High resolution combined molecular and structural optical imaging of colorectal cancer in a xenograft mouse model

With the emergence of immunotherapies for cancer treatment, there is a rising clinical need to visualize the tumor microenvironment (TME) non-invasively in detail, which could be crucial to predict the efficacy of therapy. Nuclear imaging techniques enable whole-body imaging but lack the required spatial resolution. Conversely, near-infrared immunofluorescence (immuno-NIRF) is able to reveal tumor cells and/or other cell subsets in the TME by targeting the expression of a specific membrane receptor with fluorescently labeled monoclonal antibodies (mAb). Optical coherence tomography (OCT) provides three-dimensional morphological imaging of tissues without exogenous contrast agents. The combination of the two allows molecular and structural contrast at a resolution of ~15 µm, allowing for the specific location of a cell-type target with immuno-NIRF as well as revealing the three-dimensional architectural context with OCT. For the first time, combined immuno-NIRF and OCT of a tumor is demonstrated in situ in a xenograft mouse model of human colorectal cancer, targeted by a clinically-safe fluorescent mAb, revealing unprecedented details of the TME. A handheld scanner for ex vivo examination and an endoscope designed for imaging bronchioles in vivo are presented. This technique promises to complement nuclear imaging for diagnosing cancer invasiveness, precisely determining tumor margins, and studying the biodistribution of newly developed antibodies in high detail

The human cytomegalovirus-encoded G protein- coupled receptor UL33 exhibits oncomodulatory properties

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein- coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies

89Zr-labeled compounds for PET imaging guided personalized therapy

89Zr-immuno-PET is an attractive option for the in vivo evaluation of monoclonal antibodies (mAbs). For the coupling of 89Zr to monoclonal antibodies several conjugation strategies are available all using desferrioxamine as chelate. Here we discuss the production of 89Zr, the available methods for coupling of 89Zr via desferrioxamine to mAbs, and the evaluation of 89Zr-mAb conjugates in preclinical and clinical studies

The added value of diagnostic and theranostic PET imaging for the treatment of CNS tumors

This review highlights the added value of PET imaging in Central Nervous System (CNS) tumors, which is a tool that has rapidly evolved from a merely diagnostic setting to multimodal molecular diagnostics and the guidance of targeted therapy. PET is the method of choice for studying target expression and target binding behind the assumedly intact blood–brain barrier. Today, a variety of diagnostic PET tracers can be used for the primary staging of CNS tumors and to determine the effect of therapy. Additionally, theranostic PET tracers are increasingly used in the context of pharmaceutical and radiopharmaceutical drug development and application. In this approach, a single targeted drug is used for PET diagnosis, upon the coupling of a PET radionuclide, as well as for targeted (nuclide) therapy. Theranostic PET tracers have the potential to serve as a non-invasive whole body navigator in the selection of the most effective drug candidates and their most optimal dose and administration route, together with the potential to serve as a predictive biomarker in the selection of patients who are most likely to benefit from treatment. PET imaging supports the transition from trial and error medicine to predictive, preventive, and personalized medicine, hopefully leading to improved quality of life for patients and more cost-effective care

Basic principles, applications in oncology and improved selectivity of photodynamic therapy

Photodynamic therapy (PDT) is a promising approach for the treatment of superficially localized tumors. This review starts with a summary of the basic principles of PDT, in which the current practice, the photochemical mechanisms, cellular and subcellular targets, as well as the most prominent photosensitizers are discussed. Next, the clinical results obtained with PDT for the treatment of a variety of tumor types are outlined. Unfortunately, most of these studies revealed a lack of tumor selectivity of the photosensitizers, which resulted in prolonged skin photosensitivity and severe normal tissue toxicity. The last section of this review, therefore, focuses on novel strategies designed to improve the tumor selectivity of photosensitizers

Synthesis and evaluation of 99mTc/99Tc-MAG3-biotin conjugates for antibody pretargeting strategies

Four 99mTc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99mTc-MAG3-biotin conjugates as model compounds for 186Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99Tc were added as carrier to 99mTc. The biotin derivatives used for the preparation of the conjugates - biocytin, biotin hydrazide, biotinyl-piperazine, and biotinyl-diaminosuccinic acid - differed at the site that is regarded to be susceptible to hydrolysis by biotinidase present in human plasma. All four conjugates were produced with high radiochemical purity, were stable in PBS, and demonstrated full binding capacity to streptavidin. The 99mTc/99Tc-MAG3-labeled biotinyl-piperazine and biotinyl-diaminosuccinic acid conjugates were stable in mouse as well as human plasma, whereas the corresponding biocytin and biotin hydrazide conjugates were rapidly degraded. The biodistribution in nude mice at 30 min after injection was similar for all conjugates, and a rapid blood clearance and high intestinal excretion were both observed. It is concluded that the metabolic routing of a conjugate containing biotin and MAG3 is dominated by these two moieties. For this reason, MAG3-biotin conjugates do not seem suited for pretargeted RIT, for which quantitative and fast renal excretion is a prerequisite to minimize radiation toxicity. However, in a pretargeted RIS approach the 99mTc-MAG3-biotin conjugates might have potential

High-quality 124I-labelled monoclonal antibodies for use as PET scouting agents prior to 131I-radioimmunotherapy

Purpose: Monoclonal antibodies (MAbs) labelled with 124I are an attractive option for quantitative imaging with positron emission tomography (PET) in a scouting procedure prior to 131I-radioimmunotherapy ( 131I-RIT). In this study, three important items in the labelling of MAbs with 124I were introduced to obtain optimal and reproducible product quality: restoration of radiation-induced inorganic deterioration of the starting 124I solution, radiation protection during and after 124I labelling, and synchronisation of the I/MAb molar ratio. Methods: A new method was applied, using an NaIO3/ NaI carrier mix, realising in one step >90% restoration of deteriorated 124I into the iodide form and chemical control over the I/MAb molar ratio. Chimeric MAb (cMAb) U36 and the murine MAbs 425 and E48 were labelled with 124I using the so-called lodogen-coated MAb method, as this method provides optimal quality conjugates under challenging radiation conditions. As a standardising condition, NaIO3/NaI carrier mix was added at a stoichiometric I/MAb molar ratio of 0.9. For comparison, MAbs were labelled with 131I and with a mixture of 124I, 123I, 126I and 130I. Results: Labelling with 124I in this setting resulted in overall yields of >70%, a radiochemical purity of >95%, and preservation of MAb integrity and immunoreactivity, including at the patient dose level (85 MBq). No significant quality differences were observed when compared with 131I products, while the iodine isotope mixture gave exactly the same labelling efficiency for each of the isotopes, excluding a different chemical reactivity of 124I-iodide. The scouting performance of 124I-cMAb U36 la-belled at the patient dose level was evaluated in biodistribution studies upon co-injection with 131I- labelled cMAb U36, and by PET imaging in nude mice bearing the head and neck cancer xenograft line HNX-OE. 124I-cMAb and 131I-cMAb U36 labelled with a synchronised I/MAb molar ratio gave fully concordant tissue uptake values. Selective tumour uptake was confirmed with immuno-PET, revealing visualisation of 15 out of 15 tumours. Conclusion: These results pave the way for renewed evaluation of the potential of 124I-immuno-PET for clinical applications

Comparison of aluminium (III) phthalocyanine tetrasulfonate- and meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for their efficacy in photodynamic therapy in vitro

A challenge in photodynamic therapy (PDT) is to improve the tumour selectivity of the photosensitizers by using monoclonal antibodies (MAbs). With this aim, we developed MAb-conjugates with the hydrophobic photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) and with the hydrophilic sensitizer aluminium (III) phthalocyanine tetrasulfonate (AI-PCS4). The capacity of these photoimmunoconjugates for selective targeting of squamous cell carcinoma (SCC) in vivo was demonstrated previously in SCC-bearing nude mice. Preliminary in vitro PDT studies with the vulvar SCC cell line A431 showed promising phototoxicity with both sensitizers when coupled to the internalizing MAb 42S. To rank the photosensitizers for their potential in photoimmunotherapy, we herein describe an extensive in vitro evaluation of mTHPC-MAb and AIPcS4-MAb conjugates. Both classes of conjugates were directly compared using 5 different SCC cell lines as target and 3 different MAbs (BIWA 4, E48 and 425) for tumour cell targeting. In contrast to free AIPcS4 (IC50 ≥ 700 nM), MAb-conjugated AIPcS4 was found to be highly phototoxic in PDT in all 5 cell lines. AIPcS4-BIWA 4 was most consistently effective with IC50 values ranging from 0.06-5.4 nM. mTHPC-MAb conjugates were in general hardly effective. Phototoxicity (log IC50) of the AIPcS4-MAb conjugates was found to be strongly correlated with their total cell binding capacity (internalized and surface bound) and to be less correlated with their internalization capacity. In conclusion, these data show a high potential of AIPcS4-MAb conjugates in comparison to mTHPC-MAb conjugates for use in PDT

A panel of biomarkers of carcinogenesis of the upper aerodigestive tract as potential intermediate endpoints in chemoprevention trials

Background. Patients with squamous cell carcinoma of the head and neck have a 10–30% risk of developing a second primary tumor. The concept of “field cancerization” assumes that the whole upper aerodigestive tract is affected and prone to malignant transformation. This study was undertaken to investigate the value of a panel of monoclonal antibodies to identify biomarkers in oral mucosa associated with cancer risk. Such biomarkers may be suitable candidates to serve as intermediate endpoints in cancer chemoprevention trials. Methods. As a model, the expression of antigens was assessed in cytologic preparations obtained from macroscopically normal oral mucosa of patients with tongue carcinoma and of controls. The panel consisted of antibodies against cytokeratin 8, 10, 13, and 19 and the monoclonal antibodies designated K931, K984, E48, Ki‐67, and UM‐A9. Results. Oral mucosa of cancer patients had a more than threefold increased expression of cytokeratin 19 as compared with controls (36.0 versus 11.3%; P < 0.01). Conclusion. Cytokeratin 19 is a potential intermediate endpoint in head and neck cancer chemopreventive trials