Nuclear Medicine in the Era of Genomics and Proteomics: Lessons from Annexin V Tarik Z. Belhocine,*,† Jonathan F. Tait,‡ Jean-Luc Vanderheyden,§ Chun Li,| and Francis G. Blankenberg⊥ Department of Nuclear Medicine, Jules Bordet Cancer Institute, Brussels, Belgium, Departments of Laboratory Medicine, Medicine (Medical Genetics) and Pathology, University of Washington, Seattle, Theseus Imaging Corporation, North American Scientific, Boston, Department of Molecular Imaging, MD Anderson Cancer Center, Houston, Texas, Division of Radiology, Stanford University School of Medicine, Palo Alto, California Received January 18, 2004
In the past decade, genomics and proteomics have begun to develop many new targets for potential diagnostic and therapeutic agents. Among the life sciences, nuclear medicine is also deeply involved in the field of clinical investigation. Experience with radiolabeled annexin V highlights the many steps required to translate a good basic-science concept into the clinical setting. This model also emphasizes the value of synergy between basic and medical specialties in developing and optimizing a clinically useful product initially derived from basic investigation. Keywords: annexin V • recombinant human • radiolabeling • apoptosis
Introduction In this beginning of the 21st century, the development of high-end imaging technologies converges on the bioengineering of genetically well designed molecules.1,2 The sequencing of the human genome opened new avenues in fundamental research by using tagged nucleic acids as molecular probes for the detection of various genetic diseases.3 In a logical sequence, genomics led to proteomics, a subspecialty intended to study the functions of all expressed proteins encoded by original and mutant genes; an analysis that includes both the qualitative and the quantitative profiles of proteins.4-6 Clinical applications of genomics and proteomics involve large fields of medical research, especially for the development of new drugs and the assessment of biomarkers for early diagnosis and monitoring of cancers.7-11 Such clinical end-points necessarily require the use of proteomics biotechnologies as research tools in the hands of biologists, development scientists imaging professionals, and clinicians.3,12,13 As a life science, nuclear medicine explores and measures the physiological and pathological human processes by using various radiolabeled molecules, which are increasingly peptides or proteins, glycoproteins, or lipoproteins; tracers that are dedicated to the assessment of a particular biological function or a targeted organ.14 In recent years, this medical specialty * To whom correspondence should be addressed. Tarik Belhocine, MD, Ph.D, Department of Nuclear Medicine, Jules Bordet Cancer Institute, Rue He´ger-Bordet I, 1000 Brussels, Belgium. Phone: (322)-541-3240. Fax: (322)541-3094. E-mail:
[email protected]. † Department of Nuclear Medicine, Jules Bordet Cancer Institute. ‡ Departments of Laboratory Medicine, Medicine (Medical Genetics) and Pathology, University of Washington. § Theseus Imaging Corporation, North American Scientific. | Department of Molecular Imaging, MD Anderson Cancer Center. ⊥ Division of Radiology, Stanford University School of Medicine. 10.1021/pr049968a CCC: $27.50
2004 American Chemical Society
improved the assessment of the basic phenomena underlying cell death in normal conditions or after therapeutic interventions.15,16 In this new era of imaging, the recombinant human (rh) annexin V protein may be radiolabeled with different isotopes by means of various linkers in order to capture one of the first steps of the apoptotic cascade.17,18 Although annexin V was first purified and cloned by more traditional methods based on single protein and cDNA (complementary deoxyribonucleic acid) molecules, experience with this protein provides valuable lessons for clinical development of future imaging candidates derived from the newer methods of genomics and proteomics. Recombinant Human (rh) Annexin V: A Marker of Apoptosis. The use of the annexin V as molecular probe in various types of cells undergoing the apoptosis is not new.19 For a long time, in vitro studies, either in cellular biology or in pathology, used the fluorescein-labeled annexin V for the detection of apoptotic cells. Until now, the FITC-annexin V (fluoresceine isothiocyanate) is still extensively used to provide evidence of cell death.20,21 In recent years, however, a renewed interest was directed toward the feasibility of radiolabeled annexin V agents for the in vivo imaging of sites exhibiting spontaneous apoptotic changes or following pro-apoptotic interventions.22,23 Annexins constitute a widespread family of [Ca2+]-dependent phosphatidylserine binding proteins including more than 90 chemical variants in 45 different species, which have remained highly conserved through the evolution. These proteins are mainly involved in key biological processes such as apoptosis, haemostasis, and thrombosis.24,25 As a rule, annexins consist of an N-terminal variable region followed by four or eight copies of the characteristic repeat unit of about 70 amino acids. Among them, the human annexin V is a 36 kDa protein, which is encoded by a gene located on the human chromosome 4q26 Journal of Proteome Research 2004, 3, 345-349
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Figure 1. Crystallographic analysis of human annexin V showed that the protein includes 319 amino acids folded into a planar cyclic arrangement of four repeats, each with a similar structure of five R-helical segments wound into a compact superhelix. In this particular crystal form of annexin V, three calcium ions (red spheres) have been identified in repeats I (red), II (violet), and IV (blue).
f q28 that spans a DNA locus of 28 kb in length containing 13 exons and 12 introns.26 Huber and co-workers first reported the three-dimensional structure of annexin V determined by X-ray crystallography and showed that it was organized in 4 domains (I, II, III, IV).27 Each domain incorporates five R-helical segments folded in a compact super-helix; each domain binds calcium, but the number of bound calcium ions differs widely in different crystal forms of annexin V (see Figure 1). In cells committed to apoptosis, phosphatidylserine is exposed.18 Because annexin V binds with nanomolar affinity to cells with exposed PS in the presence of calcium ions, it can detect PS exposed in apoptotic cells.17,20,28 This provided with an important rationale for the use of the radiolabeled annexin V as in vivo marker of the early redistribution of PS from the inner leaflet to the outer leaflet of cell membrane surface.16,21,27-29 The rh-annexin V was produced by several groups using recombinant techniques (i.e., expression in Escherichia coli). The protein was then purified with no detectable endotoxin. Additionally, the rh-annexin V maintained its biological activity as [Ca2+]-dependent PS binding protein, which was equivalent to that of the native annexin V isolated from human placenta. Hence, the recombinant human annexin V was ready for further clinical application via a dedicated radiochemical preparation. Radiolabeled rh-Annexin V: A Tracer for Imaging Apoptosis. In experimental and clinical research, the [99mTc]-rhannexin V was designed for early diagnosis of disease, selection of individualized treatments, assessment of therapeutic toxicity, and real-time monitoring of anticancer therapies; all these applications of genomics and proteomics were based on the imaging of the annexin V protein network changes. Experimental models such as tumor xenografts treated by Cyclophosphamide, rheumatoid arthritis, fulminant hepatitis, autoimmune myocarditis, acute and chronic cerebral hypoperfusion, acute rejections of heart and lung transplants, unstable atherosclerotic plaques were studied with the annexin V labeled with [99mTc]-technetium 99m, the most widely used radionuclide in Nuclear Medicine. In all of these models, the labeling procedure was successfully achieved, thereby, leading to a stable tracer with a high radiochemical yield.29,31-40 Still on animals, other isotopes such as [123I]-iodine 123, [124I]-iodine 124, [111In]-indium 111, [68Ga]-gallium 68, [11C]-carbon 11 and 346
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Figure 2. (A) Structure of 99mTc-BTAP-rh-annexin V; (B) Structure of 99mTc-HYNIC-rh-annexin V.
[18F]-fluorine 18 were more recently used to label the annexin V protein.41,43,46-53 In humans, however, the rh-annexin V was only labeled with [99mTc]-technetium 99m for clinical investigation in patients with cancers (untreated tumors or malignancies treated by chemotherapy) as well as in subjects with acute myocardial infarction or heart transplant rejections.16,23,54-57 In the first clinical trial, the radiolabeling of the rh-annexin V protein used the method described by Kasina and Fritzberg.23,58,59 This chemical procedure referred as the N2S2 method consists of five process steps: formulation of the phenthioate ligand, conjugation of [99mTc]-technetium ligand ester to the alanyl amino terminal or to lysine functional groups on the rh-annexin V, purification of the conjugate on a Sephadex G-25 gel filtration column, and final dilution for patient administration. In the subsequent study, however, the hydrazinonicotinamide was first convently attached to the alanyl amino terminal or to lysine functional groups on the rh-annexin V purified by gel permeation using a tricine buffer before the introduction of the [99mTc]-technetium 99m isotope.57 Figure 2, parts A and B, illustrates the anticipated chemical forms of the recombinant human annexin V used in humans for clinical investigation. In summary, by using N2S2 or N3S linkers such as HYNIC (hydrazinonicotinamide), EC (ethylenedicysteine), NIM n-limino-4-mercaptobutyl), BTAP (4.5 bis thioacetamido pentanoyl), the 99mTc-radiolabeled annexin V allowed the imaging of apoptosis in different disease models.16,23,54,58,60,61 However, if the following equation: annexin V plus linker plus isotope is proving to be chemically feasible, and experimentally implementable in the animal imaging of apaptosis, its clinical utility remains to be confirmed. Current Limitations and Future Perspectives. The human biodistribution of the [99mTc]-radiolabeled annexin V under different chemical forms (i.e., using the NIM, BTAP, and HYNIC linkers) showed a typical pattern of proteins distribution.62-64 Indeed, the molecule predominantly diffuses to the liver, and
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Figure 4. Synthesis of DTPA-PEG-annexin V.
Figure 3. Physiological distribution of 99mTc-rh annexin V in humans. As expected for proteins, the radiolabeled annexin V has been shown to mainly diffuse to the liver (L) and the spleen (S), with a predominant renal excretion (K: kidneys; B: bladder). Table 1. Chemical Properties and Biodistribution of [99mTc]-labeled Annexin V Tracers Used for Clinical Investigationa apoptosis tracer
Rh-annexin V
Rh-annexin V
Rh-annexin V
linker isotope radiochemical purity blood half-life (min) biologic half-life (h) kidneys liver spleen colon bone marrow
BTAP 99mTc 96 ( 4% 26 ( 5 16 ( 7 ++ +++ + +++ +
NIM 99mTc 82 ( 12% 14 ( 6 62 ( 13 ++ ++ + +++ +
HYNIC 99mTc 94 ( 1.7% 24 ( 3 69 ( 7 +++ ++ ++ ++
a Abbreviations: Rh-annexin V, recombinant human annexin V; BTAP, 4.5 bis thioacetamido pentanoyl; NIM, n-1-imino-4-mercaptobutyl; HYNIC, hydrazinonicotinamide; -: traces; +: low; ++: moderate; +++: high.
the spleen; its elimination is mainly renal, in addition to a high nephron (PS-annexin V) uptake (see Figure 3). So far, the radiochemical purity rates varied from a chemical form to another. Also depending on the linker used, a variable degree of vascular and intestinal background was found, which ranged from high to low. Table 1 summarizes the chemical properties as well as the biodistribution of [99mTc]-annexin V-based apoptosis tracers used for clinical investigation. The clinical feasibility of [99mTc]-radiolabeled annexin V using various chelators demonstrated mixed results.16,23,54-56 In a small series of patients with acute myocardial infarction (n ) 7), [99mTc]-NIM annexin V located within areas showing defects of [99mTc]-sestamibi tracer.54 ([99mTc]-sestamibi is a perfusion tracer commonly used for the detection of myocardial ischemia or necrosis.) Whether the labeled annexin V was taken up in necrotic or apoptotic tissues could not be ascertained because pathological correlations were not possible. In addition, the vascular background considerably impaired the quality of the images in early SPECT (single photon emission tomography) images (3-4 h post-tracer injection). In oncology patients, the
Figure 5. Structure of annexin V mutant form 117. The primary structure of annexin V-117 is shown. The protein contains six amino acids added at the N-terminus, followed by amino acids 1-320 of wild-type annexin V. Amino acid Cys-316 is also mutated to serine in this molecule. Technetium-99m chelation is thought to occur via formation of an N3S structure involving the N-terminal cysteine and the immediately adjacent amino acids.
[99mTc]-BTAP annexin V as well as the [99mTc]-HYNIC annexin V was able to localize at sites presenting with histologically proven spontaneous apoptosis or drugs-induced apoptosis. However, the contrast of images in terms of signal-tobackground ratio may still need to be improved yet. In addition, the timing for an optimal detection of dying cells in diverse clinical circumstances remains to be determined. More importantly, within the complex tumor microenvironment, the annexin V uptake cannot differentiate between apoptosis and necrosis. Also, the signal obtained in scintigraphy may reflect the number of cell death events, which is, in turn, weighted by the importance of the necrotic component.57 Besides, in normal conditions, endothelial cells, plaquettes, erythrocytes, and vascular smooth muscle cells, may physiologically expose PS during apoptosis, which may alter, at least in part, the specificity of the annexin V update.17,18,31,65-67 Hence, the characterization of tumor tissues by means of radiological or pathological techniques, is critical to correlate the tracer uptake with the tumor biology. In recent years, alternative solutions have been proposed to optimize the imaging properties of the radiolabeled annexin V. Among them, a possibility is being offered by a long-lived annexin V in parallel with the use of a PEGylated form of the protein.46,47 This consists of a one-step procedure to introduce both poly(ethylene glycol) (PEG) and the metal chelator diethylenetriaminepentaacetic acid (DTPA) to annexin V through a heterofunctional PEG precursor. The PEG precursor contains DTPA at one end and an amine-reactive isothiocyanate (SCN-) functional group at the other end (see Figure 4). Protein conjugation was readily achieved by mixing the proteins and the PEG precursor SCN-PEG-DTPA in an aqueous solution. The PEGylated annexin V may penetrate in depth, thereby, reaching superficial as well as central zones of apoptotic tissues. Additionally, the tracer is labeled with the longer lived [111In] (Indium 111, which has a half-life of 2.8 days), which allows Journal of Proteome Research • Vol. 3, No. 3, 2004 347
perspectives the imaging of the apoptosis on a longer time interval than the [99mTc]-radiolabeled annexin V. Another promising research direction relies upon the radiolabeling of mutant forms of annexin V (annexin V 117, 118, 119) with endogenous sites for the [99mTc] chelation. In mice, the annexin V-mutant 117 produced through expression in Escherichia coli was successfully labeled with [99mTc]-technetium99m.65 Radiolabeled annexin V-117 tracer showed a more favorable distribution than 99mTc-HYNIC-annexin V. Figure 5 shows the structure of the annexin V mutant form 117. In another approach, annexin V was labeled with fluorine 18 using N-succinimidyl 4-fluorobenzoate. 18F-annexin V offers an advantageous biodistribution over 99mTc-annexin V-117 with a much lower uptake in the liver, spleen, and kidney.53 Besides, the use of PET (positron emission tomography) technology could allow a better spatial resolution that may open new perspectives for optimizing the imaging of apoptosis in clinical setting.43,48,50,51 So far, lessons from clinical trials conducted with conventional SPECT nuclear medicine techniques stress the need for more documented data in order to confirm the encouraging results obtained with the 99mTc-HYNIC-annexin V. Hence, the clinical utility of annexin V labeled with positron emitters is still speculative until “proof of principle” will be definitely provided. The clinical research, however, is not a linear process leading immediately to successful results. Rather, the development of new compounds is a long term task; a laborious process that requires iterative steps in order to achieving a product of quality. Also, the theoretical rationale for any study design can be subsequently refuted by the clinical reality. Nonetheless, in the field of experimental and clinical research, once the primary concept is well validated, which is the case with the radiolabeled rh-annexin V for the imaging of apoptosis, any (un)expected result, either positive or negative, is worthwhile when the methodology and the analysis steps are rigorously conducted. Subsequently, proteomics will help refine the radiochemical tools with regard to the clinical goals.
Summary and Conclusions Growing evidence demonstrates the feasibility of the radiolabeled annexin V as potential marker for the in vivo imaging of apoptosis. While the chemistry and the radiolabeling of the protein are nowadays well handled with various isotopes and linkers, the imaging applications remain to be optimized. The annexin V model illustrates the complexity of translating newly designed molecules into the field of clinical imaging. As such, the nuclear medicine needs should be first tailored to the clinical needs before to implementing new tracers. Also, to give a sense to genomics and proteomics in any research process, the clinical goals must be the ultimate end-points derived from a comprehensive multidisciplinary approach.
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