Strategies for Site-Specific Radiolabeling of Peptides and Proteins

*Ingrid Dijkgraaf, Stijn M. Agten, Matthias Bauwens and Tilman M. Hackeng*

## **Abstract**

Although anatomical imaging modalities (X-ray, computed tomography (CT), magnetic resonance imaging (MRI)) still have a higher spatial resolution (0.1–1 mm) than molecular imaging modalities (single-photon emission computed tomography (SPECT), positron emission tomography (PET), optical imaging (OI)), the advantage of molecular imaging is that it can detect molecular and cellular changes at the onset of a disease before it leads to morphological tissue changes, which can be detected by anatomical imaging. During the last decades, noninvasive diagnostic imaging has encountered a rapid growth due to the development of dedicated imaging equipment for preclinical animal studies. In addition, the introduction of multimodality imaging (PET/CT, SPECT/CT, PET/MRI) which combines high-resolution conventional anatomical imaging with high sensitivity of tracer-based molecular imaging techniques has led to successful accomplishments in this exciting field. In this book chapter, we will focus on chemical synthesis techniques for site-specific incorporation of radionuclide chelators. Subsequently, radiolabeling based on complexation of a radionuclide with a chelator will be discussed, with focus on: diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-triacetic acid (NOTA), hexa-histidine (His-tag), and 6-hydrazinonicotinic acid (HYNIC) that allow the production of peptides labeled with 18F, 68Ga, 99mTc, and 111In – the currently most widely used isotopes.

**Keywords:** radiolabeled peptides, chelator, PET, SPECT, radionuclide, peptide synthesis, protein synthesis

### **1. Introduction**

#### **1.1 Application of peptides and proteins as molecular imaging agents**

The concept of using radiolabeled receptor-binding peptides and proteins to target receptor-(over)expressing tissues *in vivo* has stimulated a large body of research in nuclear medicine. Peptides and small proteins for receptor imaging and targeted radiotherapy have particular advantages over antibodies and antibody fragments. Peptides are small molecules and show rapid diffusion in target tissue. They rapidly clear from the blood and non-target tissues, resulting in high target-to-background ratios. Furthermore, peptides have a low toxicity and are generally not immunogenic. However, ubiquitously occurring amino- and carboxypeptidases in the circulation will rapidly degrade most peptides preventing intact imaging agents to reach the target tissue in sufficient quantities. Thus, to prevent rapid enzymatic degradation of peptide-based imaging agents, most peptides have to be modified [1, 2]. Several methods to prevent enzymatic peptide proteolysis have been developed, including substitution of L- by D-amino acids, replacement of amino moieties by imino groups, substitution of peptide bonds, insertion of unusual amino acids or side chains, amidation, cyclization, *C*-terminal amidation or reduction, *N*-terminal acylation or methylation, and use of peptidomimetics. Cyclization of peptides results not only in resistance to enzymatic degradation, it can also lead to conformationally more constrained compounds with enhanced receptor affinity and biological activity [1, 3].

Besides stability toward enzymes, lipophilicity is very important. The preferred route of clearance of a peptide-based radiopharmaceutical is via the kidneys. For targeting of tumors, cardiovascular diseases, and infections or inflammation, the lipophilicity of the compound should not be too high (log P < 1) as lipophilic compounds result in non-specific binding and slower blood clearance via mainly the hepatobiliary route. In contrast, molecular imaging tracers that target brain diseases such as Alzheimer require a higher lipophilicity (log P > 1) in order to cross the blood–brain barrier (BBB). Lipophilicity of molecular imaging tracers can be reduced by linking them to polyethylene glycol (PEG) chains, a technique called PEGylation. An alternative method to reduce lipophilicity of a tracer is attachment of carbohydrates, as this enhances the hydrophilicity, resulting in reduced hepatobiliary uptake, enhanced urinary excretion, and reduced nonspecific binding [4].

Furthermore, conjugation of chelators like DOTA (1,4,7,10-tetraazacyclododecane-tetraacetic acid), NOTA (1,4,7-triazacyclononane-triacetic acid), or DTPA (diethylenetriaminepentaacetic acid) also reduce the lipophilicity of an imaging agent. However, modification of a tracer by for example PEGylation, glycosylation or conjugation to a chelator, can also affect the affinity of a peptide for the receptor and thus the effectiveness of the radiotracer.

#### **1.2 Introduction of bifunctional chelating agents (BFCAs) into the peptide or protein**

A chelating agent will not only influence the hydrophilicity of a peptide or protein, but it will also increase the overall size of the radiotracer and thus the pharmacokinetics. To preserve biological activity and receptor-binding affinity, conjugation of a chelator must be performed at a site remote from the active and receptor-binding region of the tracer [5]. Total chemical protein synthesis enables single site-specific protein modification, which cannot be achieved through regular labeling methods of biologically obtained proteins. To prevent interference of the chelator with the active and receptor-binding region of the peptide, introduction of a linker may be necessary. These linkers (PEG chains, amino acids, aliphatic hydrocarbon chains, etc.) can be used as pharmacokinetic modifiers (PKMs) to adjust the pharmacokinetics of the probe.

Several acyclic and cyclic bifunctional chelators have been developed for both diagnostic and therapeutic applications (**Figure 1**). A bifunctional chelator is a molecule which can be covalently coupled to the targeting compound and has the ability to chelate a (radio)metal. The most widely used chelators are DTPA, DOTA, and NOTA or derivatives thereof. A chelator should effectively sequester the radionuclide in high-yields (quantitative) and with high stability. Unstable complexation of the radionuclide by the chelator can lead to trans-chelation of the radionuclide

*Strategies for Site-Specific Radiolabeling of Peptides and Proteins DOI: http://dx.doi.org/10.5772/intechopen.99422*

**Figure 1.** *Structural formula of different chelators and co-ligands for radiolabeling peptides and proteins.*

to blood proteins and enzymes (e.g. transferrin, ceruloplasmin, superoxide dismutase). For a detailed review of chelating agents and the optimal match between chelator and radionuclide see Price *et al*. 2014 [6] and references therein.

The introduction of BFCA in proteins or peptides can be achieved using bioconjugation methods based on reactive functional groups, such as amide coupling (carboxylic acids and their activated *N*-hydroxysuccinimide (NHS) esters), thiol couplings (maleimides), oxime bond formation (ketones or aldehydes with aminooxy), and Cu(I) catalyzed azide-alkyne Huisgen 1,3-dipolar cycloaddition "click reactions". We will discuss some of the methods in detail in Section 2.3.

#### **1.3 Radionuclide imaging**

Apart from planar imaging, SPECT and PET are the two main imaging modalities in nuclear medicine. SPECT imaging is much more widely available than PET imaging and the radionuclides used for SPECT are easier to prepare, financially generally more accessible, and usually have a longer half-life than those used for PET (**Table 1**). Commonly used gamma emitters are: 123I (Emax 529 keV, t1/2 13.0 h), 111In (Emax 245 keV, t1/2 67.2 h), and 99mTc (Emax 141 keV, t1/2 6.02 h). Compared to SPECT, PET has the possibility to more accurately quantitate the *in vivo* concentration of a tracer labeled with a positron emitting radionuclide, such as for (pre)clinical applications 18F (Emax 635 keV, t1/2 1.83 h), 68Ga (Emax 1.90 MeV, t1/2 68.1 min), 64Cu (657 keV, t1/2 12.7 h), and 124I (Emax 2.13 MeV; 1.53 MeV; 808 keV, t1/2 4.18 days).

PET is independent of the location depth of the reporter probe of interest and is able to detect picomolar concentrations of tracer [7]. This high sensitivity of PET can only be matched to some degree by optical imaging (OI) techniques, but not by MRI, CT or ultrasound (US). In addition, compared to MRI and conventional optical imaging techniques, PET has the advantage of being quantitative. Though, with the introduction of fluorescence mediated tomography (FMT), quantitative measurements are also possible with OI techniques [8].


*Half-life is given in hours, unless stated otherwise***.** β*<sup>−</sup> = negative beta decay,* β*<sup>+</sup> = positive beta decay,* γ *= gamma transition, IT = isometric transition, EC = electron capture.*

#### **Table 1.**

*Half-life and decay type of several radionuclides.*

Recent developments also allow semi-quantitative measurements with SPECT, but these developments are not yet widespread and still show higher uncertainties compared to PET.

The spatial resolution of PET and SPECT scanners depends on several factors: the type of isotope (PET or SPECT), the energy of the isotope emissions, and the object being scanned. The type of isotope (positron-emitting or single-photon emitting) has a strong impact, as the image reconstruction techniques for PET are superior to those of SPECT due to physical characteristics in large objects, but this is reversed for small objects. The energy of the isotope emissions is negatively correlated to the spatial resolution: the stronger the energy, the poorer the spatial resolution. The object being scanned has a substantial impact: spatial resolution in mice is vastly superior to that in humans, and even within humans spatial resolution in obese people is worse compared to healthy subjects. Some typical spatial resolutions are: 99mTc, mouse: 0.5 mm; 99mTc, human: 10 mm; 18F, mouse: 0.8 mm; 18F, human: 2 mm; 68Ga, human and mouse: both 4 mm. The spatial resolution should be taken into account when designing studies.

#### **2. Peptide and protein synthesis**

#### **2.1 Protein production by expression systems**

Nowadays, recombinant protein expression is a routine laboratory technology that enables fast and high-yield protein production. The choice of bacterial, yeast, insect or mammalian cellular-based expression system depends on several factors such as, cell growth characteristics, intracellular and extracellular expression, posttranslational modifications, and regulatory issues of proteins used as diagnostics and therapeutics. Recently, even cell-free expression systems using purified RNA polymerase, ribosomes, tRNA and ribonucleotides have been developed [9]. Each expression system has its particular advantages and disadvantages that are relevant for the purpose of use. Several review papers give a good description of the variety of expression systems and their pros and cons. However, for development

of target-specific radiotracers, fluorescent probes, or multimodality molecular imaging agents, chemical protein synthesis is the method of choice because of reasons described below. Therefore, this book chapter does not cover recombinant expression systems further.
