HER family dynamics to guide cancer therapy

University of Groningen

Preclinical evaluation and molecular imaging of HER family dynamics to guide cancer therapy Kol, Klaas Jan-Derk

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HER family dynamics to guide cancer therapy

Proefschrift

Ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op woensdag 12 april 2017 om 12.45 uur

door

Arjan Kol

geboren op 2 maart 1987 te Duiven

Kol, K.J.

Preclinical evaluation and molecular imaging of HER family dynamics to guide cancer therapy Thesis, University of Groningen, Groningen, The Netherlands

ISBN: 978-94-034-1339-6

ISBN (electronic version): 978-94-034-1338-9

© K.J. Kol, 2018

All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, mechanically, by photocopying, recording, or otherwise, without prior written permission of the author.

Cover: Photo by Danique Giesen.

Printing by: Ipskamp Printing, Enschede, The Netherlands.

The research presented in this thesis was financially supported by European Research Council (ERC) advanced grant 293445, OnQview, a Roche Innovation Fund grant, a POINTING grant of the Dutch Cancer Society and a De Cock Stichting grant.

Printing of this thesis was supported by:

Stichting Werkgroep Interne Oncologie Graduate School of

Medical Sciences

Preclinical evaluation and

molecular imaging of HER family dynamics to guide cancer therapy

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op woensdag 30 januari 2019 om 12.45 uur

door

Klaas Jan-Derk Kol geboren op 2 augustus 1988

te Ten Boer

Promotores:

Prof. dr. S. de Jong Prof. dr. E.G.E. de Vries Copromotores:

Dr. A.G.T. Terwisscha van Scheltinga Beoordelingscommissie:

Prof. dr. N.H. Hendrikse Prof. dr. H.J.M. Groen Prof. dr. F.A.E. Kruyt

Chapter 1 General introduction and outline of thesis 9

Chapter 2 ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

Oncotarget. 2017;8:45432-45446

19

Chapter 3 Extracellular domain shedding influences specific tumor uptake and organ distribution of the EGFR PET tracer ⁸⁹Zr-imgatuzumab

Oncotarget. 2016 18;7:68111-68121

49

Chapter 4 HER3, serious partner in crime: therapeutic approaches and potential biomarkers for effect of HER3-targeting

Pharmacol Ther. 2014;143:1-11

69

Chapter 5 ⁸⁹Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

MAbs. 2017;9:1370-1378

99

Chapter 6 MAPK pathway activity plays a key role in programmed death ligand-1 expression of EGFR wild-type lung adenocarcinoma cells

Submitted

121

Chapter 7 Summary, general discussion and future perspectives 149 Chapter 8 Nederlandse samenvatting (summary in Dutch) 163

Appendix 1 Dankwoord (acknowledgements) 171

CHAPTER

General Introduction 1

10

Chapter 1

BACKGROUND

Cancer remains one of the leading causes of death worldwide (1). Current cancer treatment strategies consist of surgery, radiotherapy and systemic treatment. Using conventional multimodality therapy, the survival rates for certain tumor types still could benefit from major improvements (2). Moreover, the cancer incidence is increasing globally. This demonstrates an urgent need for new, effective anti-cancer therapies.

During the past two decades, the paradigm for cancer treatment has expanded alongside relatively nonspecific chemotherapeutic drugs, such as taxanes and platinum-based anticancer drugs, to molecularly targeted therapeutics and cancer immunotherapy.

Targeted therapies aim to inhibit molecular pathways important for tumor growth and survival, whereas cancer immunotherapy stimulates a host immune response to induce tumor cell kill. Targeted therapies can also have an effect on the immune system by affecting pathways that are crucial for immune development and function. Moreover, therapeutic monoclonal antibodies targeting specific tumor antigens can induce antibody-dependent cellular cytotoxicity (ADCC). Combining these approaches are part of current treatment strategies for many types of cancer, including lung, breast, colon, gastric, head and neck and ovarian cancer as well as melanoma.

One of the first known molecular targets has been the extensively studied human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. The family comprising the epidermal growth factor receptor (EGFR) (also known as HER1), HER2, HER3 and HER4, is not only essential for the development and maintenance of normal tissues, but is also strongly involved in the development of many types of cancer (3,4). Upon ligand binding, these receptors homodimerize or interact with each other, forming heterodimers. Dimerization of the receptors results in activation of downstream signaling cascades such as the RAS-extracellular signal-regulated kinase (ERK) pathway and phosphoinositide 3-kinase (PI3K)/Akt pathway, thereby controlling many biological processes in normal tissue. However, these HER signaling pathways are frequently hyperactivated in cancer due to receptor overexpression, autocrine stimulation, crosstalk with other receptors and/or mutations in components of these pathways (5).

EGFR and HER2 are widely used targets for cancer therapy. In view of its overexpression in tumors and compensatory role in HER signaling, HER3 has gained much interest as an additional potential drug target as well. Drugs that block HER family members or inhibit downstream signaling by HER family members, including human anti-HER antibodies and tyrosine kinase inhibitors (TKIs), are increasingly becoming available. Anti-HER monoclonal antibodies can exert their action via a variety of mechanisms, including blocking (hetero)dimerization and ligand binding, as well as inducing HER endocytosis, complement dependent cytotoxicity (CDC) and ADCC (6–10). Several EGFR- and HER2-

1

targeted monoclonal antibodies and TKIs have already been approved by the Food and Drug Administration USA (11). Furthermore, a number of monoclonal antibodies that target HER3 have entered clinical trials (12). Nonetheless, intrinsic and acquired resistance against HER-targeted agents is a clinical problem, and much research focuses on optimizing targeting of the HER family (13,14).

Rapid non-invasive assessment of changes in HER protein expression as a result of treatment with HER-targeted agents would be of major interest for clinical decision making. HER-targeted agents affect many processes involved in HER protein expression, dynamics and availability, such as a) ligand binding, b) receptor dimerization, shedding, internalization, recycling to the cell surface and degradation, c) mobilization of internal receptor pools, and d) induction of HER family gene expression. In addition, the size of receptor-antibody complexes influences the amount of internalization and recycling, and subsequent degradation of receptors (15). A good example of these drug-induced changes in HER protein dynamics is the treatment of HER2-driven breast cancer with the EGFR/HER2 TKI lapatinib that has been shown to induce a transcriptional and posttranslational compensatory increase in HER3, which may attenuate antitumor efficacy of this drug (16).

Because of the dynamic HER membrane expression, a non-invasive dynamic assessment of HER tumor status in vivo will provide important supplementary information that cannot be obtained with invasive static techniques such as immunohistochemistry on biopsy material. Non-invasive molecular imaging, defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level, could potentially be used as a tool to monitor receptor dynamics (17). Molecular characteristics, such as HER cell surface expression, can be imaged with three-dimensional whole body positron emission tomography (PET) using antibodies radiolabeled with zirconium-89 (⁸⁹Zr, t½ = 78.4 h), for optimal lesion accumulation. HER antibody-based PET-imaging could play an important role in identifying patients who might benefit from anti-HER antibody therapy, as well as monitoring responses to HER-targeting agents. Several (pre-) clinical studies have assessed in vivo treatment effects of drugs targeting HER2 such as lapatinib, trastuzumab or HSP90 inhibitors by quantifying the (change in) radiolabeled tracer accumulation in tumors (18–22). Results showed that these drugs reduced tumor uptake of radiolabeled anti-HER2 antibodies.

HER-targeted therapies can also influence dynamics of other plasma membrane proteins, such as immune checkpoints. Immune checkpoints refer to multiple co-stimulatory or inhibitory protein interactions that (among others) regulate T cell responses to antigens (23,24). These protein interactions are essential in self-tolerance and limit collateral

12

Chapter 1

tissue damage when the immune system is responding to pathogenic infections.

Tumors can exploit these immune checkpoints by upregulating inhibitory proteins and downregulating stimulatory proteins. This dampens T cell effector functions by inhibiting signaling downstream of the T cell receptor and, as a consequence, protects tumors from immune-mediated rejection. Programmed cell death 1 (PD-1) and its ligand PD-L1 have emerged as critical immune checkpoint proteins in cancer. PD-L1 expressed on tumor cells causes anergy and apoptosis of activated T cells by binding to PD-1 expressed on the surface of these T cells. PD-1/PD-L1 checkpoint blockade with antibodies, which are examples of cancer immunotherapy, has resulted in significant long lasting responses in patients with advanced-stage cancers, including heavily pretreated non-small cell lung cancer (NSCLC) and melanoma patients (25–27). Recently, it has been shown that EGFR plays a role in PD-L1 expression by tumor cells. EGFR inhibition reduces PD-L1 expression in vitro, resulting in a concomitant increase in T cell activation (28,29). Therefore, EGFR inhibition may be used to improve efficacy of immunotherapy.

Taken together, these results indicate that plasma membrane protein dynamics might well be important for proper design of (combination) therapies. Therefore, the aim of this thesis is to gain more insight in the effect of HER-targeting agents on HER and PD- L1 dynamics to provide a rationale for future combination therapies. In addition, HER tracers for molecular imaging to evaluate drug tumor targeting, organ distribution and target dynamics are explored.

OUTLINE OF THESIS

Anti-EGFR monoclonal antibody combinations can effectively inhibit the EGFR signaling pathway, and have shown superior anticancer efficacy in several human tumor xenograft models (30–32). Cetuximab and imgatuzumab are both monoclonal antibodies directed against distinct, non-overlapping epitopes in EGFR extracellular domain III. Imgatuzumab, in addition, is glycoengineered to enhance ADCC responses (33). Thus, combining these antibodies is a potential strategy to target EGFR more effectively than existing treatments with single antibodies. It is unknown whether treatment with imgatuzumab or the combination with cetuximab increases EGFR internalization and/or receptor membrane recycling in cancer cells, potentially reducing ADCC responses. In chapter 2, we investigate the effects of imgatuzumab and cetuximab on EGFR dynamics and intracellular signaling. In addition, we monitor whether changes in EGFR dynamics affect ADCC responses and tumor cell growth inhibition. Effects of imgatuzumab, cetuximab and the combination of these monoclonal antibodies on EGFR are studied in a panel of NSCLC cell lines. The effect of antibody binding on EGFR cell surface dynamics is determined using flow cytometry and immunofluorescence. In addition, the effect of antibody binding on total EGFR protein levels, downstream signaling and growth are

1

studied using Western blotting and proliferation assays. Finally, ADCC assays are used to investigate effects of EGFR dynamics on ADCC responses.

Whole body analysis of EGFR expression in tumor lesions using PET imaging could support decision making during clinical development and clinical practice. Preclinical EGFR- microPET imaging revealed a mismatch between in vivo tumor EGFR expression assessed using Western blotting and tumor uptake of the radiolabeled EGFR antibody tracer (34).

Shed EGFR ectodomain, which is present in cancer patient sera, can potentially bind tracer and therefore affect tracer kinetics (35). In chapter 3, we examine the influence of shed EGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody

89Zr-imgatuzumab in varying xenograft models in order to optimize ⁸⁹Zr-imgatuzumab PET. Human A431 (EGFR overexpressing, epidermoid carcinoma of the vulva), A549 and H441 (both EGFR medium expressing, NSCLC) cancer cell lines are xenografted in mice. The A431 cell line is used, because of its known EGFR shedding potential. Tumor bearing mice receive various doses of ⁸⁹Zr-imgatuzumab, co-injected with equal doses of ¹¹¹In-IgG as internal control. MicroPET scans are performed at multiple time points post tracer injection, followed by biodistribution analysis. Shed EGFR levels in liver and plasma samples, as well as in vitro culture media are determined by ELISA.

HER3 is the only member of the HER family having impaired tyrosine kinase activity and therefore its role in cancer has long been underestimated. However, in view of its overexpression in various tumor types and its compensatory role in HER signaling, HER3 has gained much interest as a potential target in cancer treatment. A review, chapter 4, describes the biology and relevance of HER3 in cancer, as well as drugs that block HER3 or interfere with HER3 heterodimer signaling. These drugs include fully human anti- HER3 antibodies, bispecific antibodies and TKIs, and are currently becoming available for clinical use. An overview of HER3-targeting drugs in clinical trials is given. Biomarkers might be useful for prediction and monitoring of treatment effects, as well as supporting decision making during clinical development and clinical practice. Therefore, potential biomarkers for effective HER3-targeting such as tumor analysis of HER3 expression, functional assays for downstream effector molecules and molecular imaging techniques are discussed.

HER3 membrane expression is very dynamic due to its compensatory role in HER signaling, particularly in response to drugs targeting HER family members or the PI3K pathway, which may attenuate the antitumor action of these inhibitors (13, 16, 36, 37).

Preclinically, combining HER3 with HER2 inhibitors augmented tumor growth inhibition, in vivo (38). This might advocate for a more dynamic assessment of HER3 tumor status than immunohistochemistry. Therefore, in chapter 5, we explore in vivo HER3 tumor

14

Chapter 1

status assessment with ⁸⁹Zr-labeled anti-HER3 antibody mAb3481 PET after treatment with the dual EGFR/HER2 inhibitor lapatinib. The effect of lapatinib on HER3 cell surface expression and mAb3481 internalization is evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, mice bearing BT474 or N87 xenografts receive lapatinib daily for 9 days. PET-scans of xenografts with ⁸⁹Zr- mAb3481 for visualization of HER3 are performed after 9 days lapatinib treatment.

PET imaging and ex vivo organ distribution data are compared with Western blot and immunohistochemical stainings of tumor tissue.

Recently, it was shown that constitutively activated mutant EGFR induces PD-L1 expression in NSCLC cell lines (29). EGFR inhibition reduces PD-L1 expression of these EGFR mutant cell lines, resulting in a concomitant increase in T cell activation However, in the patient setting, EGFR mutant NSCLC tumors do not respond to PD-1 blockade, even when PD-L1 expression is high. This is due to several factors such as lack of CD8+ T cell infiltration and low tumor mutational burden (39). In contrast, PD-1 blockade has improved survival of EGFR wild-type NSCLC patients (39–41). Surprisingly, there is only limited data about the regulation of PD-L1 expression in EGFR wild-type NSCLC (42). Improved understanding of PD-L1 regulation may provide a rationale to combine immune checkpoint inhibitors with other targeted agents. Therefore, in chapter 6, we aim to identify pathways that regulate PD-L1 expression in EGFR wild-type NSCLC by using RNA-sequencing data from The Cancer Genome Atlas (TCGA) lung adenocarcinoma and squamous cell lung carcinoma data sets. We functionally validate our findings using a panel of EGFR wild- type lung adenocarcinoma cell lines and cocultures with peripheral blood mononuclear cells. We assess the effect of the combination interferon gamma (IFNγ), the most potent inducer of PD-L1 expression (43), and EGF, an activator of EGFR-mediated signaling, on PD-L1 mRNA, protein and cell surface expression. Next, we investigate the effect of direct targeting of EGFR with cetuximab and erlotinib, or targeting of downstream signaling with XL147 (PI3K), everolimus (mammalian target of rapamycin 1 (mTORC1) and selumetinib (MAP/ERK kinase 1/2 (MEK1/2)) on PD-L1 expression. Effects on PD-L1 expression and downstream signaling are studied using qPCR, Western blotting and flow cytometry.

In chapter 7, the findings of this thesis are summarized, followed by a general discussion with future perspectives.

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CHAPTER

ADCC responses and blocking of EGFR- mediated signaling and cell growth by

combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

Arjan Kol¹ Anton G.T. Terwisscha van Scheltinga² Martin Pool¹ Christian A. Gerdes³ Elisabeth G.E. de Vries¹ Steven de Jong¹

1Department of Medical Oncology, ²Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands, ³Roche Pharma Research & Early Development, Roche Innovation Center Zürich, Schlieren, Switzerland.

Oncotarget 2017;8:45432-45446.

2

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Chapter 2

ABSTRACT

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.

2

INTRODUCTION

The epidermal growth factor receptor (EGFR), a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is an important regulator of cell growth and differentiation. Upon ligand binding, EGFR homodimerizes or interacts with other HER members, i.e. HER2 and HER3, to form heterodimers. This results in activation of downstream signaling cascades such as the RAS-ERK pathway and PI3K/Akt pathway, thereby controlling many biological processes. These pathways are frequently dysregulated via overexpression, autocrine stimulation, crosstalk with other receptors and/or mutations, and play a pivotal role in multiple tumor types (1).

Targeting EGFR is an important treatment modality for many solid tumors including non- small cell lung cancer (NSCLC). Treatment strategies to target EGFR consist of tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. At present, EGFR TKIs are standard of care for NSCLC patients with EGFR-mutated tumors as first- and second-line treatments (2). Cetuximab (IgG1 subtype) is an anti-EGFR monoclonal antibody approved by the Food and Drug Administration USA for colorectal cancer and head and neck squamous cell cancer. Anti-EGFR monoclonal antibody panitumumab (IgG2 subtype), on the other hand, is approved for treatment of colorectal cancer. These monoclonal antibodies can exert their action via a variety of mechanisms, including blocking (hetero)dimerization and ligand binding, as well as inducing EGFR endocytosis, complement dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) (3–7). ADCC is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 antibodies. Recent evidence suggests that addition of cetuximab to chemotherapy in first line improves overall survival of patients with EGFR-expressing advanced NSCLC (8). Interestingly, retrospective analyses revealed that mutational status and copy number of EGFR were not predictive for cetuximab benefit (9). Therefore, treatment of EGFR wild-type NSCLC with anti-EGFR monoclonal antibodies might be of interest. Unfortunately, the impact of cetuximab on survival is small and the prognosis of advanced NSCLC remains poor. There is a continuous need for new treatments to improve survival. A stronger inhibition of EGFR or higher level of ADCC can potentially contribute to this improvement.

Anti-EGFR monoclonal antibody combinations can enhance endocytic downregulation of EGFR, and these combinations showed superior anticancer efficacy in several human tumor xenograft models, including triple negative breast cancer and EGFR-mutant NSCLC (10–12). A higher level of ADCC can be achieved by glycoengineering the Fc region of a therapeutic antibody. Imgatuzumab (GA201) is a novel glycoengineered anti- EGFR monoclonal antibody of the IgG1 subclass, which is optimized to induce ADCC and inhibits EGFR signal transduction (13). Imgatuzumab showed superior preclinical

22

Chapter 2

in vivo efficacy compared with cetuximab and non-glycoengineered imgatuzumab in both KRAS-mutant and KRAS wild-type models (13). The clinical benefit of combining two monoclonal antibodies against EGFR is still unknown. Clinical benefit of combining antibodies has already been demonstrated for another HER family member, HER2, in breast cancer using the anti-HER2 antibodies trastuzumab and pertuzumab (14–

16). Trastuzumab binds to HER2 and suppresses its signaling capability. Pertuzumab complements the mechanism of action of trastuzumab by binding to another epitope of HER2, which inhibits the dimerization of HER2 with other HER receptors.

Imgatuzumab and cetuximab are directed against distinct, non-overlapping epitopes in EGFR extracellular domain III (13). Thus, the combination of both antibodies is a potential strategy to target EGFR more effectively than existing clinical single antibody treatments. It is unknown whether treatment with imgatuzumab or the combination with cetuximab increases EGFR internalization and/or reduces membranous turnover of EGFR in cancer cells, potentially diminishing ADCC responses. The aim of the present study was therefore to investigate the effects of imgatuzumab and cetuximab on EGFR dynamics, intracellular signaling and survival in a panel of human EGFR wild-type NSCLC cell lines. Finally, we monitored whether changes in EGFR dynamics affect ADCC responses and tumor cell growth inhibition.

RESULTS

Imgatuzumab combined with cetuximab strongly downregulates EGFR expression in NSCLC cells

All NSCLC cell lines expressed EGFR, with the highest cell surface levels found in H292 cells (Fig. 1A). Addition of cetuximab to imgatuzumab resulted in a nearly two-fold increase in mean fluorescence intensity of membranous EGFR (Fig. 1A), which is in line with previous findings that imgatuzumab and cetuximab are binding to non-overlapping epitopes in EGFR extracellular domain III (13). Next, we measured EGFR levels following incubation of cells with imgatuzumab and cetuximab alone or combined for 72 hours.

In the presence of imgatuzumab, membranous EGFR levels were diminished by 38% in SW-1573 and up to 75% for A549, whereas cetuximab had less effect (up to 26% for A549) (Fig. 1B). Treating cells with the combination of monoclonal antibodies resulted in a stronger downregulation of membranous EGFR levels ranging from 65% in SW-1573, up to 89% for A549. Similar results were observed with 24 hours incubation or twice the amount of each monoclonal antibody (Fig. S1A), which suggests an equilibrium in membranous turnover of EGFR. A non-glycoengineered GA201 (GA201_wt) was used to investigate the effect of antibody glycoengeneering on EGFR surface expression. GA201_wt and imgatuzumab had similar effects on membranous EGFR in SW-1573 and H292 cells, excluding the involvement of glycoengeneering (Fig. S1B).

2

# Cells

Fluorescence intensity SW-1573

Imgatuzumab + Cetuximab (MFI: 50.6) Imgatuzumab(MFI: 28.3)

Cetuximab (MFI: 26.5) Unspecific control

H441

Imgatuzumab + Cetuximab (MFI: 45.9) Imgatuzumab (MFI: 24.2)

Cetuximab (MFI: 23.6) Unspecific control Imgatuzumab + Cetuximab

(MFI: 49.7) Imgatuzumab (MFI: 27.4)

Cetuximab (MFI: 27.4) Unspecific control

H322

H292 A549

Imgatuzumab + Cetuximab (MFI: 82.9) Imgatuzumab (MFI: 43.5)

Cetuximab (MFI: 44.6) Unspecific control

Imgatuzumab + Cetuximab (MFI: 49.1) Imgatuzumab (MFI: 25.5)

Cetuximab (MFI: 26.4)

Unspecific control

H322

SW-1573 H441H292A549 H322

SW-1573 H441H292A549 H322

SW-1573 H441H292A549 0

20 40 60 80 100 120 140 160

$$$**

$$$*

*$

**$$

$$*

EGFR surface expression (% to control)

Imgatuzumab Cetuximab Imgatuzumab

+ Cetuximab

A

B

Figure 1:

Effect of anti-EGFR monoclonal antibody treatment on EGFR surface expression levels. A, Flow cytometric analysis of imgatuzumab and cetuximab binding alone or in combination in H322, SW-1573, H441, H292 and A549 cells. B, H322, SW-1573, H292, H441 and A549 cells were treated with the anti-EGFR monoclonal antibodies (20 µg/mL total) for 72 hours. Surface expression levels were determined using flow cytometry. The surface expression in untreated control cells was set at 100% both for the single antibodies and the combination.

(*P < 0.05, **P < 0.01 combination vs imgatuzumab; $P < 0.05, $$P < 0.01, $$$P < 0.001 combination vs cetuximab; unpaired t-test). Data points are mean + SD (n = 3).

24

Chapter 2

Western blot analyses demonstrated that treatment of SW-1573, H292 and A549 cells with imgatuzumab alone or combined with cetuximab led to a decrease in total cellular EGFR protein levels as well (Fig. 2). Both single agents and the combination efficiently inhibited EGF-induced phosphorylation of downstream signaling molecules such as Akt and ERK1/2 (Fig. 2 and Fig. S2). In H292, only the combination was able to completely inhibit EGF-induced Akt and ERK1/2 phosphorylation. Interestingly, treatment with imgatuzumab or cetuximab increased EGFR phosphorylation at Tyr1068 and Tyr1173, but did not lead to increased phosphorylation of Akt or ERK1/2. Despite a lower level of phosphorylated EGFR at both phosphorylation sites, EGF was able to activate EGFR downstream signaling in contrast to imgatuzumab or cetuximab. Addition of cetuximab to imgatuzumab counteracted the increase in EGFR phosphorylation. GA201_wt and imgatuzumab had similar effects on EGFR protein levels and phosphorylation in SW- 1573 and H292 cells (Fig S3A).

EGFR p-EGFR^(Y1068) p-EGFR^(Y1173) ERK1/2 p-ERK1/2 AKT p-AKT(T308)

Actin EGF:Imgatuzumab:

Cetuximab:

− − − − + + + +

− + − + − + − +

− − + + − − + +

− − − − + + + +

− + − + − + − +

− − + + − − + +

− − − − + + + +

− + − + − + − +

− − + + − − + +

0.0 0.5 1.0 1.5

EGFR/actin

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5

0 2 4 6 8 10

pEGFR(Y1068)/actin

0 5 10 15 20

0 2 4 6 8

Figure 2:

Western blot analysis of the effect of 24 hours anti-EGFR monoclonal antibody treatment on EGFR total protein levels and downstream signaling under normal growth conditions or 15 min EGF stimulation (10 ng/mL). The immunoreactive spots were quantified by densitometric analysis and normalized using actin (see also Fig. S2).

Values are expressed as fold increase versus control + SD. All experiments were performed in triplicate.

SW-1573 H292 A549

2

Taken together these results indicate a strong reduction in EGFR cell surface expression and total cellular EGFR expression by continuously exposing cells to imgatuzumab, which was not observed with cetuximab. Combining imgatuzumab with cetuximab augments the reduction in EGFR expression.

Non-internalized

0 1 2 3 4

0 20 40 60 80 100 120

Cetuximab Imgatuzumab

Imgatuzumab + Cetuximab

$$$**

$$$ $$

Time at 37 °C (hours) Non-internalized EGFR-antibody complexes (% to control)

Surface expression

0 1 2 3 4

0 20 40 60 80 100 120

Cetuximab Imgatuzumab

Imgatuzumab + Cetuximab

***$$$

***$$$

$$$**

Time at 37 °C (hours)

EGFR surface expression (% to control)

Non-internalized

0 1 2 3 4

0 20 40 60 80 100 120

Imgatuzumab

Imgatuzumab + Cetuximab

Cetuximab ^***$$$

***$$$

***

Time at 37 °C (hours) Non-internalized EGFR-antibody complexes (% to control)

Surface expression

0 1 2 3 4

0 20 40 60 80 100 120

Imgatuzumab Cetuximab

Imgatuzumab + Cetuximab

***$$$

***$$$

***$$

Time at 37 °C (hours)

EGFR surface expression (% to control)

SW-1573

H292 SW-1573

H292

A

B

Figure 3:

Influence of monoclonal antibody binding on EGFR internalization and surface expression. SW-1573 (A) and H292 cells (B) were surface labeled with the anti-EGFR monoclonal antibodies on ice and incubated at 37 °C for the indicated times, and then analyzed for the non-internalized EGFR-antibody complexes (left) and cell surface expression (right). The lower the amount of non-internalized EGFR-antibody complexes, the higher the amount of internalized antibody-EGFR complexes. The surface expression at t=0 was set at 100%. The non- internalized and surface expression were determined as described in Materials and Methods (n=3). Data points are mean ± SD. (**P < 0.01, ***P < 0.001 combination vs imgatuzumab; $$P < 0.01, $$$P < 0.001 combination vs cetuximab; two-way ANOVA followed by Bonferroni post-test).

26

Chapter 2

Imgatuzumab combined with cetuximab enhances EGFR internalization and reduces EGFR turnover at the plasma membrane

Flow cytometry was used to determine whether the downregulation of EGFR is due to enhanced internalization of this receptor. SW-1573 and H292 cells were pre-loaded with imgatuzumab and cetuximab on ice and subsequently incubated at 37 °C for the indicated time points. Experiments with radiolabeled monoclonal antibodies showed a minimal release of monoclonal antibodies from cells during 4 hours (results not shown), indicating that antibody internalization is a measurement of EGFR internalization.

Imgatuzumab and cetuximab were internalized at similar rates in SW-1573 (Fig. 3A) and H292 cells (Fig. 3B), as demonstrated by a decrease in non-internalized EGFR-antibody complexes. GA201_wt and imgatuzumab had similar effects on EGFR internalization and surface levels in SW-1573 and H292 cells (Fig. S3B). Despite the rapid internalization of these complexes, overall EGFR cell surface levels marginally changed by single antibody treatment, indicating that membranous turnover of EGFR was not rate limiting within this timeframe. Imgatuzumab in combination with cetuximab, however, accelerated EGFR internalization in SW-1573 and H292 cells, which resulted in a strong reduction of overall EGFR cell surface levels. Pretreatment with the protein synthesis inhibitor cycloheximide reduced basal EGFR surface levels. However, the percentage internalized antibody–EGFR complexes and the percentage EGFR surface expression on SW-1573 and H292 cells after 2 hours incubation with antibodies at 37 °C were proportionally similar to the results in antibody-treated cells without cycloheximide pretreatment (results not shown). These findings indicate that, within this 2 hours timeframe, the absolute level of membranous EGFR was affected by cycloheximide treatment, whereas the kinetics of antibody-induced internalization and (re)appearance of EGFR on the cell surface was predominantly determined by the antibody used.

EGFR tyrosine kinase inhibition by erlotinib does not affect EGFR internalization and degradation by imgatuzumab combined with cetuximab

We sought to determine whether imgatuzumab-induced EGFR phosphorylation is instrumental in the strong reduction in EGFR protein levels. SW-1573, H292 and A549 cells were treated with erlotinib to inhibit EGFR tyrosine kinase activity. Co-treatment of cells with erlotinib and the monoclonal antibodies individually for 24 hours showed that erlotinib indeed reduced p-EGFR levels (Fig. 4A). Interestingly, erlotinib inhibited imgatuzumab-induced EGFR protein degradation, but had no effect on EGFR degradation by the antibody combination. It should be noted that erlotinib treatment alone increased EGFR expression in H292 and A549 cells, which makes these results more difficult to interpret. To investigate the influence of EGFR kinase activity on monoclonal antibody- induced internalization in a short-term experiment, SW-1573 cells were pretreated for 24 hours with erlotinib. Erlotinib did not reduce internalization of antibody-EGFR

2

complexes (Fig. 4B). In fact, total EGFR surface expression was significantly reduced when the antibody combination was added to erlotinib-pretreated cells. These results demonstrate that p-EGFR inhibition is not antagonizing EGFR internalization and degradation induced by the antibody combination.

Figure 4:

Effects of TKI treatment on monoclonal antibody-induced internalization and degradation. A, Western blot analysis of the effect of 24 hours anti-EGFR monoclonal antibody treatment (20 µg/mL total) with or without erlotinib (10 µM) on EGFR total protein levels in SW-1573, H292 and A549. Data points are mean + SD. (*P <

0.05, **P < 0.01, ***P < 0.001 compared to untreated control; $P < 0.05, $$P < 0.01, $$$P < 0.001 compared to erlotinib treated control). B, SW-1573 cells were pretreated for 24 hours with the EGFR TKI inhibitor erlotinib (10 µM). Cells were subsequently surface labeled with the anti-EGFR monoclonal antibodies (20 µg/mL total) on ice and incubated at 37 °C for 4 hours, and then analyzed for the non-internalized EGFR-antibody complexes (NI) and cell surface expression (SE). The lower the amount of non-internalized EGFR-antibody complexes, the higher the amount of internalized antibody-EGFR complexes. Surface expression of the control and erlotinib treated cells were both set at 100%. 24 hours erlotinib treatment resulted in a slight upregulation (10 – 15 % increase). Data points are mean ± SD (n = 3). (*P < 0.05).

NI SE NI SE NI SE NI SE NI SE NI SE

0 25 50 75

100

*

Membranous EGFR antibody (% to surface expression at t=0)

− + − + − + − +

− − + + − − + +

− + − + − + − +

− − + + − − + + Erlotinib

− + − + − + − +

− − + + − − + + Erlotinib

SW-1573 A549

Erlotinib Control Imgatuzumab Cetuximab Imgatuzumab

+ Cetuximab

Erlotinib

EGFR p-EGFR(Y1068)

Actin

H292

Imgatuzumab:

Cetuximab:

0.0 0.5 1.0 1.5

EGFR/actin

0.0 0.5 1.0 1.5 2.0

0.0 0.5 1.0 1.5 2.0

** ***

**

* **

**

**

$$

$$ $

$$$

$ $

$$$

A

B

28

Chapter 2

Imgatuzumab combined with cetuximab increases clustering of EGFR-antibody complexes and targets EGFR to the lysosomal degradation pathway

The intracellular localization of EGFR after antibody treatment was investigated with immunofluorescent microscopy, focusing on the endocytic and lysosomal degradation pathways. Staining of SW-1573 cells with imgatuzumab and/or cetuximab as primary antibodies clearly showed membranous EGFR fluorescence (Fig. 5A). SW-1573 and H292 cells were subsequently incubated with the anti-EGFR monoclonal antibodies on ice and then chased for 4 hours at 37 °C. Imgatuzumab and cetuximab localized to internal vesicles (Fig. 5A and S4B). To exclude the possibility that the incubation with secondary antibodies triggers cluster formation due to crosslinking of monoclonal antibodies, SW-1573 cells were incubated with imgatuzumab directly labeled with Dylight 633.

Cluster formation was still observed when imgatuzumab Dylight 633 was combined with cetuximab (Fig. S4A). We then stained SW-1573 and H292 cells for EEA1, a marker for early endosomes. Imgatuzumab and cetuximab mostly co-localized with EEA1-positive vesicles, indicating internalized monoclonal antibodies (Fig. 5A and S4B). Following incubation of cells for 4 hours at 37 °C, the antibody combination localized to large clusters at the surface and/or intracellular vesicles (Fig. 5A and S4B). In cells treated with imgatuzumab combined with cetuximab, antibody-positive vesicles were only partially co-localizing with EEA1-positive vesicles, suggesting that the EGFR antibody combination complexes are localized in a different compartment. Therefore, cells were stained for LAMP1 to identify the lysosomal compartment. The monoclonal antibodies did not co- localize with lysosomes (data not shown), which may be due to degradation of receptor antibody-complexes in lysosomes. Adding additional anti-EGFR monoclonal antibodies after fixation showed that EGFR predominantly co-localized with lysosomes in cells treated with both imgatuzumab and cetuximab (Fig. 5A and S4B). Blocking lysosomal proteolysis by bafilomycin A1 largely prevented monoclonal antibody combination- induced EGFR degradation in SW-1573 (Fig. 5B) and A549 cells (Fig. S4C). However, EGFR degradation by monoclonal antibody combination treatment was only partially prevented in H292 cells (Fig. S4C). Taken together, these results show that the strong downregulation after treatment with imgatuzumab alone and combined with cetuximab is at least partially the result of directing EGFR to lysosomes. These observations are consistent with findings of Ferraro et al. showing that anti-EGFR monoclonal antibody mixtures accelerate lysosomal degradation of EGFR (12).

Imgatuzumab combined with cetuximab effectively inhibits EGF-dependent cell proliferation

Next, the antiproliferative effect of imgatuzumab and cetuximab on SW-1573, H292, H322, H358 and A549 cells was investigated. Under normal culture conditions (media with 10% FCS), both single agents and the combination strongly inhibited H292 and H322

2

− + − +

− − + +

− + − +

− − + +

Bafilomycin

EGFR mAb, EEA1, DAPI

EGFR (total + mAb), Lamp1, DAPI

Imgatuzumab (4 hours) Cetuximab (4 hours) Imgatuzumab + Cetuximab (4 hours)

Imgatuzumab (4 hours) Cetuximab (4 hours) Imgatuzumab + Cetuximab (4 hours)

EGFR Actin Imgatuzumab:

Cetuximab:

Control Imgatuzumab

Cetuximab Imgatuzumab + Cetuximab

Control Imgatuzumab

Cetuximab Imgatuzumab + Cetuximab 0.0

0.5 1.0 1.5

Control Bafilomycin

EGFR/actin

**

EGFR (total + mAb), Lamp1, DAPI

Imgatuzumab (0 hours) Cetuximab (0 hours) Imgatuzumab + Cetuximab (0 hours)

Lamp1 Lamp1 EEA1 EEA1 (4 hours) (0 hours) (4 hours) (0 hours)

Imgatuzumab (0 hours) Cetuximab (0 hours) Imgatuzumab + Cetuximab (0 hours)

Figure 5:

Cellular localization and endocytic trafficking of anti-EGFR monoclonal antibodies in SW-1573. A, Cells were surface labeled with the anti-EGFR monoclonal antibodies (mAbs) on ice. After staining with the primary antibody cells were washed with ice-cold FACS buffer and incubated for 4 hours at 37 ˚C. After fixation, permeabilization, and incubation with anti-EEA1 and anti-Lamp1 antibodies, cells were incubated with fluorescent secondary antibodies (anti-human Alexa 488 against imgatuzumab and cetuximab (green color), Alexa 647-labeled antibodies for endosomal markers (red color)). For the Lamp1 staining additional anti-EGFR monoclonal antibodies were added to visualize total EGFR. Colocalization appears as yellow (merged images).

Images were acquired using a confocal microscope. White scale bars represent 10 µm. B, Western blot analysis of the effect of lysosomal inhibition on monoclonal antibody-induced EGFR degradation. SW-1573 cells were pretreated for 2 hours with the lysosomal inhibitor bafilomycin A1 (100 nM). Cells were subsequently treated with the anti-EGFR monoclonal antibodies (20 µg/mL total) for 4 hours. Data points are mean + SD. (**P < 0.01 compared to control).

A

B

30

Chapter 2

cell proliferation (Fig. 6). The antiproliferative effects in H292 correlated with strong inhibition of downstream signaling after monoclonal antibody treatment (Fig. 2). To investigate the growth inhibitory potential of the monoclonal antibodies in the presence of additional EGF, H292, H322 and A549 cells were incubated with imgatuzumab, cetuximab or the combination under normal culture conditions supplemented with physiological concentrations of EGF. Imgatuzumab was more effective than cetuximab in inhibiting proliferation in media supplemented with 1 ng/mL EGF. Addition of 10 ng/mL EGF to media induced complete resistance to the single antibody treatments, whereas the antibody combination was still able to almost completely inhibit proliferation. As expected, no clear effects of imgatuzumab and cetuximab on proliferation were observed in KRAS-mutant A549 (Fig. 6) and SW-1573 cells (results not shown). Interestingly, under normal growth conditions and supplemented with 1 ng/mL EGF, imgatuzumab alone and combined with cetuximab partially inhibited proliferation of KRAS-mutant H358 cells (Fig. S5). Under conditions of high EGF, only the combination inhibited proliferation.

Both single antibody treatments and the combination completely inhibited EGF- dependent migration of H292 and A549 cells as measured with the wound-healing assay (Fig. S6).

Imgatuzumab combined with cetuximab induces strong ADCC but no CDC responses against NSCLC cells

Imgatuzumab combined with cetuximab is the most potent in downregulating EGFR surface expression. However, effective EGFR downregulation might impair imgatuzumab-dependent ADCC responses. Alternatively, imgatuzumab and cetuximab are noncompetitively binding to EGFR (Fig. 1A), which may even result in stronger ADCC responses. We performed an in vitro ADCC assay, in which human peripheral blood mononuclear cells (PBMCs) were used as effector cells and H441, H292 or A549 cells as target cells. When applied as single agent, imgatuzumab caused superior ADCC against NSCLC cells compared with cetuximab (Fig. 7A-C). No increase in ADCC responses was observed when the two monoclonal antibodies were combined. Next, the effect of EGFR downregulation on the ADCC response after long-term treatment with imgatuzumab was investigated in A549 cells. These cells were chosen based on the strong downregulation of membranous EGFR (Fig. 1B) and their resistance to anti-EGFR monoclonal antibody treatment under normal growth conditions to avoid anti-proliferative effects (Fig. 6).

Pretreatment of cells with imgatuzumab for 72 hours did not alter ADCC responses despite the strong reduction in membranous EGFR (Fig. 7D). Similar results were observed when cells were treated with imgatuzumab combined with cetuximab for 72 hours (Fig. 7D).

Next, we investigated whether imgatuzumab and cetuximab induce CDC in our NSCLC