Bioconjugate Chem. 1999, 10, 447−453
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Chemical Modification to Reduce Renal Uptake of Disulfide-Bonded Variable Region Fragment of Anti-Tac Monoclonal Antibody Labeled with 99mTc In S. Kim,† Tae M. Yoo,† Hisataka Kobayashi,† Meyoung-kon Kim,† Nhat Le,† Q-c. Wang,‡ Ira Pastan,‡ Jorge A. Carrasquillo,† and Chang H. Paik*,† Nuclear Medicine Department, Warren G. Magnuson Clinical Center, Building 21, Room 136, and Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. Received October 26, 1998; Revised Manuscript Received February 4, 1999
The anti-Tac disulfide-bonded variable region fragment (dsFv) is a genetically engineered, 25 kDa, murine monoclonal antibody fragment that recognizes the R subunit of the interleukin-2 receptor (IL-2RR). The dsFv radiolabeled with the tetrafluorophenyl ester (TFP) of [99mTc]mercaptoacetyltriglycine ([99mTc]MAG3-TFP) showed rapid tumor uptake and fast blood clearance in mice, resulting in high tumor-to-nontumor background ratios. However, its high renal uptake was a problem. In this study, we tested the effect of lowering the isoelectric point (pI) of dsFv to 9.3, thus highly positively charged at physiological pH. The radiolabeled dsFv, due to its small size, is believed to be filtered readily through the glomerulus, but is absorbed by proximal tubular cells via an electrostatic attraction between the positive charge of dsFv and the negative charge of phospholipid bilayers of parenchymal cells of the tubules, resulting in the high renal concentration of radiolabeled dsFv. In this study, we lowered the pI of dsFv by chemical modifications to test whether this lowered pI decreased its renal uptake. The pI of dsFv can be lowered chemically either by the acylation of the amino group of lysine residues or by the addition of acidic amino acids to the carboxy terminus. We chose the acylation reaction because dsFv has 12 lysine residues whose amino group is readily acylated. The acylation of amino groups to the corresponding amides is a common method of neutralizing the positive charges on the proteins (26). We used TFP-glycolate as an acylating agent because it is a small molecule with a polar hydroxy group that is expected to cause only a small perturbation to the tertiary structure of dsFv. The conversion of an amino group to an amide by acylation with one glycolate molecule reduces one positive charge from the native dsFv, adds one hydroxyl group, and increases the molecular weight by 58. Thus, the maximum glycolation of all 12 lysine residues would increase the molecular weight of the 25 kDa dsFv by 696, only 2.8% increase in the molecular weight. This study demonstrates that the acylation of the amino groups of dsFv with glycolate molecules lowers its pI inversely proportional to the molar ratio of glycolate-TFP to dsFv (Table 1), and that this lowering of the pI decreases the renal uptake (Figure 3) and the whole-body retention (Figure 4). The effect was especially pronounced at 15 min. At this time, the renal uptake of the labeled, glycolated dsFv with a pI range below 7 was reduced to about 50% ID/g, or 25% of the renal uptake of nonglycolated [99mTc]MAG3-dsFv, and the whole-body retention of the glycolated dsFv was decreased to 50% ID. These results suggest that one-half of the injected dose was excreted via the kidneys within 15 min for the glycolated dsFv conjugate. These results are consistent with our observation that the [99mTc]99m activity excreted in urine was all bound to dsFv up to 15 min for the [99mTc]99m MAG3-dsFv-glycolate prepared at a molar ratio of 73 (data not shown). Since lowering the pI of dsFv reduced renal uptake without altering the blood activity, it is likely that the glomerular filtration was unaffected and that it is the tubular reabsorption that was blocked by the lowering of the pI. The effect of the lowered pI on the biodistribution of dsFv was somewhat different from that on the biodistribution of 111In-labeled Fab′ (50 kDa), especially in blood concentrations. Tarburton et al. (27) reported that the acetylation with acetic anhydride lowered the pI of 111Inlabeled Fab′ and that the acetylated [111In]Fab′ showed
Kim et al.
lower kidney and higher blood concentrations than the nonacetylated [111In]Fab′. A similar result was also reported by Fagnani et al. (28) for the [111In]Fab′ that had its pI reduced by conjugation with low-molecularmass dextran (6000 ( 500 Da). We recently demonstrated that 125I-labeled Fab of anti-Tac monoclonal antibody increased blood concentration, but reduced renal uptake when its pI was lowered by acylation with TFP-glycolate (29). The effect of pI on tumor uptake was investigated with [99mTc]MAG3-dsFv-glycolate prepared at a TFP-glycolateto-dsFv reaction molar ratio of 73 because this reaction ratio produced a glycolated dsFv with a pI range below 7 at the expense of only 20% deactivation of immunoreactivity. This glycolated [99mTc]MAG3-dsFv accumulated selectively into the receptor-positive SP2/Tac tumor with the tumor uptake similar to that of [125I]dsFv at 90 and 180 min. In contrast, the kidney accumulation of the glycolated conjugate was decreased to only 25% of the control [125I]dsFv at 15 min, due to its rapid excretion into urine (Figures 5 and 6), as reflected by a low wholebody retention, with 60% ID remaining at 15 min, compared to 97% ID remaining with [125I]dsFv. In conclusion, this study demonstrated that TFP-glycolate is an efficient acylating agent for lowering the pI of dsFv. This new approach holds promise for enhancing the renal clearance of radiolabeled variable region fragments without altering their tumor uptake. ACKNOWLEDGMENT
The authors thank Dr. Thomas A. Waldmann for providing SP2/Tac and SP/0 cell lines, David S. Paik for his computational and editorial assistance, and Sue Kendall for her editorial assistance. LITERATURE CITED (1) Wolf, R. E., and Brelsford, W. G. (1988) Soluble interleukin-2 receptors in systemic lupus erythematosus. Arthritis Rheum. 31, 729-735. (2) Nakanishi, K., Taniguchi, Y., and Ohta, Y. (1991) Increased soluble interleukin-2 receptor levels in autoimmune thyroid disease. Acta Endocrinol. 125, 253-258. (3) Anasetti, C., Hansen, J. A., Waldmann, T. A., Appelbaum, F. R., Davis, J., Deeg, H. J., Doney, K., Martin, P. J., Nash, R., Storb, R., Sullivan, K. M., Witherspoon, R. P., Binger, M.H., Chizzonite, R., Hakimi, J., Mould, D., Satoh, H., and Light, S. E. (1994) Treatment of acute graft-versus-host disease with humanized anti-Tac: an antibody that binds to the interleukin-2 receptor. Blood 84, 1320-1327. (4) Kay, N. E., Burton, J., Wagner, D., and Nelson, D. L. (1988) The malignant B cell from B-chronic lymphocytic leukemia patients release Tac-soluble interleukin-2 receptors. Blood 72, 447-450. (5) Richards, J., Mick, R., Latta, J. M., Daly, K., Ratain, M. J., Vardiman, J. W., and Colomb, H. M. (1990) Serum soluble interleukin-2 receptor is associated with clinical and pathologic disease status in hairy cell leukemia. Blood 76, 19411945. (6) Motoi, T., Uchiyama, T., Hori, T., Itoh, K., Uchino, H., and Ueda, R. (1989) Elevated serum-soluble interleukin-2 receptor (Tac antigen) levels in chronic myelogenous leukemia patients with blastic crisis. Blood 74, 1052-1057. (7) Ambrosetti, A., Semenzato, G., Prior, M., Chilosi, M., Vinante, F., Vincenzi, C., Zanotti, R., Trentin, L., Portuese, A., Menestrina, F., Perona, G., Agostini, C., and Todeschini, G. (1989) Serum levels of soluble interleukin-2 receptor in hairy cell leukaemia: a reliable marker of neoplastic bulk. Br. J. Haematol. 73, 181-186. (8) Webber, K. O., Reiter, Y., Brinkmann, U., Kreitman, R., and Pastan, I. (1995) Preparation and characterization of a
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