Review articleIrreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting
Introduction
Cancer therapies that discriminate between malignant and normal tissues are preferred for the treatment of cancer over less selective conventional strategies [1], [2], [3], [4], [5], [6], [7]. Many targeted therapies rely on the use of monoclonal antibodies (mAbs1) designed to bind specifically to extracellular receptors that are overexpressed or found only on malignant cells. Normally, engineered mAbs are not able to effectively recruit the immune system to destroy cancer cells. Rituximab (Rituxan), a chimeric monoclonal antibody that binds the CD-20 antigen expressed on the surface of certain B-cell lymphomas, is a rare exception that effectively destroys non-Hodgkin’s lymphoma (NHL) positive cells by antibody-dependent cell-mediated cytotoxicity (ADCC) [8], [9]. More commonly a cytotoxic agent such as a radionuclide or immunotoxin is attached to the antibody to aid in the destruction of the cancer tissue (Fig. 1).
Radioiodination of targeting antibodies with the β-emitting isotope 131I was a popular option initially; Bexxar®, a 131I labeled anti-CD-20 binding antibody, was recently approved by the US Food and Drug Administration for the treatment of NHL [10], [11], [12], [13], [14], [15]. Radiometal ions became an attractive alternative to iodine for radiolabeling antibodies because of their diverse properties (range of half-lives, particle emissions and decay energies), which can be selected to suit a particular application. Metal ions are attached to targeting antibodies via bifunctional chelating agents (BCAs) such as (S)-2-(4-(2-bromo-acetamido)-benzyl)-DOTA (BAD, Fig. 1), which incorporate an electrophilic arm for attachment to proteins and a chelating moiety that binds β- or α-emitting metal ions (therapy) or γ or positron emitters (imaging) [10], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. Even the therapeutic efficacy of Rituximab is significantly improved when the related murine IgG is conjugated with the therapeutic radionuclide, yttrium-90 (Ibritumomab, also called Zevalin®) [26], [27].
Because monoclonal antibodies bind their ligands reversibly, an antibody retaining high affinity for its ligand is desirable to maximize the bound lifetime. Antibodies of the IgG type possess two identical binding sites in a single molecule (Fig. 1). The bivalent nature of IgGs increases the effective affinity (“avidity”) of these molecules when bound to a cell, virus or other entity containing many copies of a particular ligand [28].
In practice, first generation radioimmunotherapeutic (RIT) strategies have fallen short of significantly improving the therapeutic ratio (target:non-target) of conventional therapies. Antibodies have circulating half-lives in the bloodstream that can be on the order of several days, attributable to their large size, which largely exempts them from removal by glomerular filtration in the kidneys (Fig. 2) [29]. Consequently, radionuclide carrying antibody molecules that have not localized to tumors continue to circulate in the bloodstream, delivering radiation to non-target tissues. An additional complication of using whole IgG antibodies for radioimmunotherapy is that hepatocytes in the liver express Fc binding receptors on their surfaces [30], which leads to accumulation of radioactive antibodies and possible radiation toxicity to the liver.
One alternative to the use of whole IgG antibodies is to use IgG fragments such as Fab, Fab′, scFv, or other engineered molecules, which maintain the antigen binding capabilities of whole antibodies but eliminate all or part of the Fc region, avoiding undesirable binding to Fc receptors in the liver (Fig. 3) [31], [32], [33], [34], [35]. These lower molecular weight antibody fragments (approximately 50 kDa for Fab fragments, slightly larger for Fab′, and ∼28 kDa for scFv compared to 150 kDa for whole IgG) have decreased circulating lifetimes in the bloodstream, thereby decreasing the radiation dose to non-target tissues. Unfortunately, the shorter circulating lifetime also translates to decreased antibody uptake in the tumor, decreasing the amount of radiation delivered [36]. Another negative consequence to using antibody fragments is their monovalent nature; the bivalency of whole IgGs significantly enhances the bound lifetime of an antibody to a multivalent target [37], [38]. Unique constructs that fuse Fab, Fab′ or scFv domains can improve the in vivo pharmacokinetics and bound tumor lifetimes (Fig. 3) [28], [36], [39], [40], [41], [42], [43], [44].
Section snippets
Pretargeting – a shift in strategy
Manipulation of antibody binding domains has led to a variety of unique structures possessing pharmacokinetics properties that differ from those observed for whole IgGs. Although decreases in toxicity have been achieved using engineered antibody fragments, the total amount of radiation delivered to targeted tumors has often been less than that achieved with bivalent whole IgGs. Although much was learned from manipulating the size and valency of antibodies, a shift in strategy was required to
Anti-metal chelate antibodies: second generation receptors for pretargeting
Second-generation pretargeting strategies incorporating anti-metal chelate antibody fragments that bind their haptens with reasonably high affinity were designed to replace streptavidin-based receptors [44], [45], [61], [62], [63], [64]. In contrast to streptavidin, antibodies can be humanized to reduce their immunogenicity, and synthetic metal chelates are small molecules that display practically no immunogenicity. Complexes of Gd3+ have been widely used in vivo for years at high
Engineering covalent ligand–receptor interactions
Historically, the inhibition of protein-based enzyme targets has been a high priority of pharmaceutical research. Promising drug candidates often bind their targets with high affinity and are effective at low concentrations. Ideally, the best of the high affinity drug candidates bind their targets permanently, eliminating the activity of the bound target by deactivating the catalytic center. Enzymes possessing catalytic cysteine residues are well-suited to this type of inhibition [72], [73],
Irreversibly binding anti-metal chelate antibodies: artificial receptors for pretargeted imaging and therapy
Chmura et al. introduced the concept of “infinite affinity” to characterize the highly specific permanent bond formed between an engineered single-cysteine mutant of antibody CHA255 and the ligand In-AABE1 upon binding. AABE is an EDTA1 derivative incorporating a weakly electrophilic acrylamide substituent. The crystal structure of CHA255 bound to a reversible ligand, In-EOTUBE1 [89], was used to identify serine 95 (light chain, CDR3) as a promising site for mutagenesis due to its proximal
Conclusion
Much has been expected of antibody-based therapies for some time now. After many years of research only a handful of antibody-based drugs have been approved for therapeutic use. Conventional antibody targeting efforts were historically patterned after better understood small molecule-based approaches, partly because much was yet to be discovered regarding the in vivo pharmacokinetic behavior of protein-based drugs. While protein-based treatments present new challenges, there are advantages.
Abbreviations
- AABD
(S)-2-(4-acrylamidobenzyl)-DOTA
- AABD-tBu
(S)-2-(4-acrylamidobenzyl)-DOTA tetra-tertbutyl ester
- AABE
(S)-2-(4-acrylamidobenzyl)-EDTA
- BAD
(S)-2-(4-bromoacetamidobenzyl)-DOTA
- CDR
complementarity determining region
- CH1 and CL
regions of the heavy chain and light chain comprising the constant domains of the Fab
- DOTA
1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid
- EDTA
ethylenediaminetetraacetic acid
- EOTUBE
Acknowledgements
Supported by Research Grant CA016861 from the National Cancer Institute, National Institutes of Health, and by NIH Shared Instrumentation Grant RR014701.
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