Linking the Radionuclide to Protein

Research has sought to balance the conditions required to achieve a radiolabeled product with adequate stability of the resulting complex. However, such scales must be balanced within the constraints imposed by isotope chemistry and half-life. As such, the options of realistic isotopes and chelating agents have become narrowed and focused as this field has matured. However, refinement of bifunctional chelating agents (BCAs) remains an active area of endeavor (Packard et al. 1999).

All metallic radionuclides require chelation chemistry for attachment to a mAb (Fig. 6.4). BCAs are chelates possessing specific functional groups that permit the conjugation of proteins to stable metallic radionu-clide complexes.

Since suitable radio-metals are diverse in their properties and coordination chemistry, no single BCA is

6.4 Linking the Radionuclide to Protein

Fig. 6.4. A general view of the conjugation of a bifunctional chelating agent to a monoclonal antibody. Specifically, a bifunctional chelating agent possesses two functionalities. One portion binds (crab = chelos = chelate) metallic radionuclides while the other portion bearing a reactive functional group reacts and covalently binds to N-terminal and E-amines from lysine on the protein. Generally, the metallic radionuclide is added last in this sequence prior to purification of the final product; however, a pre-formed radio-metal complex can also be conjugated to protein (Milenic et al. 2004)

suitable for all (Packard et al. 1999). A selection of examples is provided in Fig. 6.5. The laudable goal of "instant" radionuclide complex formation with infinite stability has proven non-trivial. Numerous chemical criteria must be considered in the choice of chelating agent including its design and actual use. Characteristics of the metal, such as coordination number, ionic radius, binding character (hard vs soft), and reactivity (hydrolysis vs complexation) must also be considered with respect to chelate design (Packard et al. 1999). A BCA may form and maintain an adequately stable metal complex, but the formation kinetics may render a BCA impractical for an intended radionuclide. For example, DOTA (1,4, 7,10-tetra-azacylcododecane-N, N', N", N"'-tetraacetic acid) forms highly stable and kinetically inert complexes with 212Bi and 213Bi (Michel et al. 2003). However, Bi(III) complexation kinetics with DOTA require 15-45 min for reaction completion. The half-lives of the radionuclides are 60 or 46 min, respectively, making this particular combination wasteful and highly impractical (Ruegg et al. 1990). Higher temperatures traverse this in part, but are limited by denaturation of the protein vehicle. In contrast to macrocyclic BCAs, acyclic BCAs tend to possess far faster complex formation rates (Fig. 6.5). However, these are not quite as stable, representing another forced compromise. The acyclic CHX-A'', a cyclohexyl-DTPA (diethylenetriamine pentaacetic acid) (Fig. 6.5), has been shown to be a viable alternative to DOTA for labeling of mAbs with Bi(III) isotopes (Brechbiel and Gansow 1992; Milenic et al. 2001). This BCA complexes bismuth "instantaneously" (t1/2 = 0.27 s) and is sufficiently stable for clinical trials (Jurcic et al. 2002). In addition, it was reported to have similar stability with the |3-emitter 177Lu versus DOTA and PA-DOTA, the lat ter of which has been employed in clinical trials in combination with 177Lu (Mulligan et al. 1995; Alvarez et al. 1997; Schott et al. 1994). In summary, this ligand not only provides considerable versatility for radiolabeling mAbs with the a-emitters 213Bi and 212Bi, but also with (3-emitters, such as 90Y and 177Lu, permitting a wide range of clinical applications (Milenic et al. 2001; Rosel-li et al. 1999; Milenic et al. 2002).

Although 67Cu copper isotopes have been investigated, the choice of BCA remains an open and unresolved topic (Novak-Hofer and Schubiger 2002). Several different macrocyclic chelating agents have been reported as stable and inert with 67Cu, despite reports of its transchelation to superoxide dismutase and detection in patients' ceruloplasmin and liver (DeNardo et al. 1998; Bass et al. 2000; Rogers et al. 19 9 6). 67Cu remains an interesting candidate for therapy with regards to emission energy, half-life, and imageable emissions (Novak-Hofer and Schubiger 2002). Production and availability may limit 67Cu and as such 64Cu may eventually be deemed more viable (Wu et al. 2000).

The chemistry for linking 211At to proteins has been dominated by aryl active ester reagents that have advanced to clinical trials (Vaidyanathan and Zalutsky 1996; Zalutsky et al. 2002). Issues of inadequate in vivo stability for general application in clinical settings are as yet unresolved. Such concerns may be addressed with a better understanding of the chemistry of 211At itself.

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