N-succinimidyl 3-[211At]astato-4-guanidinomethylbenzoate: an acylation agent for labeling internalizing antibodies with α-particle emitting 211At

doi:10.1016/S0969-8051(03)00005-2

Nuclear Medicine and Biology

Volume 30, Issue 4, May 2003, Pages 351-359

https://doi.org/10.1016/S0969-8051(03)00005-2 Get rights and content

Abstract

The objective of this study was to develop a method for labeling internalizing monoclonal antibodies (mAbs) such as those reactive to the anti-epidermal growth factor receptor variant III (EGFRvIII) with the α-particle emitting radionuclide ²¹¹At. Based on previous work utilizing the guanidine-containing acylation agent, N-succinimidyl 4-guanidinomethyl-3-[¹³¹I]iodobenzoate ([¹³¹I]SGMIB), we have now investigated the potential utility of its astato analogue for labeling the anti-EGFRvIII mAb L8A4. N-succinimidyl 3-[²¹¹At]astato-4-guanidinomethylbenzoate ([²¹¹At]SAGMB) in its Boc-protected form was prepared from a tin precursor in 61.7 ± 13.1% radiochemical yield, in situ deprotected to [²¹¹At]SAGMB, which was coupled to L8A4 in 36.1 ± 1.9% yield. Paired-label internalization assays demonstrated that tumor cell retention of radioactivity for L8A4 labeled using [²¹¹At]SAGMB was almost identical to L8A4 labeled using [¹³¹I]SGMIB, and 3-4-fold higher than for mAb radioiodinated using Iodogen. Paired-label biodistribution of L8A4 labeled using [²¹¹At]SAGMB and [¹³¹I]SGMIB in athymic mice hosting U87MGΔEGFR xenografts resulted in identical uptake of both ²¹¹At and ¹³¹I in tumor tissues over 24 h. Although higher levels of ²¹¹At compared with ¹³¹I were sometimes seen in tissues known to sequester free astatide, these ²¹¹At/¹³¹I uptake ratios were considerably lower than those seen with other labeling methods. These results suggest that [²¹¹At]SAGMB may be a useful acylation agent for labeling internalizing mAbs with ²¹¹At.

Introduction

Monoclonal antibodies (mAbs) and their fragments that react specifically with molecules present on malignantly transformed cells are attractive vehicles for targeting radionuclides to tumors. The clinical potential of this approach is exemplified by the emergence of radioimmunotherapy as a potentially valuable treatment for patients with lymphomas and leukemias [6], [8], [29]. Of particular interest for targeted radiotherapy are mAbs that undergo rapid internalization after their binding to cell surface antigens [4], [15]. One advantage of utilizing mAbs that undergo internalization is that this process can increase the radiation absorbed dose to cell nuclei, the cell locus generally considered to be the most radiosensitive. For example, dosimetry calculations have suggested that even with the multi-cellular range β-emitter ¹³¹I, shifting the site of decay from the cell membrane to cytoplasmic vesicles could increase the dose received by the cell nucleus by a factor of two [7].

Many of the antigens and receptors that have served as molecular targets for the generation of tumor-selective mAbs are molecules that are rapidly internalized. One of these, the wild type epidermal growth factor receptor (EGFR), is present in a variety of cancers including gliomas and squamous cell carcinomas [2], [5]; however, it is also present on many normal tissues causing problems for RIT. A mutant form of EGFR, EGFRvIII has been found on cancers such as gliomas, breast and non-small cell lung carcinomas [9], [12], [19], [30], [32]. Unlike the wild type receptor, EGFRvIII is not present in normal tissues [31], making it an attractive carrier for radioimmunotherapy.

Because internalization is a process which can subject the mAb to additional catabolic processes including lysosomal proteolysis, it is imperative that the labeled catabolites that are generated are retained intracellularly. Direct radioiodination techniques are not suitable for labeling internalizing proteins because of the rapid escape of iodotyrosine from tumor cells after proteolysis of the mAb [13]. To circumvent this problem, we and others have developed a number of radiolabeled prosthetic groups including those composed of d-amino acid peptides [10], [14] and positively charged organic molecules [20].

Recently we have evaluated a guanidine substituted acylation agent for the radioiodination of mAbs that undergo rapid internalization [26], [27]. Our hypothesis was that the high pKa of the guanidine would interfere with the transport of labeled catabolites out of the lysosome, trapping the radioiodine in the tumor cell. When the anti-EGFRvIII mAb L8A4 was labeled with N-succinimidyl 4-guanidinomethyl-3-[¹³¹I]iodobenzoate ([¹³¹I]SGMIB), intracellular retention of radioiodine was 3-4-fold higher than seen for the same mAb labeled via direct electrophilic iodination or that labeled with a less basic positively charged template, N-succinimidyl 3-[¹²⁵I]iodo-5-pyridinecarboxylate ([¹²⁵I]SIPC). Use of SGMIB for labeling the L8A4 mAb also resulted in considerably higher retention in EGFRvIII-expressing tumor xenografts.

The objective of this study was to determine whether it would be possible to adapt this labeling approach for use with the α-particle emitting radiohalogen ²¹¹At. Alpha-particles offer considerable radiobiological advantages for targeted radiotherapy, and might be ideal for treating micrometastatic disease, hematologic malignancies such as lymphoma, and compartmentally-spread cancers [34]. Astatine-211 has a half life of 7.2 h, can be produced on a medium energy cyclotron, and α-particle emission is associated with each of its decays. Herein we describe the preparation of N-succinimidyl 3-[²¹¹At]astato-4-guanidinomethylbenzoate ([²¹¹At]SAGMB; Fig. 1) and its use for radiolabeling anti-EGFRvIII mAb L8A4. In vitro internalization assays and biodistribution measurements were performed using U87MGΔEGFR cells as the EGFRvIII-expressing target to evaluate the potential of [²¹¹At]SAGMB as a reagent for labeling internalizing mAbs with ²¹¹At.

Section snippets

General

All reagents were obtained from Aldrich or Sigma unless otherwise indicated. Sodium [¹³¹I]iodide, with a specific activity of about 1200 Ci/mmol was obtained from Perkin Elmer Life Sciences. The tin precursor 1 (Fig. 1), ¹³¹I-labeled SGMIB, and 4-guanidinomethyl-3-[¹³¹I]iodobenzoic acid (GMIBA) were prepared as reported before [26].

The ²¹¹At activity was produced on the Duke University CS-30 cyclotron via the ²⁰⁹Bi(α, 2n)²¹¹At reaction by bombarding natural bismuth metal targets with 28 MeV

Acknowledgements

This work was supported by Grants CA42324, CA78417, CA91927, and CA11898 from the National Institutes of Health. The authors would like to thank Kevin Alston, Phil Welsh, and Holly LeGrand for their excellent technical assistance.

References (34)

S.A. Ali et al.
Synthesis and radioiodination of tyramine cellobiose for labeling monoclonal antibodies
Nucl. Med. Biol.
(1988)
J. Baselga et al.
Receptor blockade with monoclonal antibodies as anti-cancer therapy
Pharmacol. Ther.
(1994)
L.W. Brady et al.
Malignant astrocytomas treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor receptora phase II trial
Int. J. Radiat. Oncol. Biol. Phys.
(1992)
R.H. Larsen et al.
Evaluation of an internal cyclotron target for the production of astatine-211 via the ²⁰⁹Bi(α, 2n)²¹¹At reaction
Appl. Radiat. Isot.
(1996)
T. Lindmo et al.
Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess
J. Immunol. Methods
(1984)
K.M. Murud et al.
Synthesis, purification, and in vitro stability of ²¹¹At- and ¹²⁵I-labeled amidobisphosphonates
Nucl. Med. Biol.
(1999)
C.J. Reist et al.
In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibodycomparison with its murine parent
Nucl. Med. Biol.
(1997)
C.J. Reist et al.
Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody using N-succinimidyl 5-[²¹¹At]astato-3-pyridinecarboxylate
Nucl. Med. Biol.
(1999)
G. Vaidyanathan et al.
Improved xenograft targeting of tumor-specific anti-epidermal growth factor receptor variant III antibody labeled using N-succinimidyl 4-guanidinomethyl-3-iodobenzoate
Nucl. Med. Biol.
(2002)
G.W.M. Visser et al.
The preparation and stability of astatotyrosine and astato-iodotyrosine
Int. J. Appl. Radiat. Isot.
(1979)

M.R. Zalutsky et al.

Tissue distribution and radiation dosimetry of astatine-211-labeled chimeric 81C6, an α-particle-emitting immunoconjugate

Nucl. Med. Biol.

(1997)

K.-F. Becker et al.

Mutant cell surface receptors as targets for individualized cancer diagnosis and therapy

Curr. Cancer Drug Targets

(2002)

T.M. Behr et al.

Therapeutic advantages of Auger electron over beta-emitting radiometals or radioiodine when conjugated to internalizing antibodies

Eur. J. Nucl. Med.

(2000)

J.M. Burke et al.

Radioimmunotherapy for acute leukemia

Cancer Control

(2002)

F. Daghighian et al.

Enhancement of radiation dose to the nucleus by vesicular internalization of iodine-125-labeled A33 monoclonal antibody

J. Nucl. Med.

(1996)

G.L. DeNardo et al.

Radiolabeled anti-lymphoma antibodies

Cancer Chemother. Biol. Response Modifiers

(2001)

A.J. Ekstrand et al.

Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails

Proc. Natl. Acad. Sci. U.S.A.

(1992)

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Regular articleN-succinimidyl 3-[211At]astato-4-guanidinomethylbenzoate: an acylation agent for labeling internalizing antibodies with α-particle emitting 211At

Abstract

Introduction

Section snippets

General

Acknowledgements

Nucl. Med. Biol.

Pharmacol. Ther.

Int. J. Radiat. Oncol. Biol. Phys.

Appl. Radiat. Isot.

J. Immunol. Methods

Nucl. Med. Biol.

Nucl. Med. Biol.

Nucl. Med. Biol.

Nucl. Med. Biol.

Int. J. Appl. Radiat. Isot.

Nucl. Med. Biol.

Mutant cell surface receptors as targets for individualized cancer diagnosis and therapy

Curr. Cancer Drug Targets

Therapeutic advantages of Auger electron over beta-emitting radiometals or radioiodine when conjugated to internalizing antibodies

Eur. J. Nucl. Med.

Radioimmunotherapy for acute leukemia

Cancer Control

Enhancement of radiation dose to the nucleus by vesicular internalization of iodine-125-labeled A33 monoclonal antibody

J. Nucl. Med.

Radiolabeled anti-lymphoma antibodies

Cancer Chemother. Biol. Response Modifiers

Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails

Proc. Natl. Acad. Sci. U.S.A.

Regular article
N-succinimidyl 3-[²¹¹At]astato-4-guanidinomethylbenzoate: an acylation agent for labeling internalizing antibodies with α-particle emitting ²¹¹At