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Supplementary Figure from A Novel EGFRvIII T-Cell Bispecific Antibody for the Treatment of Glioblastoma

PDF file - 414KB, Supplementary Figure 2 - Fluorescence imaging after i.v. application of anti-DR5-Cy5 shows no specific antibody accumulation in the right brain hemisphere.

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. Cancer Res; 74(7); 1913–23. ©2014 AACR.

PDF file - 68KB

PDF file - 186KB, Supplementary Figure 4 - Antibody penetration is enhanced in U87-Luc and U251 brain xenograft tumors indicating BBB disruption.

PDF file - 936KB, Supplementary Figure 3 - Apoptosis and fluorescence imaging reveal that 3.9-7.0% of anti-DR5-Cy5 given i.v. reaches subcutaneous D54-Caspase-3/7 GloSensor tumors.

PDF file - 75KB, Supplementary Figure 1 - Highly sensitive apoptosis imaging allows tumor detection and randomization at early time points.

PDF file - 63KB, Fluorescence imaging - Fluorescence imaging was used for monitoring anti-DR5-Cy5 distribution in the mouse. Cell culture and cell inoculation of U87-Luc and U251 cell lines - U87-Luc and U251 were used for additional BBB penetration studies and were inoculated as described for D54-Caspase-3/7 GloSensor cell line.

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 have 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 choroid plexi in the brain and duodenal papillae as unexpected locations of high EpCAM-expression. These tissue locations may be considered as particularly sensitive sites due 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. Conclusively, 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.

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.

Summary: We present a novel light sheet fluorescence microscopy (LSFM)-based imaging platform and corresponding set of procedures for greatly improved preclinical investigation of the biodistribution of novel drug candidates by the example of an EpCAM-targeting antibody. Methods: An anti-EpCAM antibody (G.8.8R) was applied intravenously to mice. Whole mice or individual organs were optically cleared following novel protocols, which we termed Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS). The procedures enabled processing of mouse organs and entire bodies for LSFM. For the GI tract, we developed 3D-Swiss rolls, a method that enables fixation, clearing and imaging of intestines in a compact form as a whole. The new procedures allowed us to investigate the biodistribution of G.8.8R in the entire mouse body and highly detailed in individual organs. Results: The ROCKETS concept achieved full transparency of any mouse organ, the entire GI tract and whole mouse bodies. All positive organs were easily identified in scans of whole mice, thereby providing excellent guidance for scanning of positive individual organs at higher magnifications. We detected G8.8R bound to all normal cuboidal and columnar epithelia, as well as in lymphoid organs and were able to reproducibly score binding levels. All reported EpCAM+ tissues in mice were accessed by G8.8R after intravenous application and binding was restricted to basolateral membranes of epithelia, as expected from published expression analyses. Moreover, we detected G8.8R in tissues that were reported EpCAM- or were not investigated, e.g. gustatory papillae of the tongue, choroid plexi in the brain or lingual mucous salivary glands. 3D-Swiss rolls of the GI tract revealed a highly heterogeneous binding pattern in the stomach, while the pattern along the small intestine was overall homogeneous. All binding was restricted to basolateral membranes of epithelial cells. We detected a general gradient of signals, decreasing from the bottom of the crypts to differentiated cells in all tissues, corresponding to described EpCAM downregulation with progressing differentiation. Furthermore, we first describe significantly increased signals at the common bile duct, major and minor duodenal papillae, as well as the mucosa in proximity of Peyer's patches (PP). Conclusions: The developed ROCKETS toolbox allowed for simple preparation of mouse organs and bodies for high-quality LSFM imaging. Our investigations of the highly heterogeneous biodistribution of G8.8R revealed previously unknown EpCAM binding locations, which may have far-reaching implications for EpCAM-targeting therapeutics in general, many of which failed in clinical studies due to dose limiting toxicities. In the future, the developed LSFM-imaging platform may contribute valuable data to preclinical drug development studies of any targeted therapeutic. Citation Format: Joerg P. Mueller, Nils O'Brien, Franz Osl, Frank Herting, Christian Klein, Pablo Umana, Sara Colombetti, Thomas Poeschinger, Andreas Beilhack. ROCKETS Science: A novel processing toolbox for light sheet microscopy reveals unknown binding sites for EpCAM-targeted antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1876.

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.

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.

Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling.

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 × 106 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.

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. Cancer Res; 74(7); 1913–23. ©2014 AACR.

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.

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.