Optimized indirect 76br-bromination of antibodies using n-succinimidyl para-[76br]bromobenzoate for radioimmuno PET

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Abstract

Monoclonal antibody 38S1 was radiobrominated with the positron emitter 76Br (T1/2 = 16.2 h). Indirect labeling was performed using N-succinimidyl para-(tri-methylstannyl)benzoate (SPMB) as the precursor molecule. SPMB was labeled using Chloramine-T yielding N-succinimidyl para-[76Br]bromobenzoate, which was then conjugated to the antibody. Optimization of the labeling conditions and further conjugation gave a total yield ( mean±max error) of 49±2%. The immunoreactivity of the antibodies was retained after labeling. Thus, antibodies intended for positron emission tomography can be labeled with 76Br, which gives high yields and preserved immunoreactivity when using the SPMB technique described.

Introduction

Radionuclear imaging plays an important role in the detection and characterization of tumors and metastases as well as for determining the eligibility of cancer patients for therapy. The most advanced metabolic imaging technique, positron emission tomography (PET) using whole-body 18FDG, yields such information with high sensitivity and spatial resolution. However, diagnostic proceedings can also utilize labeled monoclonal antibodies (MAbs) or their fragments, which bind to tumor-specific antigens on the cell surface. So far this type of immunodetection has been performed mainly with nonoptimal measuring techniques such as gamma camera imaging of 131I-labeled and 99mTc-labeled compounds. A possible way to improve the sensitivity could be the use of positron-emitting labels for MAbs in combination with PET. For this reason, interest in MAbs labeled with positron emitters for PET has intensified during recent decades [e.g., see Goldenberg (4)].

A particular problem with the use of MAbs as tracers is their slow blood clearance rate, which requires labels with a half-life longer than that of conventional positron emitters (between 2 and 110 min). Long-lived positron emitters 66Ga ( T1/2=9.48 h), 64Cu ( T1/2=12.7 h), 55Co ( T1/2=17.5 h), 89Zr ( T1/2=3.27 d), and 124I ( T1/2=4. 2 d) have been suggested as labels for MAbs [e.g., see refs 3, 6, 16, 18, 20, respectively]. The radio-halogen 76Br ( T1/2=16.2 h, 54% β+-emission) is also a highly suitable candidate for labeling of MAbs, since its half-life is in the same order of magnitude as the blood clearance half-life of most MAbs (14). Moreover, 76Br can be produced with a low energy cyclotron by the nuclear reaction 76Se(p,n)76Br (24), thus making this radionuclide available at most PET centers.

Radiobromine labeling of proteins has been carried out in numerous ways using direct methods [for example, with Chloramine-T (CAT) 19, 21 or enzymes such as myelo- (15), chloro- (8), and bromoperoxidase (13)] and indirect methods [for example, with N-succinimidyl-3-(4-hydroxyphenyl) propionate (SHPP) (7) or N-succinimidyl-4-(4-tri-n-butylstannyl) benzoate (27)].

These various methods are associated with different problems. Direct bromination of MAbs using CAT has generally been associated with too harsh labeling conditions (i.e., low pH and the use of high concentrations of oxidants), which have damaged sensitive antibodies or resulted in low labeling yields. An exception, in this respect, has been a study where four different proteins were labeled with 76Br using CAT (22) and where an overall labeling yield of some 75% was achieved with preserved MAb immunoreactivity. With an enzymatic method using bromoperoxidase, high labeling yields up to 75% (13) were achieved. However, a drawback of enzymatic labeling techniques is the difficulty of ensuring a final product free of enzyme after purification (25).

During direct halogenation at neutral pH, the halogen attaches mostly to the tyrosine residues (9). Unfortunately, many antibodies have an elevated fraction of tyrosine residues in the complementarity determining regions (CDR) (17), and halogenation of these may thus reduce the immunoreactivity of the proteins. Also, a common problem with directly radiobrominated proteins is the unfavorable distribution of labeled catabolites. In a rat model, intravenous injection of directly radiobrominated MAbs was followed by whole-body extracellular accumulation of radiobromine (12), resulting in elevated background levels and thus deteriorated tumor to nontumor ratios.

A possible way to overcome these problems could be the use of an indirect labeling technique whereby a labeled linker molecule is attached to the more evenly distributed lysine residues of the protein. In this manner a better preserved immunoreactivity of the labeled antibodies would be achieved. Previous results of indirect labeling of MAbs, where N-succinimidyl 3-(tri-n-butylstannyl) benzoate (30) was used as precursor to produce an iodinated linker molecule, have been encouraging. In that study there was less radioiodine accumulation in thyroid and stomach and a faster whole-body clearance of labeled MAb catabolites than when MAbs were labeled directly using Iodogen. We surmised that these advantages regarding in vivo biodistribution and whole-body clearance of catabolites would also hold true for brominated MAbs labeled by the same indirect technique.

Furthermore, indirect labeling of a MAb with 77Br ( T1/2=57.1 h) has been reported (27). In that study, N-succinimidyl para-(tri-n-butylstannyl) benzoate (SPBB) was labeled, yielding N-succinimidyl para-[77Br]bromobenzoate ([77Br]SPBrB). The [77Br]SPBrB was further conjugated with an antimelanoma antibody, giving an overall yield of some 22%. In that experiment the labeling of the precursor molecule and the further conjugation with the MAb was not fully optimized, however. Moreover, preparation of N-succinimidyl para-[76Br]bromobenzoate ([76Br]SPBrB) with a 45–60% yield and its conjugation with human serum albumin, chromogranin, and deoxyoligonucleotides with 12-61% efficiency has been reported (28).

The aim of the present study was to optimize the conditions for 76Br labeling of SPMB, producing [76Br]SPBrB and the subsequent conjugation with the anticarcinoembryonic antigen (anti-CEA) antibody MAb 38S1 (5).

Section snippets

Chemicals

Dichlorobis(triphenylphosphine)palladium(II) [(P(Ph3))2Pd(II)Cl2], 4-iodobenzoic acid (IC6H4COOH), disuccinimidyl carbonate (DSC), and hexamethylditin [(CH3)6Sn2] were purchased from Aldrich (Stockholm, Sweden). Acetic acid, citric acid, methanol, hexane, and ethyl acetate were purchased from Merck (Darmstadt, Germany). CAT was from Sigma Chemical Company (St Louis, MO, USA). All chemicals were of analytical grade or better.

Acetonitrile, 1,4-dioxane, and pyridine were anhydrous (freshly

Labeling of SPMB with 76Br yielding [76br]spbrb

The amount of CAT was varied between 10 and 80 μg to optimize the labeling. The result is shown in Figure 1a. The influence on the labeling yield of the amount of SPMB varied between 0.2 and 10 μg and is shown in Figure 1b. The reaction time, lasting between 1 and 10 min, showed optimal labeling at 5 min, as shown in Figure 1c. This resulted in a labeling yield ( mean±max error) of 76±2% under optimal labeling conditions (i.e., 80 μg of CAT, 10 μg of SPMB, and 5 min reaction time). The HPLC

Discussion

A preparation of N-succinimidyl para-[77Br]bromobenzoate ([77Br]SPBrB) was made earlier (27), using N-succinimidyl para-(tri-n-butylstannyl) benzoate (SPBB) as precursor. Without further purification the [77Br]SPBrB was then conjugated to an antimelanoma antibody giving an overall yield of 22%. According to the authors this was a preliminary study based on a single batch of 77Br. Synthesis of N-succinimidyl para-[76Br]bromobenzoate giving a 45–60% yield has been described earlier (28). The

Acknowledgements

The authors would like to thank the staff at the Uppsala University PET Centre for their assistance with the radiobromine production. We also gratefully acknowledge The Svedberg Laboratory for allowing us to use their facilities and Pharmacia, Uppsala, Sweden, for supplying purified antibodies and antigens. This work was supported by the Swedish Cancer Foundation (grant no. 2971-B97-08XAB).

References (30)

  • D.S Wilbur et al.

    Radiolabeling of a monoclonal antibody with N-succinimidyl para-[77Br]bromobenzoate

    Int. J. Rad. Appl. Instrum. B

    (1991)
  • M.R Zalutsky et al.

    A method for the radiohalogenation of proteins resulting in decreased thyroid uptake of radioiodine

    Int. J. Rad. Appl. Instrum. [A]

    (1987)
  • Browne E. and Firestone R. (1986) In: Section III, Table of Radioactive Isotopes. (Edited by Shirley V. S.), pp....
  • P Goethals et al.

    Production of carrier-free 66Ga and labeling of antimyosin antibody for positron imaging of acute myocardial infarction

    Eur. J. Nucl. Med.

    (1990)
  • D.M Goldenberg

    Cancer imaging with CEA antibodiesHistorical and current perspectives

    Int. J. Biol. Markers

    (1992)
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