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Introduction: Molecular imaging of HER2-expression in malignant tumors provides potentially important information for patient management. Affibody molecules have shown to be suitable tracers for imaging applications using SPECT or PET. Results from an earlier evaluation of the application of site specific ^99mTc-labeling on the Affibody molecule, Z_HER2:2395-C, were favorable.

Methods: As a preparation for clinical application of this tracer we have developed and evaluated a robust single-vial freeze-dried kit, allowing labeling of the Affibody molecule, Z_HER2:2395-C, with ^99mTc.

Results: The composition of the kit (containing glucoheptonate, EDTA and tin(II)-chloride), as well as the protein amount and the pertechnetate volume were optimized for a high labeling yield (> 90 %) and minimal presence of reduced hydrolyzed technetium colloids (< 1 %). The specificity to HER2 receptors, the binding competence and the stability in PBS and murine serum were verified in vitro. The shelf-life was also evaluated in vitro, showing no reduction in labeling yield or binding capacity to HER2-expressing cells after over 400 days of storage of the single-vial freeze-dried kit.

Conclusions: Z_HER2:2395-C labeled with ^99mTc using the lyophilized kit was stable and resulted in a favorable biodistribution in an in vivo evaluation in normal NMRI mice.

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.

The current way to increase efficacy of cancer therapy is the use of molecular recognition of aberrantly expressed gene products for selective treatment. However, only a fraction of the patients have tumors with a particular molecular target. Radionuclide imaging of molecular targets might help to stratify patient for cancer treatment. Affibody molecules are scaffold proteins, which can be selected for high affinity recognition of proteinaceous molecular targets. The capacity to re-fold under physiological conditions allows labeling of Affibody molecules in a broad range of pH and temperatures with preserved binding properties. Peptide synthesis or introduction of a unique cysteine enables site-specific labeling of Affibody molecules, resulting in uniform conjugates with well-defined pharmacological characteristics. The small size (7 kDa) of Affibody molecules provides rapid extravasation, rapid tumor penetration, and rapid clearance of unbound tracer from healthy organs and tissues. In combination with sub-nanomolar affinity, this results in high contrast in vivo imaging a few hours after injection. Excellent targeting has been demonstrated in pre-clinical studies with HER2-targeting Affibody molecules labeled with (99m)Tc and (111)In for single photon computed tomography (SPECT), and (18)F, (64)Cu, (124)I and (68)Ga for positron emission tomography (PET). Pilot clinical data confirm the high potential of Affibody molecules.

The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. METHODS: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. RESULTS: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

The humanized antibody A33 binds to the A33 antigen, expressed in 95% of primary and metastatic colorectal carcinomas. The restricted pattern of expression in normal tissue makes this antigen a possible target for radioimmunotherapy of colorectal micrometastases. In this study, the A33 antibody was labeled with the therapeutic nuclide ²¹¹At using N-succinimidyl para-(tri-methylstannyl)benzoate (SPMB). The in vitro characteristics of the ²¹¹At-benzoate-A33 conjugate (²¹¹At-A33) were investigated and found to be similar to those of ¹²⁵I-benzoate-A33 (¹²⁵I-A33) in different assays. Both conjugates bound with high affinity to SW1222 cells (K_d = 1.7 ± 0.2 nM, and 1.8 ± 0.1 nM for ²¹¹At-A33 and ¹²⁵I-A33, respectively), and both showed good intracellular retention (70% of the radioactivity was still cell associated after 20 hours). The cytotoxic effect of ²¹¹At-A33 was also confirmed. After incubation with ²¹¹At-A33, SW1222 cells had a survival of approximately 0.3% when exposed to some 150 decays per cell (DPC). The cytotoxic effect was found to be dose-dependent, as cells exposed to only 56 DPC had a survival of approximately 5%. The ²¹¹At-A33 conjugate shows promise as a potential radioimmunotherapy agent for treatment of micrometastases originating from colorectal carcinoma.

Radioimmunotherapy (RIT) could be a possible adjuvant treatment method for patients with colorectal carcinoma. The A33 antigen is a promising RIT target, as it is highly and homogenously expressed in 95% of all colorectal carcinomas. In this study, the humanized monoclonal antibody A33 (huA33), targeting the A33 antigen, was labeled with the therapeutic nuclide 211At, and the biodistribution and in vivo targeting ability of the conjugate was investigated in an athymic mouse xenograft model. There was an accumulation of 211At in tumor tissue over time, but no substantial accumulation was seen in any organ apart from the skin and thyroid, indicating no major release of free 211At in vivo. At all time points, the uptake of 211At-huA33 was higher in tumor tissue than in most organs, and at 8 hours postinjection (p.i.), no organ had a higher uptake than tumor tissue. The tumor-to-blood ratio of 211At-huA33 increased with time, reaching 2.5 after 21 hours p.i. The highest absorbed dose was found in the blood, but the tumor received a higher dose than any organ other than the thyroid. An in vivo blocking experiment showed that 211At-huA33 binds specifically to human tumor xenografts in athymic mice. In conclusion, the favorable biodistribution and specific in vivo targeting ability of 211At-huA33 makes it a potential therapeutic agent for the RIT of metastatic colorectal carcinoma.

INTRODUCTION: The humanized monoclonal antibody A33 (huA33) is a potential targeting agent against colorectal carcinoma since the A33 antigen is highly and homogenously expressed in >95% of all colorectal cancers, both primary tumors and metastases. The aim of this study was to determine the biodistribution and tumor-targeting ability of (177)Lu-labeled huA33. METHODS: huA33 was labeled with the beta-emitting therapeutic nuclide (177)Lu using the chelator CHX-A"-DTPA, and the properties of the (177)Lu-CHX-A"-huA33 ((177)Lu-huA33) conjugate was determined both in vitro and in vivo in a biodistribution study in nude mice xenografted with colorectal SW1222 tumor cells. RESULTS: The (177)Lu-huA33 conjugate bound specifically to colorectal cancer cells in vitro (with a K(D) value of 2.3+/-0.3 nM, determined by a saturation assay) and in vivo. The tumor uptake of (177)Lu-huA33 was very high, peaking at 134+/-21%ID/g 72 h postinjection (pi). Normal tissue uptake was low; radioactivity concentration in blood (which had the second highest radioactivity concentration) was lower than in tumor at all time points studied (8 h to 10 days). The tumor-to-blood ratio increased with time, reaching 70+/-30, 10 days pi. Throughout the study, the uptake of (177)Lu in bone (known to accumulate free (177)Lu) was low, and the fraction of protein-bound (177)Lu in plasma samples was high (95% to 99%). This indicates high stability of the (177)Lu-huA33 conjugate in vivo. CONCLUSION: The (177)Lu-huA33 conjugate shows a very favorable biodistribution, with an impressively high tumor uptake and high tumor-to-organ ratios, indicating that the conjugate may be suitable for radioimmunotherapy of colorectal cancer.

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry.

Paper I and II report the comparative evaluation of the anti-HER2 Z_HER2:2395 affibody molecule site specifically labelled with ¹¹¹In (suitable for SPECT imaging) and ⁶⁸Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized.

Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 Z_HER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of Z_HER2:2395 molecules in the kidneys.

Paper IV reports the optimization and development of in vivo targeting properties of ¹⁸⁸Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of ¹⁸⁸Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties.

Paper V reports the preclinical evaluation of ¹⁸⁸Re-Z_HER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using ¹⁸⁸Re-Z_HER2:V2 may be feasible.

It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.