With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragments retain the targeting specificity of whole mAbs but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Antibody fragments have been forged into multivalent and multispecific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents. Single-domain antibodies are anticipated to significantly expand the repertoire of antibody-based reagents against the vast range of novel biomarkers being discovered through proteomics. As this review aims to show, there is tremendous potential for all antibody fragments either as robust diagnostic reagents (for example in biosensors), or as nonimmunogenic in vivo biopharmaceuticals with superior biodistribution and blood clearance properties.

Engineered antibody fragments and the rise of single domains

The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties associated with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a molecule can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addition, 18F has been established as a useful positron emitting isotope for use with in vivo imaging technology that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compounds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compounds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug molecules and applications in po...

Applications of Fluorine in Medicinal Chemistry

Successful development of ultra-sensitive molecular imaging nanoprobes for the detection of targeted biological objects is a challenging task Although magnetic nanoprobes have the potential to perform such a role, the results from probes that are currently available have been far from optimal Here we used artificial engineering approaches to develop innovative magnetic nanoprobes, through a process that involved the systematic evaluation of the magnetic spin, size and type of spinel metal ferrites These magnetism-engineered iron oxide (MEIO) nanoprobes, when conjugated with antibodies, showed enhanced magnetic resonance imaging (MRI) sensitivity for the detection of cancer markers compared with probes currently available Also, we successfully visualized small tumors implanted in a mouse Such high-performance, nanotechnology-based molecular probes could enhance the ability to visualize other biological events critical to diagnostics and therapeutics

Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging.

Immunoconjugates--monoclonal antibodies (mAbs) coupled to highly toxic agents, including radioisotopes and toxic drugs (ineffective when administered systemically alone)--are becoming a significant component of anticancer treatments. By combining the exquisite targeting specificity of mAbs with the enhanced tumor-killing power of toxic effector molecules, immunoconjugates permit sensitive discrimination between target and normal tissue, resulting in fewer toxic side effects than most conventional chemotherapeutic drugs. Two radioimmunoconjugates, ibritumomab tiuxetan (Zevalin) and tositumomab-131I (Bexxar), and one drug conjugate, gemtuzumab ozogamicin (Mylotarg), are now on the market. For the next generation of immunoconjugates, advances in protein engineering will permit greater control of mAb targeting, clearance and pharmacokinetics, resulting in significantly improved delivery to tumors of radioisotopes and potent anticancer drugs. Pre-targeting strategies, which separate the two functions of antibody-based localization and delivery or generation of the toxic agent into two steps, also promise to afford superior tumor targeting and therapeutic efficacy. Several challenges in optimizing immunoconjugates remain, however, including poor intratumoral mAb uptake, normal tissue conjugate exposure and issues surrounding drug potency and conditional release from mAb carriers. Nonetheless, highly promising results from preclinical models will continue to drive the clinical development of this therapeutic class.

Arming antibodies: prospects and challenges for immunoconjugates

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

Dual variable domain immunoglobulins and uses thereof

Small biomolecules are typically radiolabeled with 18F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein 18F is first attached to aluminum as Al18F, which is then bound to a chelate attached to a peptide, forming a stable Al18F-chelate-peptide complex in an efficient 1-pot process. Methods: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl3·6H2O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of 18F to form the Al18F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH2) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100°C for 15 min. In vitro and in vivo stability and targeting ability of the Al18F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). Results: The radiolabeled peptide was produced in 5%−20% yield with an estimated specific activity of 18,500–48,100 GBq (500–1,300 Ci)/mmol. The Al18F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al18F had higher tissue uptake, particularly in the bones, than the chelated Al18F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al18F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. Conclusion: The ability to bind highly stable Al18F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

https://jnm.snmjournals.org/content/jnumed/50/6/991.full.pdf

A Novel Method of 18F Radiolabeling for PET

Improved 18F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex

The use of antibodies against tumor-associated cell surface antigens for the targeted delivery of radionuclides was introduced >20 years ago. Although encouraging results have been achieved with radiolabeled antibodies in the management of hematopoietic malignancies, there remains a need for successfully treating solid tumors with this modality. One promising approach involving pretargeted delivery of radionuclides has been shown to be capable of significantly increasing the radioactive uptake in tumor relative to normal organs, thereby potentially improving the efficacy of both detection and therapy of cancer. Uncoupling of the radionuclide from the tumor-targeting antibody allows the relatively slow process of antibody localization and clearance to occur before a very rapid and highly specific delivery of the radioactive payload carried on a small molecule, such as a peptide. This minireview discusses the various strategies and advancements made since the concept of pretargeting was proposed in the mid-1980s, with emphasis on those comprising bispecific antibodies for cancer therapy. Critical aspects of these pretargeting systems for achieving higher tumor:nontumor ratios are considered. In addition, both preclinical and clinical results obtained from a pretargeting method known as the Affinity Enhancement System are presented. Future directions of pretargeting technology are also suggested.

https://mct.aacrjournals.org/content/molcanther/1/7/553.full.pdf

Molecular advances in pretargeting radioimunotherapy with bispecific antibodies.

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody.

The article reviews the background and current status of pretargeting for cancer imaging and therapy with radionuclides. Pretargeting procedures were introduced approximately 20 years ago as an alternative to directly radiolabeled antibodies. Because they were multistep processes, they were met with resistance but have since progressed to simple and improved procedures that could become the next generation of imaging and therapy with radionuclides. The separation of the radiolabeled compound from the antibody-targeting agent affords pretargeting procedures considerable flexibility in the radiolabeling process, providing opportunities for molecular imaging using gamma- or positron-emitting radionuclides and a variety of beta- and alpha-emitting radionuclides of therapeutic applications. Pretargeting methods improve tumor/nontumor ratios, exceeding that achieved with directly radiolabeled Fab' fragments, particularly within just a few hours of the radionuclide injection. In addition, tumor uptake exceeds that of a Fab' fragment by as much as 10-fold, giving pretargeting a greatly enhanced sensitivity for imaging. Advances in molecular biology have led to the development of novel binding proteins that have further improved radionuclide delivery in these systems. Studies in a variety of hematologic and solid tumor models have shown advantages of pretargeting compared with directly radiolabeled IgG for therapy, and there are several clinical studies under way that are also showing promising results. Thus, the next generation of targeting agents will likely employ pretargeting approaches to optimize radionuclide delivery for a wide range of applications.

https://clincancerres.aacrjournals.org/content/clincanres/11/19/7109s.full.pdf

Habibe Karacay

Papers

A Novel Method of 18F Radiolabeling for PET

Improved 18F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex

Molecular advances in pretargeting radioimunotherapy with bispecific antibodies.

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody.

Improving the Delivery of Radionuclides for Imaging and Therapy of Cancer Using Pretargeting Methods