JULY/AUGUST 1999 Volume 10, Number 4 © Copyright 1999 by the American Chemical Society
COMMUNICATIONS Enzymatic Cleavage of Peptide-Linked Radiolabels from Immunoconjugates James J. Peterson and Claude F. Meares* Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616-5295. Received January 25, 1999
We have incorporated peptides selected by combinatorial library [Peterson, J. J., and Meares, C. F. (1998) Bioconjugate Chem. 9, 618-626) into peptide-linked radiolabeled immunoconjugates of the form DOTA-peptide-antibody. Decapeptide linkers -GFQGVQFAGF- and -GFGSVQFAGF-, selected for cleavage by human liver cathepsin B, were rapidly digested in vitro when compared to the simple model tetrapeptide motif of the prototype -GGGF- [Li, M., and Meares, C. F. (1993) Bioconjugate Chem. 4, 275-283]. Cleavage properties of these library-selected substrates for cathepsin B compared favorably with decapeptide linkers -GLVGGAGAGF- and -GGFLGLGAGF-, which incorporate two of the most labile extended cathepsin B substrates from the literature. The decapeptide linker -GFGSTFFAGF-, selected from the library for cleavage by human liver cathepsin D, was rapidly digested by cathepsin D while the others were not.
INTRODUCTION
Radioimmunotherapy and imaging use cancer-selective radiolabeled monoclonal antibodies (mAbs) to deliver therapeutic or imaging doses of radiation to the site of disease (1-3). Undesired accumulation of radioactivity in healthy tissues continues to be an impediment to this approach (4-8). One possible source of radiation in nontarget organs could be radiometal that has transchelated to proteins such as transferrin (9) or albumin (10), but this problem has been practically overcome through the use of reagents with high metal-chelate stability (1114). Elimination of radioactivity from the liver, for example, is a complex process involving both enzymatic liberation * To whom correspondence should be addressed. Phone: 530752-0936. Fax: 530-752-8938. E-mail:
[email protected].
of low molecular weight radioactive fragments from the targeting molecule (usually an antibody or antibody fragment) and intracellular processing of these fragments (15-18). One strategy for improved clearance is to insert a metabolizable linker between the tumor-targeting moiety and the radiolabel. Linkers may be designed to mimic natural substrates for enzymes endogenous to the cells of interest, and may include features designed to take advantage of appropriate intracellular pathways of elimination of the resulting low molecular weight fragments (19-21). The exact conditions in vivo where either cleavage or subsequent metabolite processing will become rate limiting are still unclear, partly because potential linkers are subject to multiple lysosomal enzymes (2225) and metabolic pathways (26, 27)sall of which are sensitive to variations in linker structure (8, 16, 17). Exploration of multiple structures with oligomeric linkers
10.1021/bc990010t CCC: $18.00 © 1999 American Chemical Society Published on Web 05/20/1999
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Peterson and Meares
Table 1. DOTA-Peptide Isothiocyanatesa
a
F*, p-isothiocyanatophenylalanine.
of readily modified sequence offers a natural approach to further understanding the problem. Metabolizable linkers explored for reducing radiation in healthy organs during radioimmunotherapy include esters, thioethers, disulfides, amides, and even hydrocarbon chains (16, 17, 20, 21, 28). Although ester linkers exhibit rapid cleavage in the liver, peptide linkers are preferred for their relative stability in circulation (20, 21). It has been demonstrated in vitro that simple tetrapeptides can serve as cleavable linkers for radioimmunotherapy, where cleavage by endoproteases can occur over a period of hours at levels of enzyme expected to be present in vivo (29, 30). The great differences in susceptibility of different peptides to enzymatic cleavage, and the range of possible metabolites resulting from cleavage of polypeptides by different enzymes, afford an opportunity to improve the rate at which radioactive metabolites are eliminated. Previously, we selected peptide linkers from a combinatorial library using cleavage by specific lysosomal enzymes implicated in hepatic protein turnover: human liver cathepsins B and D (31). We have chosen for further
study two substrates for cathepsin B and one for cathepsin D, which matched the consensus sequences deduced in that work. Using a convenient solid-phase procedure for the N-terminal functionalization of peptides with the DOTA1 macrocycle (32), we prepared and conjugated peptide-linked chelators to Lym-1, a murine IgG2a mAb used in cancer therapy (6). These conjugates were radiolabeled and treated with endoproteases to explore their cleavage properties in vitro compared to the simple prototype DOTA-tetrapeptide-Lym-1 (30). In addition, we inserted labile peptide substrates from literature sources (33, 34) into the DOTA-peptide-Lym-1 format for comparison with conjugates derived from our combinatorial selection. RESULTS AND DISCUSSION
Table 1 gives the formula for each substrate studied. Methods of preparation and comjugation are described in detail in Supporting Information. Thin layer chro1 Abbreviations: DOTA, 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid.
Communications
Bioconjugate Chem., Vol. 10, No. 4, 1999 555
Figure 1. Digestions of 90Y-DOTA-peptide-Lym-1 conjugates with human liver cathepsin B at 37 °C, 0.11 units/mL, 37 min. Experiments were performed in triplicate; error bars represent 1 standard deviation.
Figure 2. Digestions of 90Y-DOTA-peptide-Lym-1 conjugates with human liver cathepsin D at 37 °C, 0.22 units/mL, 65 min. Experiments were performed in triplicate; error bars represent 1 standard deviation.
matograms showed that the stability of the unconjugated nitro form of each DOTA-peptide-90Y complex was not adversely affected by the enzymatic cleavage buffer. All peptide linkages were stable in cleavage buffer lacking enzyme. Under these conditions [ca. 10-fold lower in enzyme concentration than those previously used (30)], no significant cleavage of the prototype DOTA-peptide#0Lym-1 occurred, while substantial cleavage of all literature and library-based decapeptide conjugates was observed for cathepsin B (Figure 1). The conjugate most labile to cathepsin B (no. 5) was derived from our combinatorial library (31). The lability of conjugate 1, also derived from the library for cleavage by cathepsin B, compared favorably with literature-based conjugate 15 (34). All decapeptide conjugates were cleaved to a greater extent than conjugate no. 18. Surprisingly, even conjugate 13, selected from the combinatorial library for cleavage by cathepsin D, was substantially cleaved during treatment with cathepsin B. Cathepsin D was able to significantly cleave conjugate 13, while none of the other conjugates were significantly cleaved by this enzyme under these conditions (Figure 2). In our attempts to prepare a literature-based substrate for cathepsin D (35), the product was not sufficiently soluble at the DOTA-peptide stage to be readily isolated. Linkers for radioimmunotherapy that consist of unnatural amide bonds have been referred to as “peptidyl” in the sense that the backbone of the folded antibody flanking each modified lysine may serve as a set of arbitrary peptidyl enzyme substrates (36, 37). It has been found that the potential cleavage sites on Lym-1 provide relatively poor substrates for lysosomal endoproteases when compared to shortsor even arbitraryspeptide linkers attached to -amines on Lym-1 (29, 30). Until very recently (21), ester-based linkers (despite their relative
lack of stability in plasma) have continued to find favor when compared to simple mono- or diamide linkages, due to their superior elimination properties in healthy tissues (19, 20, 36-39). However, it is unlikely that lysosomal endoproteases are able to participate in catabolic processes involving unnatural amide-based linkers. Cathepsin D, for example, requires an extended substrate (>5 amino acid residues) for cleavage (40, 41). The cathepsin D results in Figure 2 emphasize the importance of fine structural detail. We and others have found cathepsin D to be a more selective enzyme than cathepsin B (31, 42-44). It is clear from the results in Figure 1 that all conjugates derived from specific substrates for cathepsin B were efficiently cleaved by this enzyme in their bioconjugate form. The effect of linker length on cathepsin B cleavage is still unclear, although the activity of this enzyme may be expected to be hindered by the proximity of the intended substrate to the targeting macromolecule (31). Conjugates derived from our combinatorial library were cleaved more quickly than extended cathepsin B substrates from literature that might be considered appropriate candidates for cleavable linkers. The sensitivity of conjugate 13 (a library substrate selected for cleavage by cathepsin D) to cathepsin B is notable. Our stock of cathepsin B was assayed by the supplier to be over 92% pure by SDS-PAGE, and the method used for its purification was designed to remove cathepsin D (35). However, we did not include any specific inhibitors of cathepsin D during cathepsin B cleavage experiments; thus, we cannot rule out the possibility that our stock of cathepsin B was contaminated with traces of cathepsin D. In conclusion, highly labile cathepsin substrates were derived from a combinatorial library which tapped only
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a modest fraction of the potential of this approach (having only four variable positions and nine variable amino acids, or 94 ) 6561 unique peptide sequences). It should be possible to make use of a much larger set of amino acid side chains, to incorporate features that may expedite elimination of the fragments as well (21, 27). Subsequent experiments by others have confirmed that the reagents studied here are able to stably link 90Y to Lym-1 for extended periods in human plasma under physiological conditions (David Kukis, personal communication). ACKNOWLEDGMENT
This work was supported by NIH Research Grants CA16861 (C.F.M.) and CA47829 (G.L. DeNardo P.I.). We thank David Kukis and Drs Gerald and Sally DeNardo for helpful discussions. Supporting Information Available: Detailed methods of preparation and conjugation, and cathepsin digestion. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Sands, H. (1990) Experimental Studies of Radioimmunodetection of Cancer: An Overview. Cancer Res. 50 (Suppl.), 809s-813s. (2) Subramanian, R., and Meares, C. F. (1991) Bifunctional Chelating Agents for Radiometal-labeled Monoclonal Antibodies. In Cancer Imaging with Radiolabeled Antibodies (D. M. Goldenberg, Ed.) Kluwer Academic Publications, Boston. (3) Goldenberg, D. M. (1995) Cancer Therapy with Radiolabeled Antibodies. (D. M. Goldenberg, Ed.) CRC Press, Boca Raton. (4) Hnatowich, D. J. (1990) Antibody Radiolabeling, Problems and Promises. Nucl. Med. Biol. 17, 49-55. (5) Pimm, M. V., Perkins, A. C., and Baldwin, R. W. (1985) Differences in Tumor and Normal Tissue Concentrations of Iodine and Indium Labeled Monoclonal Antibody. II. Biodistribution Studies in Mice with Human Tumor Xenografts. Eur. J. Nucl. Med. 11, 300-304. (6) DeNardo, S. J., DeNardo, G. L., Deshpande, S. V., Adams, G. P., Macey, D. J., Mills, S. L., Epstein, A. L., and Meares, C. F. (1988) The Design of Radiolabeled Monoclonal Antibody for Radioimmunodiagnosis and Radioimmunotherapy. In Radiolabeled Monoclonal Antibodies for Imaging and TherapyPotential Problems and Prospects (S. C. Srivastava, Ed.) Plenum Publishing Corp., New York. (7) Motta-Hennessy, C., Sharkey, R. M., and Goldenberg, D. M. (1990) Metabolism of Indium-111-Labeled Murine Monoclonal Antibody in Tumor and Normal Tissue of the Athymic Mouse. J. Nucl. Med. 31, 1510-1519. (8) Arano, Y., Mukai, T., Akizawa, H., Uezono, T., Motonari, H., Wakisaka, K., Kairiyama, C., and Yokoyama, A. (1995) Radiolabeled Metabolites of Proteins Play a Critical Role in Radioactivity Elimination from the Liver. Nucl. Med. Biol. 22, 555-564. (9) Hnatowich, D. (1986) Label Stability in Serum of Four Radionuclides on DTPA-coupled Antibodies. An Evaluation. Int. J. Nucl. Med. Biol. 13, 353-358. (10) Cole, W. A., DeNardo, S. J., and Meares, C. F., et al. (1986) Serum Stability of 67Cu Chelates: Comparison with 111In and 57Co. Int. J. Nucl. Med. Biol. 13, 363-368. (11) Deshpande, S. V., Subramanian, R., McCall, M. J., DeNardo, S. J., DeNardo, G. L., and Meares, C. F. (1990) Metabolism of Indium Chelates Attached to Monoclonal Antibody: Minimal Transchelation of Indium from BenzylEDTA Chelate in Vivo. J. Nucl. Med. 31, 218-224. (12) Paxton, R. J., Jakowatz, J. G., Beatty, J. D., Beatty, B. G., Vlahos, W. G., Williams, L. E., Clark, B. R., and Shively, J. E. (1985) High Specific-Activity 111In-labeled Anticarcinoembryonic Antigen Monoclonal Antibody: Improved Method for the Synthesis of Diethylenetriaminepentaacetic Acid Conjugates. Cancer Res. 45, 5694-5699.
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