PurposeAll clinical 89Zr-immuno-PET studies are currently performed with the chelator desferrioxamine (DFO). This chelator provides hexadentate coordination to zirconium, leaving two coordination sites available for coordination with, e.g., water molecules, which are relatively labile ligands. The unsaturated coordination of DFO to zirconium has been suggested to result in impaired stability of the complex in vivo and consequently in unwanted bone uptake of 89Zr. Aiming at clinical improvements, we report here on a bifunctional isothiocyanate variant of the octadentate chelator DFO* and the in vitro and in vivo comparison of its 89Zr-DFO*-mAb complex with 89Zr-DFO-mAb.MethodsThe bifunctional chelator DFO*-pPhe-NCS was prepared from previously reported DFO* and p-phenylenediisothiocyanate. Subsequently, trastuzumab was conjugated with either DFO*-pPhe-NCS or commercial DFO-pPhe-NCS and radiolabeled with Zr-89 according to published procedures. In vitro stability experiments were carried out in saline, a histidine/sucrose buffer, and blood serum. The in vivo performance of the chelators was compared in N87 tumor-bearing mice by biodistribution studies and PET imaging.ResultsIn 0.9 % NaCl 89Zr-DFO*-trastuzumab was more stable than 89Zr-DFO-trastuzumab; after 72 h incubation at 2-8 °C 95 % and 58 % intact tracer were left, respectively, while in a histidine-sucrose buffer no difference was observed, both products were ≥ 92 % intact. In vivo uptake at 144 h post injection (p.i.) in tumors, blood, and most normal organs was similar for both conjugates, except for skin, liver, spleen, ileum, and bone. Tumor uptake was 32.59 ± 11.95 and 29.06 ± 8.66 % ID/g for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, respectively. The bone uptake was significantly lower for 89Zr-DFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. At 144 h p.i. for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, the uptake in sternum was 0.92 ± 0.16 and 3.33 ± 0.32 % ID/g, in femur 0.78 ± 0.11 and 3.85, ± 0.80 and in knee 1.38 ± 0.23 and 8.20 ± 2.94 % ID/g, respectively. The uptake in bone decreased from 24 h to 144 h p.i. about two fold for the DFO* conjugate, while it increased about two fold for the DFO conjugate.ConclusionsZr-DFO*-trastuzumab showed superior in vitro stability and in vivo performance when compared to 89Zr-DFO-trastuzumab. This makes the new octadentate DFO* chelator a candidate successor of DFO for future clinical 89Zr-immuno-PET.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-016-3499-x) contains supplementary material, which is available to authorised users.

Greater control is desirable in the stochastic conjugation technology used to synthesize antibody-drug conjugates (ADC). We have shown recently that a fluorescent dye can be stably conjugated to a mAb using a bifunctional platinum(II) linker. Here, we describe the general applicability of this novel linker technology for the preparation of stable and efficacious ADCs. The ethylenediamine platinum(II) moiety, herein called Lx, was coordinated to Desferal (DFO) or auristatin F (AF) to provide storable "semifinal" products, which were directly conjugated to unmodified mAbs. Conjugation resulted in ADCs with unimpaired mAb-binding characteristics, DAR in the range of 2.5 to 2.7 and approximately 85% payload bound to the Fc region, presumably to histidine residues. To evaluate the in vivo stability of Lx and its effect on pharmacokinetics and tumor targeting of an ADC, Lx-DFO was conjugated to the HER2 mAb trastuzumab, followed by radiolabeling with Zr. Trastuzumab-Lx-DFO-Zr was stable in vivo and exhibited pharmacokinetic and tumor-targeting properties similar to parental trastuzumab. In a xenograft mouse model of gastric cancer (NCI-N87) or an ado-trastuzumab emtansine-resistant breast cancer (JIMT-1), a single dose of trastuzumab-Lx-AF outperformed its maleimide benchmark trastuzumab-Mal-AF and FDA-approved ado-trastuzumab emtansine. Overall, our findings show the potential of the Lx technology as a robust conjugation platform for the preparation of anticancer ADCs. Cancer Res; 77(2); 257-67. ©2016 AACR.

Background: The non-homogenous distribution of antibody-drug conjugates (ADCs) within solid tumors is a major limiting factor for their wide clinical application. Nanobodies have been shown to rapidly penetrate into xenografts, achieving more homogeneous tumor targeting. However, their rapid renal clearance can hamper their application as nanobody drug conjugates (NDCs). Here, we evaluate whether half-life extension via non-covalent interaction with albumin can benefit the efficacy of a HER2-targeted NDC. Methods: HER2-targeted nanobody 11A4 and the irrelevant nanobody R2 were genetically fused to an albumin-binding domain (ABD) at their C-terminus. Binding to both albumin and tumor cells was determined by ELISA-based assays. The internalization potential as well as the in vitro efficacy of NDCs were tested on HER2 expressing cells. Serum half-life of iodinated R2 and R2-ABD was studied in tumor-free mice. The distribution of fluorescently labelled 11A4 and 11A4-ABD was assessed in vitro in 3D spheroids. Subsequently, the in vivo distribution was evaluated by optical molecular imaging and ex vivo by tissue biodistribution and tumor immunohistochemical analysis after intravenous injection of IRDye800-conjugated nanobodies in mice bearing HER2-positive subcutaneous xenografts. Finally, efficacy studies were performed in HER2-positive NCI-N87 xenograft-bearing mice intravenously injected with a single dose (250 nmol/kg) of nanobodies conjugated to auristatin F (AF) either via a maleimide or the organic Pt(II)‑based linker, coined L x ® . Results: 11A4-ABD was able to bind albumin and HER2 and was internalized by HER2 expressing cells, irrespective of albumin presence. Interaction with albumin did not alter its distribution through 3D spheroids. Fusion to ABD resulted in a 14.8-fold increase in the serum half-life, as illustrated with the irrelevant nanobody. Furthermore, ABD fusion prolonged the accumulation of 11A4-ABD in HER2-expressing xenografts without affecting the expected homogenous intratumoral distribution. Next to that, reduced kidney retention of ABD-fused nanobodies was observed. Finally, a single dose administration of either 11A4-ABD-maleimide-AF or 11A4-ABD- Lx -AF led to long-lasting tumor remission in HER2-positive NCI-N87 xenograft-bearing mice. Conclusion: Our results demonstrate that genetic fusion of a nanobody to ABD can significantly extend serum half-life, resulting in prolonged and homogenous tumor accumulation. Most importantly, as supported by the impressive anti-tumor efficacy observed after a single dose administration of 11A4-ABD-AF, our data reveal that monovalent internalizing ABD-fused nanobodies have potential for the development of highly effective NDCs.

Dozens of monoclonal antibodies (mAbs) have been approved for clinical use, and hundreds more are under development. To support these developments and facilitate a personalized medicine approach, PET imaging and quantification of mAbs, after chelation with desferrioxamine B (DFO) and radiolabeling with 89 Zr, has become attractive. Also, the use of 89 Zr-mAbs in preclinical and clinical studies is expanding rapidly. Despite these rapid developments, 89 Zr radiolabeling is still performed manually. Therefore, we aimed to develop a simple, fully automated, good-manufacturingpractice (GMP)-compliant production procedure for the 89 Zr labeling of mAbs. Such procedures should increase the robustness and capacity of 89 Zr-mAb production while minimizing the radiation dose to the operator. Here, the procedures for fully automated 89 Zr-mAb production are described and applied to produce batches of 89 Zr-DFO-N-suc-cetuximab and 89 Zr-DFO-N-suc-rituximab suitable for clinical use. Both products had to meet the GMP-compliant quality standards with respect to yield, radiochemical purity, protein integrity, antigen binding, sterility, and endotoxin levels. Methods: Automated 89 Zr labeling of mAbs was developed on a Scintomics GRP 2V module and comprised the following steps: reagent transfer to the 89 Zr-containing reaction vial, mixing of the reagents followed by a 60-min reaction at room temperature to obtain optimal radiolabeling yields, and product purification using a PD-10 desalting column. Results: Radiochemical yields of 89 Zr-DFO-N-suc-cetuximab and 89 Zr-DFO-N-suc-rituximab were all more than 90% according to instant thin-layer chromatography. Isolated yields were 74.6% ± 2.0% and 62.6% ± 3.0% for 89 Zr-DFO-N-suc-cetuximab and 89 Zr-DFO-N-suc-rituximab, respectively, which are similar to isolated yields obtained using GMP protocols for manual 89 Zr labeling of mAbs. To meet the GMP-compliant quality standards, only the radiochemically pure fractions were collected from PD-10, resulting in a lower isolated yield than the radiochemical yield according to instant thin-layer chromatography. The radiochemical purity and protein integrity were more than 95% for both products, and the antigen binding was 95.6% ± 0.6% and 87.1% ± 2.2% for 89 Zr-DFO-N-suc-cetuximab and 89 Zr-DFO-N-suc-rituximab, respectively. The products were sterile, and the endotoxin levels were within acceptable limits, allowing future clinical production using this procedure. Conclusion: Procedures for fully automated GMP-compliant production of 89 Zr-mAbs were developed on a commercially available synthesis module, which also allows the GMP production of other radiolabeled mAbs. AUTOMATED 89 ZR-MAB LABELING • Poot et al. Zr Labeling and Purification of Antibodies 89 Fully Automated http://jnm.snmjournals.org/content/60/5/691 This article and updated information are available at: http://jnm.snmjournals.org/site/subscriptions/online.xhtml Information about subscriptions to JNM can be found at: http://jnm.snmjournals.org/site/misc/permission.xhtml

Introduction: Compared to the antibody and drug components of an ADC, the linker part has been somewhat neglected. However, its importance for the reduction of failures in ADC approvals is increasingly recognized. Next of being a stable glue between drug and antibody, an ideal linker should improve the manufacturability and widen the therapeutic window of ADCs. Areas covered: The biopharmaceutical company LinXis started an ADC development program in which platinum(II) is the key element of the first metal-organic linker. The cationic complex [ethylenediamineplatinum(II)] 2+ , herein called 'Lx®', is used successfully for conjugation of drugs to antibodies. Expert opinion: Based on lessons learned from ADC development, Lx linker technology fulfills most of the desirable linker characteristics. Lx allows large-scale cost-effective manufacturing of ADCs via a straightforward two-step 'plug-and-play' process. First clinical candidate trastuzumab-Lx-auristatin F shows favorable preclinical safety as well as outstanding in vivo tumor targeting performance and therapeutic efficacy.