Phase II Trial Assessing the Repeatability and Tumor Uptake of [68Ga]Ga-HER2 Single-Domain Antibody PET/CT in Patients with Breast Carcinoma.

Human epidermal growth factor receptor 2 (HER2) status is used for decision-making in breast carcinoma treatment. The status is obtained through immunohistochemistry or in situ hybridization. These two methods have the disadvantage of necessitating tissue sampling, which is prone to error due to tumor heterogeneity or interobserver variability. Whole-body imaging might be a solution to map HER2 expression throughout the body. Methods: Twenty patients with locally advanced or metastatic breast carcinoma (5 HER2-positive and 15 HER2-negative patients) were included in this phase II trial to assess the repeatability of uptake quantification and the extended safety of the [68Ga]Ga-NOTA-anti-HER2 single-domain antibody (sdAb). The tracer was injected, followed by a PET/CT scan at 90 min. Within 8 d, the procedure was repeated. Blood samples were taken for antidrug antibody (ADA) assessment and liquid biopsies. On available tissues, immunohistochemistry, in situ hybridization, and mass spectrometry were performed to determine the correlation of HER2 status with uptake values measured on PET. If relevant preexisting [18F]FDG PET/CT images were available (performed as standard of care), a comparison was made. Results: With a repeatability coefficient of 21.8%, this imaging technique was repeatable. No clear correlation between PET/CT uptake values and pathology could be established, as even patients with low levels of HER2 expression showed moderate to high uptake. Comparison with [18F]FDG PET/CT in 16 patients demonstrated that in 7 patients, [68Ga]Ga-NOTA-anti-HER2 shows interlesional heterogeneity within the same patient, and [18F]FDG uptake did not show the same heterogeneous uptake in all patients. In some patients, the extent of disease was clearer with the [68Ga]Ga-NOTA-anti-HER2-sdAb. Sixteen adverse events were reported but all without a clear relationship to the tracer. Three patients with preexisting ADAs did not show adverse reactions. No new ADAs developed. Conclusion: [68Ga]Ga-NOTA-anti-HER2-sdAb PET/CT imaging shows similar repeatability to [18F]FDG. It is safe for clinical use. There is tracer uptake in cancer lesions, even in patients previously determined to be HER2-low or -negative. The tracer shows potential in the assessment of interlesional heterogeneity of HER2 expression. In a subset of patients, [68Ga]Ga-NOTA-anti-HER2-sdAb uptake was seen in lesions with no or low [18F]FDG uptake. These findings support further clinical development of [68Ga]Ga-NOTA-anti-HER2-sdAb as a PET/CT tracer in breast cancer patients.

Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I).

Macrophages play an important role throughout the body. Antiinflammatory macrophages expressing the macrophage mannose receptor (MMR, CD206) are involved in disease development, ranging from oncology to atherosclerosis and rheumatoid arthritis. [68Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206. This first-in-human study, as its primary objective, evaluated the safety, biodistribution, and dosimetry of this tracer. The secondary objective was to assess its tumor uptake. Methods: Seven patients with a solid tumor of at least 10 mm, an Eastern Cooperative Oncology Group score of 0 or 1, and good renal and hepatic function were included. Safety was evaluated using clinical examination and blood sampling before and after injection. For biodistribution and dosimetry, PET/CT was performed at 11, 90, and 150 min after injection; organs showing tracer uptake were delineated, and dosimetry was evaluated. Blood samples were obtained at selected time points for blood clearance. Metabolites in blood and urine were assessed. Results: Seven patients were injected with, on average, 191 MBq of [68Ga]Ga-NOTA-anti-CD206-sdAb. Only 1 transient adverse event of mild severity was considered to be possibly, although unlikely, related to the study drug (headache, Common Terminology Criteria for Adverse Events grade 1). The blood clearance was fast, with less than 20% of the injected activity remaining after 80 min. There was uptake in the liver, kidneys, spleen, adrenals, and red bone marrow. The average effective dose from the radiopharmaceutical was 4.2 mSv for males and 5.2 mSv for females. No metabolites were detected. Preliminary data of tumor uptake in cancer lesions showed higher uptake in the 3 patients who subsequently progressed than in the 3 patients without progression. One patient could not be evaluated because of technical failure. Conclusion: [68Ga]Ga-NOTA-anti-CD206-sdAb is safe and well tolerated. It shows rapid blood clearance and renal excretion, enabling high contrast-to-noise imaging at 90 min after injection. The radiation dose is comparable to that of routinely used PET tracers. These findings and the preliminary results in cancer patients warrant further investigation of this tracer in phase II clinical trials.

Photothermal nanofibres enable safe engineering of therapeutic cells.

Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR-Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.

Non-viral transfection technologies for next-generation therapeutic T cell engineering.

Genetically engineered T cells have sparked interest in advanced cancer treatment, reaching a milestone in 2017 with two FDA-approvals for CD19-directed chimeric antigen receptor (CAR) T cell therapeutics. It is becoming clear that the next generation of CAR T cell therapies will demand more complex engineering strategies and combinations thereof, including the use of revolutionary gene editing approaches. To date, manufacturing of CAR T cells mostly relies on γ-retroviral or lentiviral vectors, but their use is associated with several drawbacks, including safety issues, high manufacturing cost and vector capacity constraints. Non-viral approaches, including membrane permeabilization and carrier-based techniques, have therefore gained a lot of interest to replace viral transductions in the manufacturing of T cell therapeutics. This review provides an in-depth discussion on the avid search for alternatives to viral vectors, discusses key considerations for T cell engineering technologies, and provides an overview of the emerging spectrum of non-viral transfection technologies for T cells. Strengths and weaknesses of each technology will be discussed in relation to T cell engineering. Altogether, this work emphasizes the potential of non-viral transfection approaches to advance the next-generation of genetically engineered T cells.

Engineering and crystal structure of a monomeric FLT3 ligand variant.

The overarching paradigm for the activation of class III and V receptor tyrosine kinases (RTKs) prescribes cytokine-mediated dimerization of the receptor ectodomains and homotypic receptor-receptor interactions. However, structural studies have shown that the hematopoietic receptor FLT3, a class III RTK, does not appear to engage in such receptor-receptor contacts, despite its efficient dimerization by dimeric FLT3 ligand (FL). As part of efforts to better understand the intricacies of FLT3 activation, we sought to engineer a monomeric FL. It was found that a Leu27Asp substitution at the dimer interface of the cytokine led to a stable monomeric cytokine (FLL27D) without abrogation of receptor binding. The crystal structure of FLL27D at 1.65 Šresolution revealed that the introduced point mutation led to shielding of the hydrophobic footprint of the dimerization interface in wild-type FL without affecting the conformation of the FLT3 binding site. Thus, FLL27D can serve as a monomeric FL variant to further interrogate the assembly mechanism of extracellular complexes of FLT3 in physiology and disease.

Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells.

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

Photothermally Triggered Endosomal Escape and Its Influence on Transfection Efficiency of Gold-Functionalized JetPEI/pDNA Nanoparticles.

Plasmonic nanoparticles for drug delivery have attracted increasing interest over the last few years. Their localized surface plasmon resonance causes photothermal effects on laser irradiation, which allows for delivering drugs in a spatio-temporally controlled manner. Here, we explore the use of gold nanoparticles (AuNP) as carriers for pDNA in combination with pulsed laser irradiation to induce endosomal escape, which is currently considered to be one of the major bottlenecks in macromolecular drug delivery on the intracellular level. In particular, we evaluate nanocomplexes composed of JetPEI (polyethylenimine)pDNA and 10 nm AuNP, which do not exhibit endosomal escape by themselves. After incubating HeLa cells with these complexes, we evaluated endosomal escape and transfection efficiency using low- and high-energy laser pulses. At low laser energy heat is produced by the nanocomplexes, while, at higher laser energy, explosive vapour nanobubbles (VNB) are formed. We investigated the ability of heat transfer and VNB formation to induce endosomal escape and we examine the integrity of pDNA cargo after inducing both photothermal effects. We conclude that JetPEI/pDNA/AuNP complexes are unable to induce meaningful transfection efficiencies because laser treatment causes either dysfunctionality of the cargo when VNB are formed or forms too small pores in the endosomal membrane to allow pDNA to escape in case of heating. We conclude that laser-induced VNB is the most suitable to induce effective pDNA endosomal escape, but a different nanocomplex structure will be required to keep the pDNA intact.