The positron emitter zirconium-89 ((89)Zr) has very attractive properties for positron emission tomography (PET) imaging of intact monoclonal antibodies (mAbs) using immuno-PET. This protocol describes the step-by-step procedure for the facile radiolabeling of mAbs or other proteins with (89)Zr using p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). First, Df-Bz-NCS is coupled to the lysine-NH(2) groups of a mAb at pH 9.0 (pre-modification), followed by purification using gel filtration. Next, the pre-modified mAb is labeled at room temperature by the addition of [(89)Zr]Zr-oxalic acid solution followed by purification using gel filtration. The entire process of pre-modification, radiolabeling and purification steps will take about 2.5 h.

PurposeImmuno-PET is an emerging imaging tool for the selection of high potential antibodies (mAbs) for imaging and therapy. The positron emitter zirconium-89 (89Zr) has attractive characteristics for immuno-PET with intact mAbs. Previously, we have described a multi-step procedure for stable coupling of 89Zr to mAbs via the bifunctional chelate (BFC) tetrafluorophenol-N-succinyldesferal (TFP-N-sucDf). To enable widespread use of 89Zr-immuno-PET, we now introduce the novel BFC p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS) and compare its performance in 89Zr-immuno-PET with the reference BFC TFP-N-sucDf.MethodsThree mAbs were premodified with Df-Bz-NCS and labeled with 89Zr at different pHs to assess the reaction kinetics and robustness of the radiolabeling. Stability of both 89Zr-Df-Bz-NCS- and 89Zr-N-sucDf-conjugates was evaluated in different buffers and human serum. Comparative biodistribution and PET studies in tumor-bearing mice were undertaken.ResultsThe selected conjugation conditions resulted in a chelate:mAb substitution ratio of about 1.5:1. Under optimal radiolabeling conditions (pH between 6.8–7.2), the radiochemical yield was >85% after 60 min incubation at room temperature, resulting in radioimmunoconjugates with preserved integrity and immunoreactivity. The new radioimmunoconjugate was very stable in serum for up to 7 days at 37°C, with <5% 89Zr release, and was equally stable compared to the reference conjugate when stored in the appropriate buffer at 4°C. In biodistribution and imaging experiments, the novel and the reference radioimmunoconjugates showed high and similar accumulation in tumors in nude mice.ConclusionsThe novel Df-Bz-NCS BFC allows efficient and easy preparation of optimally performing 89Zr-labeled mAbs, facilitating further exploration of 89Zr-immuno-PET as an imaging tool.

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies (mAbs) have been successfully used, among which cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In the present study, we aimed to improve on these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single bi-paratopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or of cetuximab to EGFR and that did not compete for each others binding. A combination of nanobodies from both epitope groups into the bi-paratopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this bi-paratopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, mono-specific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of bi-paratopic nanobody-based anti-cancer therapeutics may yield potent lead molecules for further development.

Purpose: Immuno^positron emission tomography (PET), the combination of PET with monoclonal antibodies (mAb), is an attractive option to improve tumor detection and to guide mAb-based therapy. The long-lived positron emitter zirconium-89 ( 89 Zr) has ideal physical characteristics for immuno-PET with intact mAbs but has never been used in a clinical setting. In the present feasibility study, we aimed to evaluate the diagnostic imaging performance of immuno-PET with 89 Zr-labeled-chimeric mAb (cmAb) U36 in patients with squamous cell carcinoma of the head and neck (HNSCC), who were at high risk of having neck lymph node metastases. Experimental Design: Twenty HNSCC patients, scheduled to undergo neck dissection with or without resection of the primary tumor, received 75 MBq 89 Zr coupled to the anti-CD44v6 cmAb U36 (10 mg). All patients were examined by computed tomography (CT) and/or magnetic resonance imaging (MRI) and immuno-PET before surgery. Six patients also underwent PET with 18 F-fluoro-2-deoxy-D-glucose. Immuno-PETscans were acquired up to 144 hours after injection. Diagnostic findings were recorded per neck side (left or right) as well as per lymph node level (six levels per side), and compared with histopathologic outcome. For this purpose, the CT/MRI scores were combined and the best of both scores was used for analysis. Results: Immuno-PETdetected all primary tumors (n = 17) as well as lymph node metastases in 18 of 25 positive levels (sensitivity 72%) and in 11of 15 positive sides (sensitivity 73%). Interpretation of immuno-PET was correct in 112 of 121 operated levels (accuracy 93%) and in 19 of 25 operated sides (accuracy 76%). For CT/MRI, sensitivities of 60% and 73% and accuracies of 90% and 80% were found per level and side, respectively. In the six patients with seven tumorinvolved neck levels and sides, immuno-PETand 18 F-fluoro-2-deoxy-D-glucose PETgave comparable diagnostic results. Conclusion: In this study, immuno-PET with 89 Zr-cmAb U36 performed at least as good as CT/MRI for detection of HNSCC lymph node metastases.

Antibody-PET imaging might be of value for the selection of radioimmunotherapy (RIT) candidates to confirm tumor targeting and to estimate radiation doses to tumor and normal tissues. One of the requirements to be set for such a scouting procedure is that the biodistributions of the diagnostic and therapeutic radioimmunoconjugates should be similar. In the present study we evaluated the potential of the positron emitters zirconium-89 ((89)Zr) and iodine-124 ((124)I) for this approach, as these radionuclides have a relatively long half-life that matches with the kinetics of MAbs in vivo (t(1/2) 3.27 and 4.18 days, respectively). After radiolabeling of the head and neck squamous cell carcinoma (HNSCC)-selective chimeric antibody (cMAb) U36, the biodistribution of two diagnostic (cMAb U36-N-sucDf-(89)Zr and cMAb U36-(124)I) and three therapeutic radioimmunoconjugates (cMAb U36-p-SCN-Bz-DOTA-(88)Y-with (88)Y being substitute for (90)Y, cMAb U36-(131)I, and cMAb U36-MAG3-(186)Re) was assessed in mice with HNSCC-xenografts, at 24, 48, and 72 hours after injection. Two patterns of biodistribution were observed, one pattern matching for (89)Zr- and (88)Y-labeled cMAb U36 and one pattern matching for (124)I-, (131)I-, and (186)Re-cMAb U36. The most remarkable differences between both patterns were observed for uptake in tumor and liver. Tumor uptake levels were 23.2 +/- 0.5 and 24.1 +/- 0.7%ID/g for the (89)Zr- and (88)Y-cMAb U36 and 16.0 +/- 0.8, 15.7 +/- 0.79 and 17.1 +/- 1.6%ID/g for (124)I-, (131)I-, and (186)Re-cMAb U36-conjugates, respectively, at 72 hours after injection. For liver these values were 6.9 +/- 0.8 ((89)Zr), 6.2 +/- 0.8 ((88)Y), 1.7 +/- 0.1 ((124)I), 1.6 +/- 0.1 ((131)I), and 2.3 +/- 0.1 ((186)Re), respectively. These preliminary data justify the further development of antibody-PET with (89)Zr-labeled MAbs for scouting of therapeutic doses of (90)Y-labeled MAbs. In such approach (124)I-labeled MAbs are most suitable for scouting of (131)I- and (186)Re-labeled MAbs.

(89)Zr-Zevalin and (88)Y-Zevalin showed a very similar biodistribution in mice, implying that (89)Zr-Zevalin-PET might be well suited for prediction of (90)Y-Zevalin biodistribution in a myeloablative setting.

Hepatocyte growth factor (HGF) and its receptor c-Met are associated with increased aggressiveness of tumors and poor prognostic outcome of patients with cancer. Here, we report the development and characterization of therapeutic anti-HGF (aHGF)-Nanobodies and their potential for positron emission tomographic (PET) imaging to assess HGF expression in vivo. Two aHGF-Nanobodies designated 1E2 and 6E10 were identified, characterized, and molecularly fused to an albumin-binding Nanobody unit (Alb8) to obtain serum half-life extension. The resulting Nanobody formats were radiolabeled with the positron emitter zirconium-89 ( 89 Zr, t1/ 2 ¼ 78 hours), administered to nude mice bearing U87 MG glioblastoma xenografts, and their biodistribution was assessed. In addition, their therapeutic effect was evaluated in the same animal model at doses of 10, 30, or 100 mg per mouse. The 89 Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showed decreased blood levels of 12.6%ID/g AE 0.6%ID/g, 7.2%ID/g AE 1.0%ID/g, 3.4%ID/g AE 0.3%ID/g, and 0.3%ID/g AE 0.1%ID/g at 1, 2, 3, and 7 days after injection, whereas tumor uptake levels remained relatively stable at these time points: 7.8%ID/g AE 1.1%ID/g, 8.9%ID/g AE 1.0%ID/g, 8.7%ID/g AE 1.5%ID/g, and 7.2%ID/g AE1.6%ID/g. Uptake in normal tissues was lower than in tumor, except for kidneys. In a therapy study, all Nanobody-treated mice showed tumor growth delay compared with the control saline group. In the 100-mg group, four of six mice were cured after treatment with 1E2-Alb8 and 73 days follow-up, and three of six mice when treated with 6E10-Alb8. These results provide evidence that Nanobodies 1E2-Alb8 and 6E10-Alb8 have potential for therapy and PET imaging of HGFexpressing tumors.

Background:The atypical chemokine receptor CXCR7 is highly expressed in various types of cancer. Results: CXCR7 Nanobodies were generated and show inhibition of ␤-arrestin2 signaling and secretion of angiogenic CXCL1 in vitro. Anti-CXCR7 Nanobodies reduce tumor growth by inhibiting angiogenesis. Conclusion: CXCR7 inhibition by Nanobodies inhibit head and neck tumor formation. Significance: Anti-CXCR7 therapies are potential novel treatments against head and neck cancer.