The specificity and affinity of recombinant proteins and antibodies targeted towards antigens make them highly attractive as a basis for radiopharmaceuticals for molecular imaging. To retain these key attributes, it is essential not to compromise the recognition function of the protein when radiolabelling. This can be achieved using site-specific labelling methods that exert maximum control over the number and site of modification(s) to the molecule, while still maintaining protein function. Such methodologies should give rise to homogeneous conjugates with reproducible chemical and pharmacological properties. They must ensure that the conjugation of the radiochelate is outside of the target binding site or at a distinct site known not to affect antigen binding. Ideally, radiolabelling would be achieved rapidly, under mild conditions, to a high specific activity and preferably in a simple one-pot kit-based method without the need for subsequent purification steps.
Waibel et al. [
1] developed an elegant method that permits radiolabelling of proteins engineered with sequences of additional histidine residues known as His-tags. The His-tag was originally developed to facilitate purification of recombinant proteins using metal chelate-based affinity chromatography. Radiolabelling of His-tags could be achieved with technetium-99 m (
99mTc) in its stable + I oxidation state in the form of
99mTc-tricarbonyl ([
99mTc(CO)
3]
+). This organometallic complex is produced in the form of its aqua ion [
99mTc(CO)
3(H
2O)
3]
+ in a one-step reaction by reduction of the generator-eluted form of
99mTc, sodium pertechnetate ([
99mTcO
4]
-). Histidine has been demonstrated to be the favoured [
99mTc(CO)
3]
+-binding ligand among amino acids and labelling efficiency and stability increase with increased number of engineered histidines [
2]. Direct labelling of non-His-tag proteins with [
99mTc(CO)
3]
+ has previously resulted in poor stability and low labelling efficiency and specific activity, indicating that other potential amino acid side chains donor groups such as thiol, thioether, carboxylate and amine do not make a significant contribution in the absence of histidines [
3‐
9]. Modification of the His-tag sequence from HHHHHH to HEHEHE in order to improve tracer biodistribution also resulted in reduced labelling efficiency [
10,
11]. In contrast, by engineering an additional cysteine seven amino acids downstream of the His-tag, Tavaré et al. demonstrated an improvement in labelling efficiency and specific activity compared to His-tag alone [
12] and showed for the first time that the radiolabelling is indeed site-specific to the His-tagged region of the protein by carrying out tryptic digest and mass spectrometry on a His-tagged protein labelled with [Re(CO)
3]
+. The rhenium complex was only present in His-tag-containing fragments [
12]. Furthermore, we recently demonstrated that the engineered cysteine that increases radiolabelling efficiency is also involved in the coordination of the rhenium tricarbonyl [
13]. Several variants on labelling conditions have been studied to optimise the use of [
99mTc(CO)
3]
+-labelling of His-tagged proteins, and there is a general agreement in the literature that labelling at neutral pH, high protein concentration and high temperature increase the rate of radiolabelling (see Additional file
1: Table S1) [
1,
2,
12,
14‐
26]. These conditions, however, exclude proteins that are susceptible to aggregation or loss of function at high temperatures and concentrations or require particularly high specific activity [
15,
23,
25]. To make this labelling chemistry readily accessible, a kit-based formulation has until recently been distributed by Mallinckrodt (subsidiary of Covidien, Petten, The Netherlands) under the trademarked name IsoLink. Since its introduction, IsoLink has supported the development of numerous protein-based imaging tracers. Despite the intent to provide a practical, simple and versatile radiolabelling method, there has been a large variation in labelling efficiencies reported in the literature, likely due to variations in protein/peptide properties and labelling conditions (see Additional file
1: Table S1), and no clinical trial has been performed to date with proteins labelled using the tricarbonyl labelling method.
The IsoLink kit comes in a 10-mL glass vial containing lyophilised reagents and is designed to reduce up to 3.7 GBq of [
99mTcO
4]
- in a final volume of 1 mL. With the current composition, to achieve maximum specific activity (crucial for
in vivo imaging), the protein must be added to this total volume, leading to low protein concentration and hence inefficient labelling [
12] and high wastage. Recombinant proteins used in R&D are often precious, produced in low yield and available in small quantities and may be difficult to concentrate due to aggregation, precipitation or loss on columns or membranes. Furthermore, for preclinical work, volumes above 200 μL are not desirable or feasible for injection into mice (one tenth of total blood volume). The current kit necessitates an avoidable and time-consuming protein concentration step or leads to low specific activity. Thus, while labelling via the [
99mTc(CO)
3]
+ method may be one of the most promising site-specific methods currently available for recombinant protein tracer development, the IsoLink kit in its current form is not optimal for routine use in preclinical research or for future clinical imaging. It needs to be optimised to achieve reproducible high labelling efficiencies (LE) and specific activities (SA) of recombinant proteins, without wasting large amounts of protein and
99mTc.
In this paper, we address how the kit could be further optimised to increase specific activity and radiolabelling efficiency of His-tagged proteins, at suitable labelling rates with reduced wastage of protein and radioactivity. We used four different His-tagged proteins to study the effect of various radiolabelling conditions.