiah K., Hamlin D. K., Wilbur D. S., Johnson T., Theodore L., Yau E., Mallett R., Meyer D. L., Axworthy D. A comparative evaluation of conventional and pretargeted radioimmunotherapy of CD-20 expressing lymphoma xenografts. Blood, 98: 2535-2543, 2001.[Abstract/Free Full Text]
  13. Cremoneze M., Ferrari M., Chinol M., Stabin M. G., Grana C., Pisco G., Robertson C., Tosi G., Paganelli G. Three-step radioimmunotherapy with yttrium-90 biotin:dosimetry and pharmacokinetics in cancer patients. Eur. J. Nucl. Med., 26: 110-120, 1999.[CrossRef][Medline]
  14. Lindergen S., Anderson H., Jacobsson L., Bäck T., Skarnemark G., Karlsson B. Synthesis and biodistribution of 211At-labeled, biotinylated and charge modified poly-L-lysine: evaluation for use as an effector molecule in pretargeted intraperitoneal tumor therapy. Bioconjugate Chem., 13: 502-509, 2002.[CrossRef][Medline]
  15. Lindegren S., Bäck T., Jensen H. Dry-distillation from irradiated bismuth targets: a time-saving procedure with high recovery yields. Appl. Radiat. Isot., 55: 157-160, 2001.[CrossRef][Medline]
  16. Lindegren S., Andersson H., Bäck T., Jacobsson L., Karlsson B., Skarnemark G. High-efficiency astatination of antibodies using N-iodosuccinimide as the oxidising agent in labelling of N-succinimidyl 3-(trimethylstannyl)benzoate. Nucl. Med. Biol., 28: 33-39, 2001.[CrossRef][Medline]
  17. Cayot P., Tainturier G. The quantification of protein amino groups by the trinitrobenzensulfonic acid method: a reexamination. Anal. Biochem., 249: 184-200, 1996.
  18. Green N. M. A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J., 94: 23c-24c, 1965.
  19. Wilbur D. S., Hamlin D. K., Pathare P. M., Weerawarna S. A. Biotin reagents for antibody pretargeting. Synthesis radioiodination, and in vitro evaluation of biotinidase resistant biotin derivatives. Bioconjugate Chem., 8: 572-584, 1997.[CrossRef][Medline]
  20. Foulon C. F., Alston K. L., Zalutsky M. R. Astatine-211-labeled biotin conjugates resistant to biotinidase for use in pretargeted radioimmunotherapy. Nucl. Med. Biol., 25: 81-88, 1998.[Medline]
  21. Wilbur D. S., Chyan M-K., Pathatre P. M., Hamlin D. K., Frownfelter M. B., Kegley B. B. Biotin reagents for antibody pretargeting. 4. Selection of biotin conjugates for in vivo application based on their dissociation from avidin and streptavidin. Bioconjugate Chem., 12: 616-623, 2000.
  22. Wilbur D. S., Hamlin D. K., Kegley B. B., Pathatre P. M. Biotin reagents for antibody pretargeting. 5. Additional studies of biotin conjugate design to provide biotinidase stability. Bioconjugate Chem., 11: 569-583, 2001.
  23. Wilbur D. S. Potential use of {alpha} emmitting radionuclides in the treatment of cancer. Antibody Immunoconj. Radiopharm., 4: 85-97, 1991.
  24. McDevitt M. R., Sgouros G., Finn R. D., Humm J. L., Juricic J. G., Larson S. M., Scheinberg D. A. Radioimmunotherapy with {alpha}-emitting nuclides. Eur. J. Nucl. Med., 25: 1341-1351, 1998.[CrossRef][Medline]
  25. Imam S. K. Advancements in cancer therapy with {alpha}-emitters: A review. Int. J. Radiat. Oncol. Biol. Phys., 51: 271-278, 2001.[Medline]
  26. Zalutsky M. R., Bigner D. D. Radioimmunotherapy with {alpha}-particle-immunoconjugates. Acta. Oncol., 35: 373-379, 1996.[Medline]
  27. Kennel S. J., Mirzadeh S., Eckelman W. C., Waldman T. A., Garmestani K., Yordanova A. T., Stabin M. G., Brechbiel M. W. Vascular-targeted radioimmunotherapy with the {alpha}-particle emitter 211At. Radiat. Res., 157: 633-641, 2002.[CrossRef][Medline]
  28. Zalutsky M. R., Vaidayanathan G. Astatine-211-labelled radiotherapeutics: an emerging approach to targeted {alpha} particle radiotherapy. Curr. Pharm. Design, 6: 1433-1455, 2000.[CrossRef][Medline]
  29. Andersson H., Lindegren S., Bäck T., Jacobsson L., Leser G., Horvath G. Radioimmunotherapy of nude mice with intraperitoneally growing ovarian cancer xenograft utilizing 211At-labelled monoclonal antibody MOv18. Anticancer Res., 20: 459-462, 2000.[Medline]
  30. del Rosario R. B., Wahl R. L. Biotinylated iodo-polylysine for pretargeted radiation delivery. J. Nucl. Med., 34: 1147-1151, 1992.
  31. Torchilin V. P. Biotin-conjugated polychelating agent. Bioconjugate Chem., 10: 146-149, 1999.[Medline]
  32. Li L., Olafsen T., Anderson A-L., Wu A., Raubitschek A. A., Shively J. E. Reduction of kidney uptake in radiometal labeled peptide linkers conjugated to recombinant antibody fragments. Site-specific conjugation of DOTA-peptides to a cys-diabody. Bioconjugate Chem., 13: 985-995, 2002.[CrossRef][Medline]
  33. Larsen R. H., Slade S. M., Zalutsky M. R. Blocking [211At]-astatide in normal tissues: preliminary evaluation of seven potential compounds. Nucl. Med. Biol., 25: 351-357, 1998.[Medline]




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