Chemically Modified Antibodies as Diagnostic Imaging Agents

Summary

Notable new applications of antibodies for imaging involve genetically extracting the essential molecular recognition properties of an antibody, and in some cases enhancing them by mutation, before protein expression. The classic paradigm of intravenous administration of a labeled antibody to image not only its target but also its metabolism can be improved on. Protocols in which molecular targeting with an engineered unlabeled protein derived from an antibody, followed by capture of a small probe molecule that provides a signal, are being developed to a high level of utility. This is accompanied by new strategies for probe capture such as irreversible binding, incorporation of engineered enzyme active sites, and antibody-ligand systems that generate a signal only upon binding or uptake.

Introduction

Most of us think of an antibody molecule as an immunoglobulin G (IgG) protein, a Y-shaped macromolecule composed of two identical polypeptide heavy chains (each ≈440 residues) paired with two identical light chains (each ≈214 residues), with an overall molecular weight of ≈150KD (Figure 1). Well-established methods are available to prepare new antibodies that specifically bind to a chosen group of atoms as small as a dinitrophenyl group or as large as a 1,000 Ų region on another macromolecule.

Figure 1.

An example monoclonal antibody structure (pdb 1IGT, mouse IgG2a), showing 82 lysine residues in cpk spacefill, carbohydrates in yellow spacefill, and N-terminal residues in gray spacefill (visible on the right side only). Heavy chains are red and blue; light chains green and yellow. Important functional regions and fragments are also noted with single brackets.

The organic chemistry of natural antibodies begins with nucleophilic primary amines on lysine side chains, of which there may be 80–90 on the IgG surface. Because most lysines are available for reaction, it is a common strategy to statistically label a small average number of lysines per antibody with the reagent of interest and use the resulting mixture in biological experiments. This practical but untidy procedure can be replaced by site-specific chemistry as discussed below.

Even more nucleophilic than lysine are the N-terminal amines of the four polypeptide chains, but these may be blocked; for example, N-terminal glutamine can eliminate ammonia and form a cyclic amide. IgG molecules contain glycosylation sites at heavy-chain position 297, located well away from the antigen-binding sites; their distinctive chemistry makes these carbohydrates useful attachment sites for enzymes or other macromolecules. IgG molecules also have 16 or more pairs of cysteine residues, practically always occurring in disulfide bonds. Special techniques to selectively reduce some of these disulfides to yield reactive thiols are useful in preparing antibody-drug conjugates [1]. The C-terminal half of each antibody heavy chain (the Fc region), including the carbohydrate, is involved in a variety of interactions important to the behavior of the antibody in vivo [2].

It has become common practice to use molecular biology to improve properties by engineering fragments or analogs of antibodies. This generally preserves the antigen-binding site while decreasing the protein size and deleting other immunologically active sites such as the Fc region. Therefore the papers discussed below only occasionally involve intact IgG molecules. Often the antigen-binding function is expressed from genes coding for the Fv fragment (Figure 1), comprising the N-terminal regions of the heavy and light chains, with additional DNA codons for a peptide linker inserted to form a single gene coding for a single-chain Fv (scFv) protein [3]. A further refinement is pretargeting an engineered protein to a desired site on a cell or tissue, and then using it to capture a small probe molecule [4]. References [5, 6••] describe an important recent example.

Pretargeting for In Vivo Imaging

An approach for imaging has been evaluated in animal models, using an antibody-based reporter gene whose receptor product is capable of binding irreversibly to metal chelate reporter probes by Michael addition [7••, 8•]. The reporter gene, named DOTA Antibody Reporter 1 (DAbR1), consists of the scFv fragment of the mutant anti-DOTA(Y) antibody 2D12.5 G54C [9], genetically fused to the hinge region of a human IgG4 Fc fragment and the T-cell CD4 transmembrane domain (Figure 2). Transfected human glioma U-87 tumors, expressing ≈10⁶ DAbR1 sites per cell on their surface, were xenografted into scid mice [7••]. The ability of DAbR1 to capture and bind to the reporter probe ligand acrylamidobenzyl-DOTA(⁸⁶Y) (AABD(⁸⁶Y)) was studied using positron emission tomography (PET). The images revealed substantial uptake of AABD(⁸⁶Y) in DAbR1-expressing tumors versus tumors lacking the DabR1 gene, and low background in non-target tissues.

Figure 2.

Expression of the reporter gene for engineered probe-capture antibody with infinite affinity DAbR1 on the surface of glioma cells leads to excellent images of tumors implanted in scid mice [7••]. Probe binding to DAbR1 followed by attachment of cysteine thiol to the acryloyl group of the probe leads to durable labeling. Serial small-animal PET/CT images from a dynamic scan of mice bearing DAbR1-expressing tumor on the right shoulder show uptake in target tumor, bladder, and small bowel up to 1 hr after injection of 3.7 MBq AABD(⁸⁶Y) probe in the tail vein. Image after 14 hr shows the tumor is labeled while other organs are clear. Control animal at lower right was treated identically, including ⁸⁶Y probe injection, except its tumor did not receive the reporter gene. Credit: Wei LH, Olafsen T, Radu C, Hildebrandt IJ, McCoy MR, Phelps ME, Meares CF, Wu AM, Czernin J, Weber WA. Engineered Antibody Fragments with Infinite Affinity as Reporter Genes for PET Imaging. J. Nucl. Med. 2008;49:1828–1835. Copyright Society of Nuclear Medicine. Adapted with permission. PET is positron emission tomography, showing probe distribution in color; CT is computed x-ray tomography, showing anatomy in grayscale.

A recent extension of the Michael addition strategy has led to preparation of an acryloyl-bearing affibody protein that forms a covalent bond with an appropriately positioned cysteine, lysine, or histidine on its specific protein target, even in complex biological mixtures [10]. An affibody is a 58-residue peptide based on Staphylococcus aureus Protein A [11].

A novel bio-orthogonal reaction that employs an inverse-electron-demand Diels-Alder reaction involving cycloaddition of an s-tetrazine probe to a trans-cyclooctene tagged antibody, presents an alternative to the strategy above [12,13]. Antibody CC49 conjugated with trans-cyclooctene ex vivo was administered to nude mice bearing LS174T human colon cancer xenografts, followed 27 hr later by injection with a tetrazine-DOTA(¹¹¹In) probe. Single-photon tomography images indicate that the covalent adduct was formed at the surface of the tumor, while very high levels of probe are found in the bladder [14]. In contrast to the approach of [7••], the probe was attached by direct reaction with the antibody-bound cyclooctene without affinity capture at a probe-binding site.

¹⁸F is commonly used for PET imaging in the form of fluorodeoxyglucose (FDG). Due to the 110 min radioactive half-life of ¹⁸F, its applications to imaging are constrained by the time required for the synthesis and purification of ¹⁸F-labeled probes. A new method of using ¹⁸F has been demonstrated by the chelation of the aluminum fluoride ([Al¹⁸F]²⁺) complex by the macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) [5, 15••]. A NOTA-labeled peptide is able to bind the [Al¹⁸F]²⁺ stably in the presence of human serum. Starting from ¹⁸F⁻, the preparation and purification of the radiolabeled peptide can be accomplished within 1 hr, as opposed to ¹⁸FDG protocols which may take several hours [15••]. This NOTA-peptide can also be labeled with ⁶⁸Ga(III), another radionuclide used for PET imaging [6••].

Using the “dock and lock” (DNL) approach, a trivalent bispecific antibody has been constructed to bind to an antigen-bearing tumor and to an [Al¹⁸F]²⁺ labeled peptide-chelate (Figure 3). Previous work describes the dock and lock approach in detail [16]. Pretargeting involves the intravenous administration of the modified antibody, and a later injection of the Al¹⁸F-NOTA-peptide. This takes advantage of quick clearance of the excess radiolabeled peptide compared to an antibody [17].

Figure 3.

Static PET/CT imaging study of a BALB/c nude mouse with LS174T tumor (0.1 g) on the right side, which received 6.0 nmol TF2 and 0.25 nmol ¹⁸F-IMP-449 (5 MBq) intravenously with a 16-hr interval [6••]. The animal was imaged 1 hr after injection of ¹⁸F-IMP-449. The panel shows the three-dimensional volume rendering (posterior view; A) and cross-sections at the tumor region: coronal (B), sagittal (C), and transverse (D). Credit: Schoffelen R, Sharkey RM, Goldenberg DM, Franssen G, McBride WJ, Rossi EA, Chang C-H, Laverman P, Disselhorst JA, Eek A, et al.: Pretargeted Immuno-Positron Emission Tomography Imaging of Carcinoembryonic Antigen-Expressing Tumors with a Bispecific Antibody and a ⁶⁸Ga- and ¹⁸F-Labeled Hapten Peptide in Mice with Human Tumor Xenografts. Molecular Cancer Therapeutics 9:1019–1027. ©2010 American Association for Cancer Research. Adapted with permission.

Optical imaging

A method for imaging cells has recently been developed using antibody-based Fluorogen-Activating Proteins (FAPs) that bind to fluorogenic dye molecules, causing them to fluoresce. Familiar examples of fluorescence activation of dye molecules include DNA binding with the intercalating dyes ethidium bromide or thiazole orange (TO), or RNA binding with malachite green (MG). The mechanism responsible for the activation of the dye is thought to involve structural constraints on the bound fluorophore [18••].

FAPs have been isolated that bind to and activate fluorogens such as MG, TO and dimethyl indole red (DIR). FAPs displayed on yeast or mammalian cell surfaces have been found to bind their respective fluorogens with nanomolar affinity, and increased fluorescence by as much as a thousand-fold (Figure 4). A “promiscuous” FAP that binds with high affinity to several fluorogenic cyanine dyes has also been reported [19]. In some cases, an entire scFv is unnecessary for fluorogen activation, and a heavy or light chain fragment may activate a fluorogen [20].

Figure 4.

(left) Surface labeling of human tumor cells with a malachite green (MG) fluorogen-activating protein (FAP). Stably transformed M21 melanoma cells expressing fluorogen-activating protein HL4-MG fused to platelet-derived growth factor receptor (PDGFR) were imaged as a confocal stack at 488-nm excitation using 10 nM impermeant malachite green derivative MG-11p [18••]. Photomicrograph is a three-dimensional reconstruction of the stack. (center) Surface labeling of fibroblasts with a thiazole orange (TO1) FAP. Stably transformed NIH3T3 cells expressing fluorogen-activating protein HL1.1-TO1 fused to PDGFR and imaged using 40 nM impermeant thiazole orange derivative TO1-2p. (right) Simultaneous surface labeling of fibroblasts with MG and TO1 FAPs. NIH3T3 cells respectively expressing the FAPs of left and center panels were mixed 1:1 and imaged using 10 nM MG-2p and 40 nM TO1-2p. The transparency of surface-labeled cells allows fine discrimination of contact surfaces between cells of different colors. Scale bars, 10 µm. Adapted by permission from Macmillan Publishers Ltd: Nature Biotechnology Vol 26 Issue 2, pp 235–240 (2008), copyright 2007.

The activatable fluorogen, indocyanine green (ICG), has been used in combination with radiolabeling to test a multimodal approach for imaging human epidermal receptors HER1 and HER2 on xenograft tumors in mice. ICG is used for in vivo imaging, with emission in the range of 700–850 nm [21••]. Antibodies have been labeled with ¹¹¹In and ICG and injected into tumor-bearing mice; comparing the optical and nuclear images may have promise for use with whole-body nuclear imaging followed by optically guided surgery.

Site-Specific Conjugation

The simplest labeling methods for antibodies can yield complex mixtures due to random reactions with subsets of cysteine or lysine residues, which can affect antibody function adversely. The following examples demonstrate recently reported ways of attaching probes while retaining antigen-binding activity. These selective conjugations are flexible, and may be extended to yield antibody molecules with imaging capabilities for in vitro assays and future in vivo studies. The copper-catalyzed azide-alkyne "click" reaction is discussed elsewhere (e.g., [22]).

SNAP-tag [23] and Covalin [24•] are small proteins that react specifically and stoichiometrically with synthetic linkers. A human DNA-repair enzyme O(6)-alkylguanine DNA alkyltransferase (SNAP-Tag) containing a reactive thiol in its binding pocket reacts with its substrate, para-substituted O(6)-benzylguanine (BG). BG can be derivatized with probes such as biotin, fluorophores, reporter proteins, or nanoparticles. Various SNAP-Tag-scFvs have been cloned to image cell surfaces using BG-derivatized probes. Covalin is a SNAP-Tag/Halo-Tag fusion protein for cross-linking orthogonally tagged molecules, small or large. The Halo-tag protein contains a reactive carboxylate in the binding pocket that forms a stable ester linkage with primary chloroalkane groups conjugated to a chosen molecule, orthogonal to the SNAP-tag reaction.

An aldehyde tag peptide sequence, genetically engineered into the Fc region of an IgG [25], facilitates the conversion of a cysteine residue to formylglycine by a formylglycine generating enzyme. The product can react covalently with aminooxy or hydrazide probes. After enzymatic removal of terminal galactose residues, a sugar residue with a functional group C2-keto-Gal (modified ketone on Galactose) can be placed on the N-glycan moiety of the IgG by a galactosyltransferase mutant [26]. An alternative strategy to attach multiple copies of C2-keto-Gal to an engineered antibody is to genetically add several threonine residues to the C terminus [27] and use human polypeptide-alpha-N-acetylgalactosaminyltransferase II to transfer C2-keto-Gal to the side-chain hydroxyl group of threonine.

Genetic insertion of selenocysteine into the C-termini of whole antibodies, Fab fragments [28] or Fc fragments [29] facilitates stoichiometric conjugation to electrophilic moieties. At mildly acidic pH the selenocysteine can be labeled selectively in the presence of free cysteine residues. In another unusual reaction, herceptin has been conjugated via the phenol side chain of tyrosine to an RGD conjugate of a cyclic diazodicarboxamide, 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione [30•]. This provides an alternative to lysine tagging, and may be useful where essential lysines must be preserved without modification.

A variation on chemically modified antibodies is to covalently attach the Fc fragment to non-antibody receptor ligands. These ligands bind to highly expressed receptors on cancer cells and are designed to direct the Fc to the receptors [31]. Antibody 38C2 produced by reactive immunization of mice with a 1,3-diketone [32] has been chemically programmed by reacting the beta-lactam conjugates of biotin or cyclic RGD peptide [25]. Similarly, a glycosylated IgG1 Fc fragment expressed with N-terminal cysteine residues has been selectively modified with a thioester-containing cyclic RGD peptide through native chemical ligation [33].

Improving Biodistribution of Engineered Antibody Fragments

Among the other functional sites in the Fc region of an IgG molecule is one that binds the FcRn receptor, which is involved in extending the half-life of the antibody in circulation [34]. Proteins that lack binding sites for the FcRn receptor often clear too quickly, before adequate target uptake has been achieved. Improvement of the pharmaceutical properties of recombinant scFv-F8 antibody fragments by extension of the serum half-life has recently been accomplished using site-specific conjugation of "Albu tag", a small organic molecule — 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate — designed to bind reversibly to the abundant, long-lived protein serum albumin [35••, 36]. Comparison of Albu-tagged and unmodified proteins in animal models shows that antibody fragments conjugated with Albu tag exhibit an ≈10-fold increase in tumor uptake and an ≈35-fold reduction in blood clearance rate. Cloning an albumin-binding peptide to an antibody fragment can also dramatically improve biodistribution properties, as shown earlier by Dennis et al. [37,38]. Aspects of this strategy have been compared to conjugation with polyethylene glycol (PEG) [39–41]. A PEGylated scFv occupies a large volume and may exhibit decreased binding affinity, while a tagged scFv complexed with albumin forms a reservoir with which free scFv is in equilibrium.