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Antibody Radiolabeling Techniques To Optimize Cellular Retention Stephen J. Archibald* Positron Emission Tomography Research Centre and Department of Chemistry, The University of Hull, Cottingham Road, Hull, HU6 7RX, U.K. ABSTRACT: Radiolabeling of antibodies and antibody fragments facilitates the development of new targeted therapeutics or tracking and validation of biosimilars. The typical metal ion chelators that can be used for radiolabeling reactions have residualizing properties in tissues/tumors. A team at Genentech has developed an elegant new technique for combining iodine radiolabeling with an azamacrocyclic chelator to confer residualizing properties on the radioiodine metabolites. Robust protocols, such as this example, are required for the future development of protein based drugs.
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Radiohalide labeling with 18F or one of a family of iodine isotopes offers a useful range of radioactive tags for imaging and therapy. The iodine isotopes (123I, 124I, 124I, and 131I) include β, γ, and positron emitters. Hence, there is a desire to combine the retention advantages of polyamino/polycarboxylate metal ion complexes with the versatility of the iodine isotopes. Boswell and co-workers report a method to conjugate an azamacrocyclic chelator with an antibody and the attachment of an iodine radioisotope (125I), while the precursor can be readily synthesized using a four-component Ugi reaction.4 Previous methods for labeling antibodies with residualizing iodine probes are complex and unsatisfactory.5 Meares and co-workers demonstrated the covalent binding of DOTA-lanthanide complexes by X-ray crystallographic studies of the covalently linked complexes with antibody 2D12.5 antigen binding fragment (Fab) (see Figure 1).6 This study clearly demonstrated that no metal−protein interactions were observed and that two of the lanthanides (yttrium(III) and gadolinium(III)) were effectively interchangeable. The commercially available ibritumomab tiuxetan utilizes a DTPA chelator for binding to a 90Y isotope for the therapeutic targeting of β-particles in B cell non-Hodgkin’s lymphoma. The patient can be assessed with an 111In analogue of the mAb drug prior to treatment. It was anticipated that this drug would result in a rapid increase in the number of therapeutic antibodies (particularly chelator conjugated mAbs and fragments) approved for clinical use. Unfortunately, developments have been slower than originally expected and improvements are required for conjugation and radiolabeling along with better methods for evaluation and monitoring. The Ugi reaction has been previously developed for the modification of azamacrocycles7,8 and is a method to introduce multiple functional groups into a single molecular structure. The utility of the method of Boswell and co-workers is further underlined by the use of commercially available reagents to increase the potential for widespread application. There have been significant advances for the attachment of chelators to biomolecules, including click techniques and other bioorthogonal reactions. The four-component Ugi reaction where an
olyamino/polycarboxylic acid chelators such as 1,4,7,10tetraacetic-1,4,7,10-tetraazacyclododecane (DOTA) and diethylenetriaminepentaacetic acid (DTPA) (see Figure 1)
Figure 1. (a, left) DOTA and DTPA chelators that form the basis of the most commonly used metal ion chelators in biomedical imaging. (b, right) Meares and co-workers’ X-ray crystal structure of a DOTA− yttrium(III) complex covalently bound to a Fab.
have long been exploited in radiolabeling of biological targeting vectors, such as proteins or peptides.1 They can be used in molecular imaging via the chelation of radiation emitting metal ions to form stable complexes in vivo and are also utilized in the formation of gadolinium(III) based magnetic resonance imaging contrast agents. Macrocyclic polyamino/polycarboxylic acid chelators are highly appropriate for these applications because they offer high thermodynamic and, in some cases, high kinetic stability of the complexes formed with the metal ion.2 There are additional advantages of these systems in antibody and protein labeling, as the metabolites are generally retained within the cells and hence tissue/tumor.3 This is advantageous for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging, as it widens the time window for obtaining useful data and for radioimmunotherapy as the α- or β-particles have a greater opportunity to damage the target tissue. © 2013 American Chemical Society
Received: November 19, 2013 Published: November 27, 2013 9415
dx.doi.org/10.1021/jm401794v | J. Med. Chem. 2013, 56, 9415−9417
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less satisfactory residualizing approaches. This allows site specific radiolabeling of modified mAbs with engineered cysteine residues and reduced disruption of mAb binding characteristics. Patent expiration for biopharmaceuticals will drive the development of biosimilars for production, evaluation, and release. This will potentially lower the cost of biologics in the clinic and widen their availability to patients worldwide. Rapid and effective methods are required for the evaluation of these compounds and to allow comparison more efficiently and more accurately.9,10 This can be effectively carried out using radioiodine labeling and preclinical imaging techniques combined with radiography. Techniques such as that developed by Boswell could have a significant impact on the future development of therapeutic antibodies and biosimilars, which is expected to expand dramatically over the next 10 years. Alternative mechanisms for the expansion of mAb or Fab imaging may arise through the use of longer lived metal ion positron emitting isotopes, such as 89Zr, and improvements in chelator design that better match coordination requirements for such isotopes. It is clear, based on existing radioiodination data, that the need for long-lived isotopes (such as 125I) for preclinical studies will require new methods such as those developed by Boswell and co-workers. Understanding the parameters that influence radiolabel retention could lead to new design paradigms for molecular imaging; however, the modifications must be simple if the impact is going to be widespread. The untapped potential of including the chelator on a radioiodinated protein is the potential to bind a metal radioisotope which provides exciting opportunities for multimodal imaging, co-registration of two SPECT isotopes, or combined therapy and diagnosis.
acid, an amine, an aldehyde, and an isocyanide react to give an acylaminoamide was originally exploited for the construction of an azamacrocyclic system by Faulkner, Snaith, and co-workers to form a range of luminescent lanthanide complexes with a DOTA precursor, L1 (Figure 2). Combination of the Ugi
Figure 2. Macrocyclic Ugi reaction components used by Faulkner and co-workers (L1), Botta and co-workers (L2 and L3), and Boswell and co-workers (L2).
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reaction enlisting macrocyclic precursors along with targeting groups provides the facile generation of a wide range of compounds for subsequent evaluation. Botta and co-workers utilized this system to produce ditopic chelators in a single step, highlighting the robustness of this method for the generation of functionalized DOTA type macrocycles. They extended the macrocyclic precursors to include both the acid and amine components of the four-component reaction mixture. The reaction was validated through the use of various aldehydes and isocyanides with yields ranging between 38% and 52%. Boswell and co-workers utilized the same amino derivative (L2) (see Figure 2) in the Ugi reaction to form the protected HIP-DOTA precursor in 58% yield, prior to the addition of the maleimide group. This was followed by radioiodination and deprotection of the HIP-DOTA acid groups as the last step prior to mAb labeling. Free thiols remaining on the mAbs were then capped to avoid dimerization. Biodistribution studies for biologics are routinely carried out using radioactive iodine isotopes. On degradation, iodine labeled proteins show rapid loss of the radiolabel from the target tissue. The iodotyrosine formed upon proteolysis diffuses out of the cell/tissue, making results difficult to interpret on an appropriate time scale for the evaluation of antibody localization. Hence, standard radioiodinated mAbs do not demonstrate the residualizing capacity of the radiometal complexes of 111In or 90Y with DOTA or DTPA. Boswell and co-workers have combined these advantages to give a system wherein switching to metal isotopes is no longer required and the iodine isotopes can be easily used in a residualizing system. An additional advancement found in this work is that the [125I]HIP-DOTA system was designed for the labeling of thiols rather than lysine residues, which have been labeled by other
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Telephone: (+44)1482 465488.
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REFERENCES
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(7) Tei, L.; Gugliotta, G.; Avedano, S.; Giovenzana, G. B.; Botta, M. Application of the Ugi four-component reaction to the synthesis of ditopic bifunctional chelating agents. Org. Biomol. Chem. 2009, 7 (21), 4406−4414. (8) Main, M.; Snaith, J. S.; Meloni, M. M.; Jauregui, M.; Sykes, D.; Faulkner, S.; Kenwright, A. M. Using the Ugi multicomponent condensation reaction to prepare families of chromophore appended azamacrocycles and their complexes. Chem. Commun. 2008, No. 41, 5212−5214. (9) Chirino, A. J.; Mire-Sluis, A. Characterizing biological products and assessing comparability following manufacturing changes. Nat. Biotechnol. 2004, 22 (11), 1383−1391. (10) Roger, S. D. Biosimilars: current status and future directions. Expert Opin. Biol. Ther. 2010, 10 (7), 1011−1018.
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