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.

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48 h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.

The reactivity and stereoselectivity of a galacturonic acid 3,6‐lactone thioglycosyl donor, previously described as a highly reactive glycosylating agent, has been investigated by using a series of competition experiments and condensation reactions with different thiophilic activator systems. It is revealed that the relative reactivity of the thioglycosides depends on the activator system used and that p‐nitrophenylsulfenyl triflate shows overall attenuated reactivity differences with respect to the commonly used N‐iodosuccinimide/triflic acid promoter system. With respect to the stereoselectivity of the studied galacturonic acid 3,6‐lactone thioglycosyl donor, it is revealed that a preactivation‐based glycosylation system gives rise to α‐selective glycosylation, whereas an in situ activation protocol leads to the formation of the β‐product with good selectivity. It is hypothesized that these opposingstereoselectivities are the result of different product‐forming intermediates. Where preactivation of the donor leads to the formation of an intermediate β‐triflate, which is substituted in a concerted fashion to provide the α‐product, a 3H4 oxocarbenium ion like species is substituted in the in situ activation experiment to provide the β‐linked product.

Linker instability and impaired tumor targeting can affect the tolerability and efficacy of antibody-drug conjugates (ADCs). To improve these ADC characteristics, we recently described the use of a metal-organic linker, [ethylenediamineplatinum(II)], herein called Initial therapy studies in xenograft-bearing mice revealed that trastuzumab--auristatin F (AF) outperformed its maleimide benchmark trastuzumab-mal-AF and the Food and Drug Administration-approved ado-trastuzumab emtansine, both containing conventional linkers. In this study, we aimed to characterize -based ADCs for in vivo stability and tumor targeting usingPt and Zr. The γ-emitter Pt was used to produce the radiolabeled [Pt]Zr-Desferrioxamine (Zr-DFO) was conjugated to trastuzumab either via [Pt] (to histidine residues) or conventionally (to lysine residues) in order to monitor the biodistribution of antibody, payload, and linker separately. Linker stability was determined by evaluating the following ADCs for biodistribution in NCI-N87 xenograft-bearing nude mice 72 h after injection: trastuzumab-[Pt]-DFO-Zr, trastuzumab-[Pt]-AF, and Zr-DFO-(Lys)trastuzumab (control), all having drug-to-antibody ratios (DARs) of 2.2-2.5. To assess the influence of DAR on biodistribution,Zr-DFO-(Lys)trastuzumab--AF with an AF-to-antibody ratio of 0, 2.6, or 5.2 was evaluated 96 h after injection. Similar biodistributions were observed for trastuzumab-[Pt]-DFO-Zr, trastuzumab-[Pt]-AF, and Zr-DFO-(Lys)trastuzumab irrespective of the isotope used for biodistribution assessment. The fact that follows the antibody biodistribution indicates that the payload- bond is stable in vivo. Uptake of the 3 conjugates, as percentage injected dose (%ID) per gram of tissue, was about 30 %ID/g in tumor tissue but less than 10 %ID/g in most healthy tissues. Trastuzumab-[Pt]-AF (DAR 2.2) showed a tendency toward faster blood clearance and an elevated liver uptake, which increased significantly to 28.1 ± 4.2 %ID/g at a higher DAR of 5.2, as revealed from the biodistribution and PET imaging studies. As shown byPt/Zr labeling, ADCs containing the linker are stable in vivo. In the case of trastuzumab--AF (DARs 2.2 and 2.6), an unimpaired biodistribution was demonstrated.