The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors.

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.

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9–EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9–EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9–EWI-F with potential CD9 homo-dimerization at either end provides a “concatenation model” for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

Purpose Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [111In]DOTA-NB/[177Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate sitespecific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation.