HER2-Positive Tumors Imaged Within 1 Hour Using a Site-Specifically ¹¹C-Labeled Sel-Tagged Affibody Molecule

Abstract

A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, Z HER2:342 , was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either 11 C or 68 Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the 11 C-labeled Affibody molecule. Results: Both the 11 C-and 68 Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (.5-fold) for the 11 C-labeled protein, and its overall absorbed dose was considerably lower. 11 C-Z HER2:342 showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P 5 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables sitespecific 11 C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, 11 C-labeled Sel-tagged Z HER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors. Overexpressi on of the human epidermal growth factor receptor 2 (HER2), found in about 20%-30% of breast cancers, is associated with poor prognosis, and HER2 is the molecular target for effective but expensive monoclonal antibody drugs such as trastuzumab (Herceptin; Roche) (1). Determination of the expression levels of HER2 is therefore important for predicting prognosis and for patient stratification for treatment. Currently, expression levels are mainly determined using biopsies for molecular assays such as in situ hybridization (2). These procedures are, however, invasive, are difficult to perform repeatedly, and risk missing overexpressing tissue due to sampling errors. Therefore, the radionuclide-based in vivo molecular imaging of HER2, primarily using polypeptide probes, is rapidly expanding, aiming for personalized medicine with image-and-treat strategies (3-6). Speed, sensitivity, and specificity are necessary for implementation in clinical routines.Comparing nuclear medicine imaging techniques, PET has better spatial resolution, higher sensitivity, and more accurate quantification than SPECT. With recent radiolabeling advances, the feasibility of using polypeptides...

Antibody Fragment and Affibody ImmunoPET Imaging Agents: Radiolabelling Strategies and Applications

“…Despite its short 20 min half‐life, carbon‐11 has been used to radiolabel a HER2 targeting affibody and a scFv. The affibody labelling route exploited a C‐terminal selenocysteine tetrapeptide Sel‐Tag, that enabled site‐specific labelling using the common 11 C‐labelling reagent, [ 11 C]methyl iodide within 45 min (Scheme ) . This labelling method, using the shorter 11 C half‐life, may be promising for the rapid and repeated monitoring of HER2 expression levels in tumours, and also for monitoring of responses to therapeutic treatment over time using lower doses of radioactivity.…”

Alternative non-antibody protein scaffolds for molecular imaging of cancer

“…The most extensively studied class of affibodies is those targeting human epidermal growth factor receptor 2 (HER2). The second generation HER2-binding affibody, Z HER2:342 , was engineered to bind with 22 pM affinity [5], and has successfully imaged HER2-expressing tumor xenografts in mice when labeled with 125 I [5], 111 In [6], 99m Tc [7], 18 F [8], 114m In [9], 124 I [10], 68 Ga [11], and 11 C [12]. Tumor targeting is effective across many mouse models with a median tumor uptake of 9 %ID/g at 4 h (range: 1 – 26 %ID/g), tumor-to-blood ratio median of 31 (range: 1.5 – 187), and tumor-to-muscle ratio median of 61 (range; 3 – 650).…”

Design and evaluation of radiolabeled tracers for tumor imaging

“…The only reported way to site‐specifically label proteins with 11 C is by utilizing a Sel‐tag, a C‐terminal tetrapeptide motif that contains a selenocysteine (Sec, U). In its reduced state, the highly reactive nucleophile Sec will react with electrophiles like [ 11 C]methyl iodide (CH 3 I) allowing for rapid, site‐specific labeling of Sel‐tagged proteins .…”