Martin Pool

Biodistribution and PET Imaging of Labeled Bispecific T Cell-Engaging Antibody Targeting EpCAM

AMG 110, a bispecific T cell engager (BiTE) antibody construct, induces T cell–mediated cancer cell death by cross-linking epithelial cell adhesion molecule (EpCAM) on tumor cells with a cluster of differentiation 3 ε (CD3ε) on T cells. We labeled AMG 110 with 89Zr or near-infrared fluorescent dye (IRDye) 800CW to study its tumor targeting and tissue distribution. Methods: Biodistribution and tumor uptake of 89Zr-AMG 110 was studied up to 6 d after intravenous administration to nude BALB/c mice bearing high EpCAM-expressing HT-29 colorectal cancer xenografts. Tumor uptake of 89Zr-AMG 110 was compared with uptake in head and neck squamous cell cancer FaDu (intermediate EpCAM) and promyelocytic leukemia HL60 (EpCAM-negative) xenografts. Intratumoral distribution in HT-29 tumors was studied using 800CW-AMG 110. Results: Tumor uptake of 89Zr-AMG 110 can be clearly visualized using small-animal PET imaging up to 72 h after injection. The highest tumor uptake of 89Zr-AMG 110 at the 40-μg dose level was observed at 6 and 24 h (respectively, 5.35 ± 0.22 and 5.30 ± 0.20 percentage injected dose per gram; n = 3 and 4). Tumor uptake of 89Zr-AMG 110 was EpCAM-specific and correlated with EpCAM expression. 800CW-AMG 110 accumulated at the tumor cell surface in viable EpCAM-expressing tumor tissue. Conclusion: PET and fluorescent imaging provided real-time information about AMG 110 distribution and tumor uptake in vivo. Our data support using 89Zr and IRDye 800CW to evaluate tumor and tissue uptake kinetics of bispecific T cell engager antibody constructs in preclinical and clinical settings.

Extracellular domain shedding influences specific tumor uptake and organ distribution of the EGFR PET tracer 89 Zr-imgatuzumab

Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 μg 89Zr-imgatuzumab, co-injected with equal doses 111In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 μg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 μg) 89Zr-imgatuzumab dose. High 89Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

(89)Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

ABSTRACT Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (89Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on89Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to 89Zr-mAb3481 administration. Subsequently, ex vivo 89Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). 89Zr-mAb3481 BT474 tumor uptake was remarkably high at SUVmean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg 89Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect 89Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. 89Zr-mAb3481 PET accurately reflected HER3 tumor status. 89Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.

Emerging Opportunities for c-MET Visualization in the Clinic

In this issue of The Journal of Nuclear Medicine, Arulappu et al. present results of c-MET–specific peptide 18F-AH113804 PET screening for local regional recurrence after surgical excision of orthotopically implanted HCC1954 human basal-like breast cancer (BLBC) xenografts. 18F-AH113804 PET could visualize this locoregional recurrence as early as 6 d postoperatively, whereas CT did so after day 20, and tumors were first palpable only by day 27. They were also able to differentiate between high and low c-MET–expressing tumors. On the basis of these results, the authors concluded that 18F-AH113804 c-MET PET has potential as a clinical screening tool for earlier detection of locoregional BLBC recurrences (1). c-MET is an interesting target for molecular imaging and treatment in solid tumors. It is a tyrosine growth factor receptor and together with ligand hepatocyte growth factor (HGF), c-MET is often dysregulated in cancers, including breast cancer. Increased activation of the c-MET/HGF pathway leads to invasion, angiogenesis, motility, and cell proliferation (2).

ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

Imgatuzumab is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and EGFR signal transduction inhibition. We investigated anti-EGFR monoclonal antibodies imgatuzumab and cetuximab–induced internalization and membranous turnover of EGFR, and whether this affected imgatuzumab–mediated ADCC responses and growth inhibition of non-small cell lung cancer (NSCLC) cells. In a panel of wild-type EGFR expressing human NSCLC cell lines, membranous and total EGFR levels were downregulated more effectively by imgatuzumab when compared with cetuximab. Imgatuzumab plus cetuximab enhanced EGFR internalization and reduced membranous turnover of EGFR, resulting in an even stronger downregulation of EGFR. Immunofluorescent analysis showed that combined treatment increased clustering of receptor-antibody complexes and directed internalized EGFR to lysosomes. The antibody combination potently inhibited intracellular signaling and epidermal growth factor (EGF)-dependent cell proliferation. More importantly, robust EGFR downregulation after 72 hours with the antibody combination did not impair ADCC responses. In conclusion, imgatuzumab plus cetuximab leads to a strong downregulation of EGFR and superior cell growth inhibition in vitro without affecting antibody-induced ADCC responses. These findings support further clinical exploration of the antibody combination in EGFR wild-type NSCLC.

Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition

Tumor responses to cancer therapeutics are generally monitored every 2–3 months based on changes in tumor size. Dynamic biomarkers that reflect effective engagement of targeted therapeutics to the targeted pathway, so-called “effect sensors”, would fulfill a need for non-invasive, drug-specific indicators of early treatment effect. Using a proteomics approach to identify effect sensors, we demonstrated MUC1 upregulation in response to epidermal growth factor receptor (EGFR)-targeting treatments in breast and lung cancer models. To achieve this, using semi-quantitative mass spectrometry, we found MUC1 to be significantly and durably upregulated in response to erlotinib, an EGFR-targeting treatment. MUC1 upregulation was regulated transcriptionally, involving PI3K-signaling and STAT3. We validated these results in erlotinib-sensitive human breast and non-small lung cancer cell lines. Importantly, erlotinib treatment of mice bearing SUM149 xenografts resulted in increased MUC1 shedding into plasma. Analysis of MUC1 using serial blood sampling may therefore be a new, relatively non-invasive tool to monitor early and drug-specific effects of EGFR-targeting therapeutics.

Abstract LB-B11: Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with the HER-targeting tyrosine kinase inhibitor lapatinib can lead to a rapid compensatory increase in expression, signaling activity and relocalization of HER3 to the plasma membrane, which may attenuate the response to lapatinib. This might imply a potential role for a more dynamic assessment of HER3 tumor status using molecular imaging techniques, such as positron emission tomography (PET), instead of immunohistochemical HER3 staining on tumor biopsies. Here, we explored the feasibility of a dynamic assessment of HER3 status during lapatinib treatment in human breast cancer xenografts using zirconium-89 labeled anti-human HER3 monoclonal antibody (mAb) as a potential tracer for animal PET imaging. The anti-human HER3 mouse mAb MAB3481 was used for all experiments. The effect of lapatinib treatment on HER3 expression and HER3 mAb internalization in human breast cancer cell lines SKBR3 and BT474 was determined using flow cytometry. Biodistribution was performed using 89Zr-anti-HER3 mAb in mice bearing BT474 or SKBR3 tumors. Mice received daily vehicle or a lapatinib dose of 25, 50 or 100 mg/kg orally. A tracer dose of 89Zr-anti-HER3 mAb combined with the aspecific tracer 111In-IgG was injected 3 days after treatment. Ex vivo organ distribution assessment of 89Zr-anti-HER3 mAb was performed 6 days after tracer injection. Ex vivo tumor analysis using western blotting, ELISA and immunohistochemistry were performed to measure HER3 levels. In vitro, lapatinib treatment resulted in a ∼2-fold increase in membranous HER3 expression and HER3 internalization in SKBR3 and BT474 tumor cells. 89Zr-anti-HER3 mAb tumor uptake was significantly higher compared to 111In-IgG uptake in BT474 (P In conclusion, HER3-specific uptake of 89Zr-anti-HER3 mAb was shown in breast cancer xenografts. HER3 upregulation after lapatinib treatment was related to an enhanced 89Zr-anti-HER3 mAb uptake in these xenografts. These promising data warrant future dynamic assessment of HER3 status with 89Zr-anti-HER3 mAb PET imaging. Citation Format: Arjan Kol, Martin Pool, Steven de Jong, Elisabeth GE de Vries, Marjolijn N. Lub-de Hooge, Anton GT Terwisscha van Scheltinga. Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B11.

Abstract A86: Extracellular domain shedding influences specific tumor uptake and kinetics of EGFR PET tracer 89Zr-imgatuzumab

Background Overexpression and mutations of epidermal growth factor receptor (EGFR) are associated with tumor cell growth, differentiation, proliferation, apoptosis and cellular invasiveness. Imgatuzumab is a novel EGFR monoclonal antibody (mAb), glycoengineered for enhanced antibody-dependent cellular cytotoxicity. Molecular imaging using radiolabeled mAbs can potentially support decision making during (pre)clinical development and clinical practice. However, preclinical EGFR imaging studies revealed a mismatch between in vivo EGFR expression levels and tumor tracer uptake. Factors suggested causing the mismatch include differences in perfusion rates, vascularity, vascular permeability, interstitial pressure and mAb plasma half-life. Another factor that might influence tracer kinetics is shed EGFR (sEGFR) extracellular domain (ECD), which is found in sera of cancer patients with EGFR expressing tumors. We radiolabeled imgatuzumab with zirconium-89 (89Zr) and determined the influence of sEGFR on 89Zr-imgatuzumab tracer kinetics and tumor uptake in xenograft models. Methods Imgatuzumab was conjugated to N-Suc-desferal and radiolabeled with 89Zr. MicroPET imaging was performed 24, 72 and 144 hours post injection of 10, 25 and 160 μg 89Zr-imgatuzumab (5 MBq). As a non-specific control, 111In-DTPA-IgG (1 MBq) was used in equal doses in the same animals. Imaging studies were performed in A431 (human epidermoid carcinoma, EGFR overexpressing) and A549 and H441 (both human non-small cell lung cancer, medium EGFR expressing) subcutaneous xenograft bearing mice. Ex vivo biodistribution analysis was performed after the last scan. sEGFR levels in liver lysates and plasma were obtained using a human EGFR ECD ELISA assay. Results Increasing 89Zr-imgatuzumab dose from 10 to 160 μg enhanced tumor uptake in A431 bearing mice from 8.7 ± 3.1 to 31.4 ± 11.6% ID/g. On contrary, dose escalation between 10 and 160 μg 89Zr-imgatuzumab lowered tumor uptake from 13.8 ± 5.9 to 6.7 ± 0.8% ID/g in A549 and from 27.6 ± 3.6 to 15.5 ± 3.2% ID/g in H441 bearing mice. High liver uptake of 22.0 ± 5.4% ID/g was observed in A431 tumors at 10 μg 89Zr-imgatuzumab, this was higher than A549 (7.4 ± 1.5% ID/g, p Conclusion 89Zr-imgatuzumab effectively accumulates in EGFR expressing tumors. A431 tumors extensively shedded EGFR, which highly influenced 89Zr-imgatuzumab kinetics in A431 bearing mice. These results support the use of shed antigen measurements and subsequent tracer dose adjustment in future EGFR imaging studies. Citation Format: Martin Pool, Arjan Kol, Marjolijn N. Lub-de Hooge, Christian A. Gerdes, Steven de Jong, Elisabeth G.E. de Vries, Anton G.T. Terwisscha van Scheltinga. Extracellular domain shedding influences specific tumor uptake and kinetics of EGFR PET tracer 89Zr-imgatuzumab. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A86.

Abstract 4511: Differential effects of GA201 and cetuximab on EGFR expression and endosomal recycling in non-small cell lung cancer cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA GA201 is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) that is optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and inhibition of EGFR signal transduction. Recently, combinations of EGFR mAbs were shown to be more potent in inducing EGFR downregulation and superior in controlling growth in several tumor types, including breast and lung cancer. Currently, it is unknown how internalization and membrane recycling of EGFR after treatment with GA201, and in combination with the anti-EGFR mAb cetuximab, will affect GA201-induced ADCC response and tumor growth inhibition. In the present study we have investigated the effects of the GA201 and cetuximab on EGFR expression and endosomal trafficking in non-small cell lung cancer cell lines (NSCLC). Effects of GA201, cetuximab and the combination of these mAbs on EGFR were studied in a panel of NSCLC cell lines (A549, H441, H322 and H292). The influence of mAb binding on EGFR membrane expression, internalization and endocytic trafficking was determined using flow cytometry and immunofluorescence. For the internalization experiments, cells were pre-incubated with the mAbs for one hour at 4 °C. The effects on total EGFR protein levels and downstream signaling were studied using Western blotting. Treatment of cells with GA201 or cetuximab at 37 °C triggered EGFR internalization as demonstrated with flow cytometry. During treatment with either antibody alone for 1, 2 and 4 hours, EGFR reappeared at the cell surface. Immunofluorescence demonstrated the presence of GA201 and cetuximab in early endosomes and recycling endosomes, which can explain the EGFR reappearance on the cell surface. Interestingly, after 24 and 72 hours incubation with GA201 membranous EGFR levels were diminished to 50%, whereas cetuximab had no effect on membranous EGFR expression levels. Treating cells with the combination of the two mAbs resulted in EGFR internalization, downregulation of membranous EGFR levels with 80% and a 2-fold reduction in cellular EGFR protein levels as shown by Western blotting. When combined, most of the internalized GA201 and cetuximab colocalized with the lysosomes, and were almost absent in early endosomes and recycling endosomes. Moreover, the combination efficiently inhibited EGF-induced phosphorylation of downstream signaling molecules. In conclusion, GA201 downregulated membranous EGFR levels, whereas cetuximab had no effect. GA201 in combination with cetuximab leads to stronger downregulation of membranous and cellular EGFR levels by inhibiting endosomal recycling and increasing EGFR degradation in the endosmal/lysosomal compartment compared to both antibodies separately. We are currently investigating the effects of the single agents and the combination on EGFR signaling, ADCC response and tumor growth. Funded by Hoffmann-La Roche AG and ERC grant OnQview Citation Format: Arjan Kol, Steven de Jong, Martin Pool, Elisabeth G.E. de Vries, Christian A. Gerdes, Anton G.T. Terwisscha van Scheltinga. Differential effects of GA201 and cetuximab on EGFR expression and endosomal recycling in non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2014-4511

Abstract 4931: ImmunoPET and fluorescence imaging with Zirconium-89 and IRDye 800CW labeled glycoengineered epidermal growth factor receptor antibody GA201

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: GA201 is a novel epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), glycoengineered for enhanced antibody dependent cell-mediated cytotoxicity (ADCC). ADCC response is influenced by the amount of antibody bound to the membrane. Therefore we investigated specific accumulation and membrane binding of GA201 on tumor cells in vitro and in human cancer xenografts in vivo using Zirconium-89 (89Zr) and IRDye 800CW (800CW ) labeled GA201. Furthermore, 89Zr-GA201 and 800CW-GA201 were tested in human tumor bearing mice in order to visualize EGFR tumors expression in vivo. Methods: GA201 was conjugated with N-Suc-TFP-desferal and labeled with 89Zr or 800CW-NHS. EGFR membrane binding and internalization of GA201 was determined in vitro using 800CW-GA201 immunofluorescence microscopy and radio-immuno assay (RIA) with 89Zr-GA201 during 4 h at 37 ° C on the human cancer cell line A431 (epidermoid; EGFR overexpressing). 89Zr-GA201 and 800CW-GA201 were validated in human tumor bearing mice for quantification and visualization of EGFR-driven tumor uptake and biodistribution using micro positron emission tomography (microPET), near infrared (NIR) imaging and microscopic evaluation of tumor distribution. Biodistribution of 89Zr-GA201 was performed 6 days post injection (pi) using 10, 25 and 100 μg doses (1 MBq) in A431 subcutaneous(s.c.) xenografts. Serial micro positron emission tomography (microPET) scans were made on day 1, 3 and 6 pi in A431 s.c. xenografts, followed by biodistribution. All mice were co-injected with an equal dose of 111In-DTPA-ITC-IgG (1 MBq) as aspecific control. NIR imaging in A431 s.c. xenografts was performed daily up to 6 days pi using 100 μg 800CW-GA201, co-injected with 100 μg IRDye 680RD-IgG as aspecific control. At 1, 3 and 6 days pi mice were sacrificed for microscopic assessment. Results: Immunofluorescence shows membrane binding and internalization of 800CW-GA201. RIA showed 41.8 ± 3.3 % of 89Zr-GA201 internalized after 4 h incubation. Biodistribution showed tumor uptake of 89Zr-GA201 (specific) vs 111In-IgG (aspecific) of 9.5 ± 4.5 vs 6.4 ± 2.5 (ns, 10 μg dose), 13.5 ± 5.5 vs 5.7 ± 1.2 (p < 0.05, 25 μg dose) and 10.2 ± 3.5 vs 4.7 ± 1.0 (p < 0.05, 100 μg dose) %ID/g. The optimal dose was selected as 25 μg. Preferential tumor uptake of 89Zr-GA201 was seen on microPET scans. 800CW-GA201 (specific) vs 680RD-IgG (aspecific) NIR Imaging 6 days pi resulted in a tumor-to-background ratio (TBR) of 2.9 ± 0.3 vs 2.1 ± 0.4 (P < 0.05). 800CW-GA201 was mainly detected on the cell membrane in tumor tissue slices. Conclusion: GA201 binds to the outer cell membrane and internalizes in vitro. 89Zr-GA201 effectively accumulates in and visualizes EGFR expressing tumors, whereas 800CW-GA201 can be used to visualize EGFR expressing tumors locally. Acknowledgments: Supported by ERC Advanced Grant, OnQview and Hoffmann-La Roche AG. Citation Format: Martin Pool, Arjan Kol, Marjolijn N. Lub-De Hooge, Christian A. Gerdes, Steven de Jong, Elisabeth G.E. de Vries, Anton G.T. Terwisscha van Scheltinga. ImmunoPET and fluorescence imaging with Zirconium-89 and IRDye 800CW labeled glycoengineered epidermal growth factor receptor antibody GA201. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4931. doi:10.1158/1538-7445.AM2014-4931