ROCKETS - a novel one-for-all toolbox for light sheet microscopy in drug discovery.

Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

A Novel EGFRvIII T-Cell Bispecific Antibody for the Treatment of Glioblastoma.

T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.

Intrinsic Respiratory Gating for Simultaneous Multi-Mouse µCT Imaging to Assess Liver Tumors.

Small animal micro computed tomography (µCT) is an important tool in cancer research and is used to quantify liver and lung tumors. A type of cancer that is intensively investigated with µCT is hepatocellular carcinoma (HCC). µCT scans acquire projections from different angles of the gantry which rotates X-ray source and detector around the animal. Motion of the animal causes inconsistencies between the projections which lead to artifacts in the resulting image. This is problematic in HCC research, where respiratory motion affects the image quality by causing hypodense intensity at the liver edge and smearing out small structures such as tumors. Dealing with respiratory motion is particularly difficult in a high throughput setting when multiple mice are scanned together and projection removal by retrospective respiratory gating may compromise image quality and dose efficiency. In mice, inhalation anesthesia leads to a regular respiration with short gasps and long phases of negligible motion. Using this effect and an iterative reconstruction which can cope with missing angles, we discard the relatively few projections in which the gasping motion occurs. Moreover, since gated acquisition, i.e., acquiring multiple projections from a single gantry angle is not a requirement, this method can be applied to existing scans. We applied our method in a high throughput setting in which four mice with HCC tumors were scanned simultaneously in a multi-mouse bed. To establish a ground truth, we manually selected projections with visible respiratory motion. Our automated intrinsic breathing projection selection achieved an accordance of 97% with manual selection. We reconstructed volumetric images and demonstrated that our intrinsic gating method significantly reduces the hypodense depiction at the cranial liver edge and improves the detectability of small tumors. Furthermore, we show that projection removal in a four mice scan discards only 7.5% more projections than in a single-mouse setting, i.e., four mouse scanning does not substantially compromise dose efficiency or image quality. To the best of our knowledge, no comparable method that combines multi-mouse scans for high throughput, intrinsic respiratory gating, and an available iterative reconstruction has been described for liver tumor imaging before.

Comparison of the Accuracy of FMT/CT and PET/MRI for the Assessment of Antibody Biodistribution in Squamous Cell Carcinoma Xenografts.

Noninvasive imaging technologies are increasingly used in preclinical drug research for the pharmacokinetic analysis of therapeutic compounds in living animals over time. The different preclinical imaging modalities available differ intrinsically in their detection principle and thus might exhibit limitations for a specific application. Here, we systematically investigated the performance of advanced fluorescence-mediated tomography (FMT)/CT in comparison to PET/MRI for quantitative analysis of the biodistribution of different antibody formats and dependence on the required imaging label in squamous cell carcinoma xenografts. Methods: Different formats of an antibody (monoclonal antibody and the antigen binding fragments F(ab')2 and Fab) targeting epidermal growth factor receptor were labeled with Alexa750 or 64Cu-NODAGA and injected intravenously into separate cohorts of nude mice bearing subcutaneous A-431 tumors. Two and 24 h after injection, the mice were measured by FMT/CT and PET/MRI. Probe accumulation was quantitatively assessed in organs and tumors. In vivo data were compared between modalities and correlated with ex vivo fluorescence, γ-counting, and electrochemiluminescence immunoassay. Results: Both imaging methods faithfully monitored the biodistribution and elimination routes of the compounds, and organ accumulation measured by FMT/CT and PET/MRI correlated significantly with ex vivo measurements. In addition, the accumulation in kidney, muscle, and tumor tissue correlated between FMT/CT and PET/MRI. However, the pharmacokinetics of the Alexa750-labeled antibody formats showed shorter blood half-times and higher liver uptake than the radiolabeled counterparts. Conclusion: FMT/CT imaging allows quantifying the biodistribution of antibodies in nude mice and provides an alternative to PET analysis in preclinical drug research. However, even for large molecules, such as monoclonal antibodies, Alexa750 labeling can change pharmacokinetics and trigger liver uptake.

Anti-tumoral, anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2.

Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.

Apoptosis imaging for monitoring DR5 antibody accumulation and pharmacodynamics in brain tumors noninvasively.

High-grade gliomas often possess an impaired blood-brain barrier (BBB), which allows delivery of large molecules to brain tumors. However, achieving optimal drug concentrations in brain tumors remains a significant hurdle for treating patients successfully. Thus, detailed investigations of drug activities in gliomas are needed. To investigate BBB penetration, pharmacodynamics, and tumor retention kinetics of an agonistic DR5 antibody in a brain tumor xenograft model, we utilized a noninvasive imaging method for longitudinal monitoring of apoptosis induction. Brain tumors were induced by intracranial (i.c.) implantation of a luciferase-expressing tumor cell line as a reporter. To quantify accumulation of anti-DR5 in brain tumors, we generated a dosage-response curve for apoptosis induction after i.c. delivery of fluorescence-labeled anti-DR5 at different dosages. Assuming 100% drug delivery after i.c. application, the amount of accumulated antibody after i.v. application was calculated relative to its apoptosis induction. We found that up to 0.20% to 0.97% of antibody delivered i.v. reached the brain tumor, but that apoptosis induction declined quickly within 24 hours. These results were confirmed by three-dimensional fluorescence microscopy of antibody accumulation in explanted brains. Nonetheless, significant antitumor efficacy was documented after anti-DR5 delivery. We further demonstrated that antibody penetration was facilitated by an impaired BBB in brain tumors. These imaging methods enable the quantification of antibody accumulation and pharmacodynamics in brain tumors, offering a holistic approach for assessment of central nervous system-targeting drugs.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice.

PURPOSE: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. PROCEDURES: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(ab')2-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). RESULTS: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F(ab')2 fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). CONCLUSIONS: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Quantification of human Alu sequences by real-time PCR--an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants.

For measuring the efficacy of new anti-metastatic drugs in preclinical models, macroscopical analysis or classical histology of secondary organs are established methods. However, macroscopical evaluation does not take into consideration intra-organ metastasis. Histological analysis is often performed in few sections of the relevant organs, and this may be misleading, since equal distribution of tumor cells within an organ is unlikely. In addition, recent studies have demonstrated that anti-tumorigenic drugs are able to promote metastasis and to change the metastatic pattern. Therefore, extensive analysis of metastasis is mandatory for the evaluation of new compounds. A feasibility study was conducted to find out if the quantification of human Alu sequences could be applied as a surrogate marker for metastasis in xenografts. Alu PCR was performed by using the LightCycler system, which allows PCR reaction and subsequent quantification of the PCR products in less than 30 min. We found that i) the equivalent of one human tumor cell in 1 x 10(6) murine cells could be detected; ii) in tumor-carrying mice, Alu signal increased over time in secondary organs; iii) this increase was more prominent using highly metastatic tumor cells; iv) Alu signal intensity in DNA extracted from tissue slides correlated with the expression of histological tumor markers; v) in three different tumor models (colon, breast and lung), treatment with Taxol or 5-fluorouracil reduced the amount of Alu in different organs. In contrast, reduction of Alu by the matrix metalloproteinase inhibitor RO 28-2653 was not significant. Taken together, quantification of Alu sequences is a fast and accurate method to evaluate the therapeutic efficacy of anti-metastatic drugs in xenografts.