Arjan Kol

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

BackgroundCigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.MethodsWe studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.ResultsWe observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.ConclusionThe observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

HER3, serious partner in crime Therapeutic approaches and potential biomarkers for effect of HER3-targeting

The human epidermal growth factor receptor (HER) family members are targeted by a growing numbers of small molecules and monoclonal antibodies. Resistance against the epidermal growth factor receptor (EGFR) and HER2-targeting agents is a clinically relevant problem forcing research on optimizing targeting of the HER family. In view of its overexpression in tumors, and compensatory role in HER signaling, HER3 has gained much interest as a potential additional target within the HER family. It is the only member of the HER family lacking intrinsic tyrosine kinase activity and therefore its role in cancer has long been underestimated. Drugs that block HER3 or interfere with HER3 dimer signaling, including fully human anti-HER3 antibodies, bispecific antibodies and tyrosine kinase inhibitors (TKIs), are currently becoming available. Several compounds have already entered clinical trial. In the meantime potential biomarkers are tested such as tumor analysis of HER3 expression, functional assays for downstream effector molecules and molecular imaging techniques. This review describes the biology and relevance of HER3 in cancer, agents targeting HER3 and potential biomarkers for effect of HER3-targeting.

Associate editor: B. TeicherHER3, serious partner in crime: Therapeutic approaches and potential biomarkers for effect of HER3-targeting

The human epidermal growth factor receptor (HER) family members are targeted by a growing numbers of small molecules and monoclonal antibodies. Resistance against the epidermal growth factor receptor (EGFR) and HER2-targeting agents is a clinically relevant problem forcing research on optimizing targeting of the HER family. In view of its overexpression in tumors, and compensatory role in HER signaling, HER3 has gained much interest as a potential additional target within the HER family. It is the only member of the HER family lacking intrinsic tyrosine kinase activity and therefore its role in cancer has long been underestimated. Drugs that block HER3 or interfere with HER3 dimer signaling, including fully human anti-HER3 antibodies, bispecific antibodies and tyrosine kinase inhibitors (TKIs), are currently becoming available. Several compounds have already entered clinical trial. In the meantime potential biomarkers are tested such as tumor analysis of HER3 expression, functional assays for downstream effector molecules and molecular imaging techniques. This review describes the biology and relevance of HER3 in cancer, agents targeting HER3 and potential biomarkers for effect of HER3-targeting.

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.

TGF-β induced CXCL13 in CD8+ T cells is associated with tertiary lymphoid structures in cancer

Coordinated immune responses against human tumors are frequently characterized by tertiary lymphoid structures (TLS) which predict improved prognosis. The development of TLS is dependent on the chemokine CXCL13, reported to be secreted by dendritic cells and follicular helper T cells only. We report the unexpected finding that CXCL13 is also secreted by activated CD8+ T cells following stimulation by transforming growth factor beta (TGF-β). Using single cell RNA sequencing we found that expression of CXCL13 in CD8+ T cells was restricted to the intraepithelial CD103+ population. Accordingly, CD8+ T cells activated in the presence of TGF-β simultaneously upregulated CD103 and secreted CXCL13. CXCL13 expression was strongly correlated with neo-antigen burden and cytolytic gene signatures in bulk tumors. In line with this, TLS were abundant in neo-antigen-high, CD103+ T cell-enriched tumors. TGF-β thus appears to play a role in coordinating immune responses against human tumors through CD8-dependent CXCL13-associated formation of TLS.

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.

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