1 Title: Premedication and Chemotherapy Agents Do Not Impair

Author Manuscript Published OnlineFirst on November 18, 2015; DOI: 10.1158/1078-0432.CCR-14-2579 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Gonzalez-Nicolini et al.

Title:

Premedication and chemotherapy agents do not impair imgatuzumab- (GA201) mediated

antibody-dependent cellular cytotoxicity and combination therapies enhance efficacy

Authors

Valeria Gonzalez-Nicolini, Sylvia Herter, Sabine Lang, Inja Waldhauer, Marina Bacac,

Michaela Roemmele, Esther Bommer, Olivier Freytag, Erwin van Puijenbroek, Pablo Umaña

and Christian A Gerdes

Affiliation

Pharma Research and Early Development, Roche Innovation Center Zurich, Schlieren,

Switzerland

Running title: Common cancer drugs do not impair imgatuzumab-mediated ADCC

Keywords: Imgatuzumab, GA201, antibody-dependent cellular cytotoxicity, combination

therapy, premedication

Financial support:

This study was funded by Roche Glycart AG, Switzerland. Support for third-party writing

assistance for this article, was provided by Prism Ideas and funded by F Hoffmann-La

Roche.

Corresponding author:

Christian A. Gerdes, Roche Glycart AG, Wagistrasse 18, CH-8952 Schlieren, Switzerland

Tel: +41 44 755 6148; Fax: +41 44 755 6165; Email: [email protected]

Conflicts of interest

All authors are employees of Roche Glycart AG, Schlieren, Switzerland

Word count:

Body text: 3413 (excl. abstract, acknowledgements, references, figure & table legends) 1

Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 18, 2015; DOI: 10.1158/1078-0432.CCR-14-2579 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Gonzalez-Nicolini et al.

Total number of figures / tables:

4 figures / 1 table

Role of the funding source:

This study and editorial support for the preparation of the manuscript were funded by Roche

Glycart AG, Switzerland.

Previous presentation of data:

Nicolini et al. GA201 (RG7160) pretreatments and combination therapies improve efficacy

without negatively affecting antitumoral ADCC. [abstract]. In: Proceedings of the AACR-NCI-

EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov

12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11

Suppl):Abstract nr A156.

Gonzalez-Nicolini et al.

Translational relevance

Therapeutic monoclonal antibodies (mAb) are frequently co-administered with chemotherapy

agents and premedication drugs. These drugs are often immunosuppressive; therefore, it is

important to establish whether the activity of novel mAbs is negatively affected by these

agents. This is particularly important for mAbs which recruit immune effector cells, such as

the anti-epidermal growth factor receptor monoclonal antibody imgatuzumab. We

demonstrate that commonly used premedication and cancer drugs have little effect on the

degree of imgatuzumab-mediated antibody-dependent cellular cytotoxicity in vitro.

Furthermore, co-administration of chemotherapy agents with imgatuzumab achieves greater

efficacy than either agent alone in orthotopic xenograft models. These data indicate that

imgatuzumab-chemotherapy regimens should not directly diminish the enhanced innate

immune effector function of imgatuzumab’s mode of action. Our findings should facilitate the

design of clinical trials of combination therapy in patients with solid tumors, which may

further improve on the efficacy achieved with single-agent imgatuzumab.

Word count: 146 words Word limit: 120–150 words

Gonzalez-Nicolini et al.

Abstract

Purpose: Imgatuzumab (GA201) is a novel anti-epidermal growth factor receptor

monoclonal antibody (mAb) that is glycoengineered for enhanced antibody-dependent

cellular cytotoxicity (ADCC). Future treatment schedules for imgatuzumab will likely involve

the use of potentially immunosuppressive drugs, such as premedication therapies to mitigate

infusion reactions characteristic of mAb therapy and chemotherapy combination partners.

Due to the strong immunological component of imgatuzumab’s mode of action, it is important

to understand whether these drugs influence imgatuzumab-mediated ADCC and impact

efficacy.

Experimental design: We performed a series of ADCC assays using human peripheral

blood mononuclear cells that were first preincubated in physiologically relevant

concentrations of commonly used premedication drugs and cancer chemotherapies. The

ability of common chemotherapy agents to enhance the efficacy of imgatuzumab in vivo was

then examined using orthotopic xenograft models of human cancer.

Results: The majority of premedication and chemotherapy drugs investigated had no

significant effect on the ADCC activity of imgatuzumab in vitro. Furthermore, enhanced in

vivo efficacy was seen with imgatuzumab combination regimens compared with single-agent

imgatuzumab, single-agent chemotherapy or cetuximab combinations.

Conclusions: These data indicate that medications currently co-administered with anti-

EGFR therapies are unlikely to diminish the ADCC capabilities of imgatuzumab. Further

studies using syngeneic models with functional adaptive T-cell responses are now required

to fully understand how chemotherapy agents will influence a long-term response to

imgatuzumab therapy. Thus, the present study and future ones can provide a framework for

designing imgatuzumab combination regimens with enhanced efficacy for investigation in

phase II trials.

Gonzalez-Nicolini et al.

Word count: 245 words Word limit: 250 words

Gonzalez-Nicolini et al.

Introduction

The Epidermal growth factor receptor (EGFR) is activated by mutation or overexpression in

a variety of epithelial cancers including colorectal cancer (CRC), non-small cell lung cancer

(NSCLC) and head and neck squamous cell carcinoma (1;2), making anti-EGFR strategies

an attractive therapeutic option for solid tumors. Imgatuzumab (GA201/RG7160) is a novel

humanized anti-EGFR monoclonal antibody (mAb) with a dual mode of action (MoA).

Binding of imgatuzumab to EGFR inhibits signaling pathways that influence proliferation,

survival and apoptosis in a similar manner to other anti-EGFR mAbs such as cetuximab (3)

and panitumumab. However, imgatuzumab is specifically glycoengineered to enhance the

binding of the Fc portion of the antibody to FcγRIIIa receptors on the surface of immune

effector cells. This results in enhanced in vitro ADCC activity compared with both the wild

type imgatuzumab antibody and with the non-glycoengineered mAb cetuximab (3). This

enhanced cell killing in vitro translated into superior in vivo efficacy compared with cetuximab

in a series of orthotopic xenograft models (3). Early clinical experience with single-agent

imgatuzumab demonstrated promising clinical efficacy in heavily pretreated patients with

advanced EGFR-positive tumors, including patients with KRAS-mutant tumors (4).

Infusion-related reactions (IRRs) are common with chimeric and humanized mAbs and

chemotherapy agents such as taxanes and platinum analogues, and premedication to

reduce the incidence and severity of these reactions is normal practice (5;6). IRRs were

observed in 76% of patients following the first infusion of single-agent imgatuzumab in a

dose-escalation trial and have been reported with 7.6–33% of cetuximab infusions (4;7). As

a fully human antibody, the incidence of infusion reactions with panitumumab is much lower

(0–4%) (7). Premedication with corticosteroids and/or antihistamines is mandated for the first

infusion of cetuximab and recommended for subsequent cycles (8); similar

recommendations are made for rituximab, alemtuzumab and infliximab (9-11). It is therefore

important to establish whether co-administration of premedication drugs might potentially

Gonzalez-Nicolini et al.

interfere with the MoA of novel mAbs. Dexamethasone, a potent glucocorticoid frequently

used to premedicate patients, reduces the efficacy of trastuzumab in vitro by reversing

trastuzumab-induced cell cycle arrest (12). Conversely, the opposite effect is seen with

rituximab, where dexamethasone exhibits a synergistic effect on the in vitro complement-

dependent cytotoxicity activity of rituximab and has minimal effect on ADCC (13).

As well as premedication drugs, treatment protocols for imgatuzumab will likely involve

combination with conventional chemotherapeutic agents. The efficacy of most mAbs is

potentiated when administered with cytotoxic or cytostatic drugs (14). Initial studies with

single-agent cetuximab in previously treated advanced CRC achieved modest response

rates (8–11.6%) and time to progression/progression-free survival (PFS) in the range of 1.4

to 2.6 months (8;15-19). When combined with first-line irinotecan- or oxaliplatin-based

therapy in KRAS wildtype patients, response rates of 57.3% and a median PFS of 9.6

months were achieved (20). Cetuximab has also demonstrated synergistic effects with

platinum compounds (21), gemcitabine (22), paclitaxel (23), and topotecan (24).

Selection of appropriate chemotherapy partners is particularly important for mAbs with a

strong immunological component to their MoA, such as imgatuzumab. Effective induction of

ADCC requires recruitment and activation of host immune effector cells including natural

killer (NK) cells, macrophages and monocytes; therefore, cytotoxic or cytostatic drugs with

minimum immunosuppressive effects would be preferable. Paclitaxel is broadly

immunosuppressive at standard doses and inhibits cell types such as macrophages, effector

T-cells and NK cells (25). Vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, and

bortezomib have been shown to inhibit NK cell-mediated cell killing in vitro, whereas NK cell-

mediated killing was unaffected by therapeutic concentrations of asparaginase,

bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea,

streptozocin, and 6-mercaptopurine (26). Differential effects of various chemotherapy agents

on the cell killing potential of cytotoxic T-cells have also been demonstrated (27).

Understanding the effect that previously and co-administered compounds may have on the

Gonzalez-Nicolini et al.

MoA of novel mAbs will be important in establishing optimal combinations for clinical

investigation.

The aim of this study was to investigate the influence of commonly used premedication

drugs and potential chemotherapy partners for imgatuzumab on the in vitro ADCC activity of

imgatuzumab and to investigate the in vivo efficacy of imgatuzumab combined with

chemotherapy in human orthotopic xenograft models.

Gonzalez-Nicolini et al.

Materials and methods

Cell lines, antibodies and drugs

Human NSCLC (A549) and colorectal (HT29 and LS174T) adenocarcinoma cell lines were

obtained from the American Type Culture Collection (ATCC). Cells obtained from ATCC are

routinely authenticated by karyotyping, short tandem repeat profiling, assessment of cell

morphology, and species verification by isoenzymology. Upon receipt cells were expanded

and frozen; cells were not passaged for more than 6 months after resuscitation. No further

authentication of cell lines was conducted. A549 cells express low levels of EGFR and,

importantly, mutant KRAS; therefore, these cells are likely to be insensitive to inhibition of

EGFR signaling but should remain sensitive to ADCC. LS174T cells are also KRAS-mutant,

whereas HT29 cells are KRAS-wildtype. Cells were routinely cultured at 37°C and 5% CO2

in a humid environment in DMEM medium (Gibco) supplemented with 10% fetal calf serum

(PAA laboratories). Commercial-grade cetuximab was obtained from Merck.

Premedication drugs investigated in this study included the immunosuppressive

glucocorticoids hydrocortisone (cortisol; Farmamondo, Switzerland), prednisolone

(Farmamondo), and dexamethasone (Sigma, Switzerland); the antiemetics granisetron

® ® hydrochloride (Kytril ; serotonin 5-HT3 receptor antagonists) and aprepitant (Emend ;

neurokinin 1 receptor antagonist); the antihistamine ranitidine hydrochloride (histamine H2-

receptor antagonist); the proton pump inhibitor pantoprazole (Pantozol®); vitamin B12, and

folic acid (all Farmamondo). Chemotherapy agents included, gemcitabine (Gemzar®, Lilly,

Switzerland), cisplatin (Hexal AG, Switzerland), carboplatin (Teva Pharma AG, Switzerland),

irinotecan (Campto®, Pfizer, Switzerland), SN-38 (the active metabolite of irinotecan,

Farmamondo), doxycycline (Farmamondo), oxaliplatin (Sanofi-Aventis, Switzerland), and

paclitaxel (Abraxane®, Celgene, UK).

Gonzalez-Nicolini et al.

ADCC methodology

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers

according to standard methods. All PBMCs employed in this study were obtained from

individuals who were homozygous for the low-affinity variant of the human IgG1 receptor

(FcγRIIIa F/F158). PBMCs were pelleted by centrifugation and resuspended in AIM-V media

at a concentration of 2 × 106 cells per flask. Cells were then incubated for 17–23 hours at

37 ºC with premedication drugs and chemotherapy agents diluted in AIM-V media at the

following concentrations: 8 µM hydrocortisone, 0.65 µg/mL prednisolone, 0.1 µg/mL

dexamethasone, 1–100 µg/mL gemcitabine, 0.1–10 µg/mL cisplatin, 28 µg/mL carboplatin,

5–2.5 µg/mL paclitaxel, 61.6 ng/mL SN-38, 10 µg/mL doxycycline, or 1.5–0.3 µg/mL

oxaliplatin. Drug cocktails consisted of: mix 1 – 1 µg/mL cisplatin, 63.8 ng/mL granisetron

hydrochloride, 100 ng/mL dexamethasone, 1.6 µg/mL aprepitant, 10 µg/mL pemetrexed,

1.25 µg/mL pantoprazole, 600 pg/mL vitamin B12, and 15 ng/mL folic acid, and mix 2 –

1 µg/mL cisplatin, 63.8 ng/mL granisetron hydrochloride, 100 ng/mL dexamethasone,

1.6 µg/mL aprepitant, 10 µg/mL gemcitabine, and 94 ng/mL ranitidine hydrochloride.

Following preincubation with investigational drugs, PBMCs were washed with AIM-V media,

check for viability with a Vi-cell counter (Beckman Coulter, Germany) and used as effector

cells in a series of ADCC assays. PBMCs (6 × 105 cells per well) were seeded in 96-well

plates and incubated with A549 target cells (2.4 × 104 cells per well) at effector:target (E:T)

ratios of 25:1 (22:1 for carboplatin) and with imgatuzumab or cetuximab concentrations

ranging from 0.01 ng/mL to 1,000 ng/mL. After 4 hours at 37 ºC, supernatants were

harvested and cell killing was detected using the Roche lactose dehydrogenase (LDH)

cytotoxicity detection kit. All ADCC assays were performed in triplicate and the percentage

cell mediated cytotoxicity was calculated as the average absorbance of the triplicates, from

which the absorbance of the background controls (untreated PBMCs) was subtracted.

Results were presented as mean values ± the standard deviation. The T-test was used to

compare cell killing by treated PBMCs with that of untreated control cells.

Gonzalez-Nicolini et al.

Mouse xenograft models

Orthotopic xenograft models were used to assess the effect of chemotherapy combinations

and the premedication hydrocortisone on the in vivo efficacy of imgatuzumab. The

production and maintenance of these models has been described previously (3). Briefly,

female SCID-beige (Taconics) or SCID-huCD16 transgenic (tg) mice (Roche-Glycart, Lyon,

France) were inoculated with 1 or 5 × 106 A549 cells injected intravenously. For the

colorectal liver metastases models, the CRC cell lines LS174T or HT29 were injected into

the spleen (3 × 106 cells). SCID-beige mice contain monocytes and macrophages that

express FcγRIV, the murine homologue of human FcγRIIIA. Murine NK cells do not express

FcγRIV, therefore the SCID/beige mouse model can only demonstrate the anti-tumoral effect

of ADCC (and/or FcγRIV-triggered antibody-dependent cellular phagocytosis and cytokine

release) mediated by monocytes and macrophages as effectors. Mice were maintained

under specific-pathogen-free conditions with daily cycles of 12 hours light/darkness

according to guidelines (GV-SOLAS; FELASA) and food and water were provided ad libitum.

Continuous health monitoring was carried out and the experimental study protocol was

reviewed and approved by the Veterinary Department of Kanton Zurich.

Mice were randomized into different treatment groups (ten mice per group) and therapy

started when evidence of tumor growth was visible in the target organ of sacrificed scout

animals (7 days or 14 days after tumor cell inoculation). All treatments were administered IV

and non-toxic efficacious doses was used as follows: imgatuzumab and cetuximab at a dose

of 25 mg/kg, gemcitabine at 150 mg/kg, cisplatin at 2 mg/kg, paclitaxel at 20 mg/kg,

carboplatin at 60 mg/kg or irinotecan at 30 mg/kg. Pretreatment with hydrocortisone

(20 mg/kg) was performed 1 hour before the first and second imgatuzumab dose. All mAbs

and drugs were given once weekly for 3 weeks (6 weeks for cisplatin). The termination

criteria for sacrificing animals was sickness with locomotion impairment, and median overall

survival (OS) was defined as the experimental day by which 50% of animals had been

sacrificed. Kaplan-Meier survival curves and the Pairwise Log-Rank test were used to

Gonzalez-Nicolini et al.

compare survival between animals treated with single-agent imgatuzumab and imgatuzumab

in combination with chemotherapy.

Gonzalez-Nicolini et al.

Results

Common cancer premedications do not negatively affect imgatuzumab-mediated in

vitro ADCC or in vivo efficacy in xenograft models

A series of ADCC experiments was performed to investigate whether commonly used

premedication therapies affected the immune effector cells responsible for performing

imgatuzumab-mediated cell killing. Incubation of human PBMC effector cells with 8 µM

hydrocortisone for 23 hours prior to performing ADCC experiments had no effect on

imgatuzumab-mediated killing of A549 lung adenocarcinoma cells (Figure 1A). The level of

imgatuzumab-mediated cell killing achieved by hydrocortisone-pretreated PBMCs was the

same as that of untreated PBMCs across a range of antibody concentrations. In contrast,

ADCC killing of A549 cells in the presence of cetuximab was decreased slightly when

PBMCs were preincubated with hydrocortisone. No significant effect on PBMCs cell viability

was observed after the incubation period with hydrocortisone, before the ADCC assay.

Pretreatment with hydrocortisone also did not affect the in vivo efficacy of imgatuzumab in a

human lung adenocarcinoma model (SCID-beige mice inoculated with A549 cells). The

median OS achieved by imgatuzumab was identical when administered as single-agent

(25 mg/kg weekly for 3 weeks) to untreated animals or to animals pretreated with 20 mg/kg

hydrocortisone 1 hour before the first and second infusion of imgatuzumab (p=0.36; Figure

1B). Both single-agent imgatuzumab and imgatuzumab administered following

hydrocortisone pretreatment achieved significantly superior median OS compared with

untreated control mice (p=0.0004 and p=0.00004, respectively).

Preincubation of ADCC effector cells with chemotherapy agents at physiologically

relevant doses does not affect imgatuzumab-mediated ADCC

The effect of chemotherapy agents on the in vitro ADCC activity and in vivo efficacy of

imgatuzumab was investigated using gemcitabine, cisplatin, carboplatin, paclitaxel and

Gonzalez-Nicolini et al.

irinotecan. These compounds are currently used clinically to treat solid tumor types in which

imgatuzumab is likely to be indicated (CRC, lung cancer, breast cancer, head and neck

cancer), and are therefore potential partners for imgatuzumab in future combination

chemotherapy regimens.

Preincubation of PBMC effector cells in 1 µg/mL gemcitabine had no effect on imgatuzumab-

or cetuximab-mediated killing of A549 lung cancer cells across the range of mAb

concentrations investigated in ADCC assays (Figure 2A). Cell killing by PBMCs pretreated

with 10 µg/mL gemcitabine was reduced at higher imgatuzumab concentrations compared

with controls and ADCC was significantly reduced for both anti-EGFR mAbs when PBMCs

were preincubated with high concentrations (100 µg/mL) of gemcitabine (approximately 33%

relative reduction in maximal cell killing seen for both mAbs). Preincubation of PBMC

effectors cells with cisplatin (0.1 µg/mL to 10 µg/mL; Figure 2B) and carboplatin (28 µg/mL;

Figure 2C) had no influence on A549 cell killing mediated by either antibody, whereas the

ADCC capacity of both anti-EGFR mAbs was reduced following preincubation of PBMCs

with paclitaxel (Figure 2D). Interestingly, SN-38 (the active metabolite of irinotecan) at a

single tested concentration of 61.6 ng/mL had no effect on imgatuzumab-mediated ADCC of

KRAS-wildtype HT29 CRC cells but reduced the degree of cell killing mediated by cetuximab

by approximately 40% (Figure 2E). No significant effect on PBMCs cell viability was

observed after the incubation period with the different chemotherapeutic agents measured

before the respective ADCC assays.

In all ADCC experiments, the degree of cell killing mediated by imgatuzumab was higher

than with cetuximab, consistent with previous findings (2).

Table 1 summarizes the EC50 and maximal cell killing values for all premedication

drugs/chemotherapies tested as single agents. Significant reductions in the maximal

percentage of ADCC were seen following pretreatment of PBMC effector cells with

dexamethasone, prednisolone, gemcitabine and paclitaxel and maximal cell killing increased

Gonzalez-Nicolini et al.

significantly when effector cells were preincubated in oxaliplatin. Pretreatment with

prednisolone and paclitaxel also significantly increased the EC50 of imgatuzumab.

The in vitro ADCC potential of PBMCs mediated by imgatuzumab is relatively

unaffected by pretreatment with complex drug mixtures

To further investigate the influence of premedication and chemotherapy agents on

imgatuzumab activity, ADCC assays were performed using PBMCs preincubated for

20 hours in the presence of two different cocktails (mix 1 and mix 2) of multiple drugs

currently used in the clinical setting. At medium to high concentrations of imgatuzumab (1–

1,000 ng/mL), mix 1 had no significant effect on imgatuzumab-mediated ADCC activity

(Figure 3). Cell killing was reduced slightly with mix 2 at higher imgatuzumab concentrations.

In contrast, cetuximab-induced ADCC was reduced following preincubation of immune

effector cells with either mix and at all concentrations of antibody. No significant effect on

PBMCs cell viability was observed after the incubation period with both cocktails (mix 1 and

mix2) measured before the ADCC assays.

Co administration of imgatuzumab with cytotoxic agents enhances in vivo efficacy

Combining imgatuzumab with chemotherapy agents significantly improved efficacy

compared with both single-agent imgatuzumab and single-agent chemotherapy. This was

seen in all combinations tested (Figure 4). Median OS of SCID-beige mice inoculated with

A549 cells was 115 days when treated with gemcitabine plus imgatuzumab compared with

95 days for single-agent imgatuzumab (p=0.05) and 69 days for single-agent gemcitabine

(p=0.05; Figure 4A). Furthermore, OS with imgatuzumab plus gemcitabine was significantly

longer than for cetuximab plus gemcitabine (98 days; p=0.05). Similar data were seen when

the same xenograft model was treated with imgatuzumab in combination with cisplatin

(Figure 4B). Median OS was longer with combination therapy (118 days) compared with

single agent imgatuzumab (84 days; p=0.003), single-agent cisplatin (70 days; p=0.001) and

a trend towards longer survival over the cetuximab plus cisplatin group (103 days; p=0.14)

Gonzalez-Nicolini et al.

was seen. Median OS of SCID/huCD16 tg mice inoculated with A549 cells was 102 days

when treated with paclitaxel plus imgatuzumab compared with 76 days for single-agent

imgatuzumab (p=0.03) and 60 days for single-agent paclitaxel (p=0.0001; Figure 4C).

Similar data were seen when the same xenograft model was treated with imgatuzumab in

combination with carboplatin (Figure 4D). Median OS was longer with combination therapy

(112 days) compared with single-agent imgatuzumab (75 days; p=0.003) or single-agent

carboplatin (48 days; p=0.001).

Combining imgatuzumab with irinotecan also improved median OS compared with either

agent alone in the CRC SCID/huCD16 tg xenograft models. In mice inoculated with KRAS-

wildtype HT29 cells median OS was 79 days when treated with irinotecan plus imgatuzumab

compared with 59 days for single-agent imgatuzumab (p=0.03) and 49 days for single-agent

irinotecan (p=0.0001; Figure 4E). In mice inoculated with KRAS-mutant LS174T cells,

median OS was longer with combination therapy (60 days) compared with single-agent

imgatuzumab (45 days; p=0.04) and single-agent irinotecan (41 days; p=0.001; Figure 4F).

Gonzalez-Nicolini et al.

Discussion

The novel anti-EGFR mAb imgatuzumab is glycoengineered for boosted ADCC (3). In this

study we demonstrated that common chemotherapy agents and medications used to reduce

the risk of therapy side effects had no significant negative effect on imgatuzumab-mediated

ADCC cell killing in vitro. Moreover, in vivo, improved efficacy was observed when

chemotherapeutic agents were combined with imgatuzumab compared with mAb therapy

alone. This in vivo effect was observed in xenograft models without an acquired immune

system but in the presence of the innate immune effector cells that mediate ADCC.

Pretreatment of human PBMCs and murine xenograft models with hydrocortisone at

concentrations similar to those used clinically had no effect on the ADCC activity or efficacy

of imgatuzumab. These data indicate that strategies to reduce the risk and severity of IRRs

in the clinic can be potentially achieved with hydrocortisone without compromising the

efficacy of imgatuzumab-based treatment regimens. Future combination regimens will also

necessitate the use of premedications designed to mitigate side-effects such as nausea and

vomiting commonly observed with the administration of cytotoxic chemotherapies. We

demonstrated that the ADCC activity of imgatuzumab was relatively unaffected by the

presence of multiple premedication drugs.

Two chemotherapy agents reduced the ADCC potential of human effector cells mediated by

imgatuzumab. Preincubation of PBMCs in paclitaxel reduced the percentage of maximal cell

mediated by imgatuzumab from 53.5% to 36.8%. This was also seen for cetuximab-

mediated in vitro cell killing, and is consistent with the known immunosuppressive effects of

paclitaxel (25). However, despite this reduction in ADCC capability, combining paclitaxel with

imgatuzumab still resulted in enhanced efficacy in SCID-beige mice models compared with

either agent alone. A slight reduction in cell killing at higher concentrations of gemcitabine

was also seen with imgatuzumab (from 92.4% to 87.7%). Interestingly, the ADCC activity of

cetuximab was reduced in the presence of the active metabolite of irinotecan (SN-38). The

Gonzalez-Nicolini et al.

ability of cetuximab to both enhance the efficacy of irinotecan and restore tumor

chemosensitivity to irinotecan is well established (15;29-32). However, no such reduction in

the ADCC activity of imgatuzumab in the presence of SN-38 was seen, which may indicate

that imgatuzumab-irinotecan combinations can further improve outcomes in previously

treated patients compared with cetuximab-irinotecan.

Cell killing mediated by imgatuzumab was superior to cetuximab-mediated cell killing in all

ADCC assays performed. This was to be expected, as the A549 cell line used in the majority

of ADCC experiments represented a particularly challenging target for anti-EGFR mAb-

mediated cell killing (low EGFR expression and mutant KRAS), and cetuximab is known to

be relatively ineffective against KRAS-mutant tumors (33). The enhanced cell killing and

superior efficacy with imgatuzumab compared with cetuximab demonstrated in this study are

consistent with our previous report, which used a variety of human tumor cell line models

with different KRAS status and EGFR expression level (3).

All chemotherapy agents tested in vivo significantly increased OS when combined with

imgatuzumab. Imgatuzumab demonstrated additive or synergistic efficacy when co-

administrated with gemcitabine and cisplatin in SCID-beige mice and with paclitaxel,

carboplatin and irinotecan in SCID-huCD16 tg mice. All imgatuzumab-chemotherapy

combinations tested to date on xenograft models achieved enhanced efficacy compared with

cetuximab plus chemotherapy.

The exact mechanisms by which conventional cytotoxic agents combine with anti-EGFR

mAbs for enhanced efficacy are not fully understood; however, there is accumulating

evidence that exposure to some chemotherapy agents can upregulate EGFR expression.

Membrane EGFR expression was upregulated following exposure of several colorectal cell

lines to gemcitabine, irinotecan, levofolinic acid, and 5-fluorouracil, and this resulted in

enhanced in vitro cetuximab-mediated ADCC and lymphocyte-assisted killing (34).

Furthermore, levels of membrane EGFR expression increased in a series of isogenic CRC

cells lines as cell lines became progressively more oxaliplatin-resistant, and the in vitro

sensitivity of the cell lines to cetuximab increased in parallel (35). The sequence of 15

Gonzalez-Nicolini et al.

treatments may also be important; platinum and taxane chemotherapy followed by anti-

EGFR therapy (both mAb and small molecule tyrosine kinase inhibitors) strongly enhanced

apoptosis and cell cycle arrest in vitro, whereas an antagonistic effect was seen when anti-

EGFR therapies were given before chemotherapy (36).

One limitation of this study is that the mouse models used lack a functional adaptive T-cell

response; therefore, it was not possible to investigate the imgatuzumab adaptive immune

related mode of action and how the tested agents affect T-cells and the influence this has on

tumor cell killing. As most chemotherapy agents affect proliferating cells, T-cell response is

likely to be affected by some of the drugs if given during previous days or in concomitant

administration with the antibody therapy; therefore, further studies using syngeneic models

with functional adaptive T-cell responses are now required as well as different schedules of

combination therapies.

In conclusion, the enhanced innate immune mediated ADCC activity of imgatuzumab

compared with cetuximab appears to be unaffected by common cancer premedication drugs

and chemotherapy agents when tested in models bearing a functional innate immune

system only. Building on these preclinical data, several imgatuzumab combination

chemotherapy regimens are under investigation. In NSCLC, the combination of

imgatuzumab plus cisplatin and gemcitabine (for squamous histology) and imgatuzumab

plus cisplatin and pemetrexed (for non-squamous histology) are investigated as first-line

treatment (NCT01185847). In CRC, second-line treatment with imgatuzumab plus FOLFIRI

are also under investigation in patients with KRAS-mutant tumors, and imgatuzumab plus

FOLFIRI and cetuximab in patients with KRAS-wildtype tumors (NCT01326000).

Gonzalez-Nicolini et al.

Acknowledgements

The authors would like to thank Priska Rueegg, Daniela Ackermann, and Michael Mauch for

quality administrative support. We appreciate the functional support of Roche Glycart

molecular biology and process biochemistry. Special thanks go to Jon Chick, and the rest of

the Roche imgatuzumab global team for discussion contributions while generating this work.

Gonzalez-Nicolini et al.

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Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2015 American Association for Cancer Research. Gonzalez-Nicolini et al. Downloaded from Author ManuscriptPublishedOnlineFirstonNovember18,2015;DOI:10.1158/1078-0432.CCR-14-2579 Tables Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited.

Table 1.

clincancerres.aacrjournals.org Imgatuzumab EC values (ng/mL) Maximal killing (%) Concentration 50 Cmax reported used Untreated Treated P-value Untreated Treated P-value

Hyrdrocortisone 8 μM 8 μM 0.28 0.44 0.0994 93.9 87.9 0.0704

Dexamethasone 0.1 μg/mL 0.06 μg/mL 5.46 4.84 0.6066 58.5 49.0 0.0001

Cisplatin 1 μg/mL n.c. 0.16 0.16 0.973 73.6 71.2 0.1047

Carboplatin 28 μg/mL 28 μg/mL 0.57 0.60 0.6499 65.6 67.1 0.5488

Paclitaxel 5–2.5 μg/mL 5 μg/mL 1.83 3.0 0.0005 53.5 36.8 <0.0001

SN 38 (metabolite of irinotecan) 0.062 μg/mL 0.062 μg/mL 1.07 1.74 0.1425 66.0 64.0 No difference

Doxycycline 10 μg/mL 7 μg/mL 0.81 0.78 0.9392 55.2 55.8 0.5235

Oxaliplatin 1.5–0.3 μg/mL 1.5–0.3 μg/mL 4.01 4.73 0.3495 29.9 31.6 0.0004

1 Author Manuscript Published OnlineFirst on November 18, 2015; DOI: 10.1158/1078-0432.CCR-14-2579 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Gonzalez-Nicolini et al.

Table and Figure legends

Table 1. Influence of pretreatments and chemotherapies on the ADCC capability of

PBMCs. PBMCs were preincubated at concentrations shown for ~24 hours after which ADCC was performed using a range of imgatuzumab concentrations (0.01–1000 ng/mL) in the absence of chemotherapy/premedication drugs (4-hour LDH release assay, different target cells used).

Bold text indicates significant differences in cell killing between pretreated and untreated PBMCs.

N= 3. n.c., not calculated

Figure 1. (A) Pretreatment of PBMC effector cells in vitro with 8 µM hydrocortisone did not affect imgatuzumab-mediated antibody-dependent killing of A549 target cells (4-hour LDH release assay, E:T ratio 25:1), whereas cetuximab-dependent killing was slightly reduced at higher antibody concentrations. PBMC viability measured as follow: before hydrocortisone preincubation

97.9%; after hydrocortisone preincubation 96.4%. N= 3. (B) In SCID-beige mice (n=10 per group) inoculated with A549 cells, premedication with hydrocortisone (20 mg/kg) 1 hour before the 1st and 2nd imgatuzumab infusion did not affect imgatuzumab efficacy; median overall survival was unchanged (p=0.36) and significantly longer than with vehicle control.

Figure 2. Antibody-dependent killing of A549 cells (A–D) or HT29 cells (E) by PBMCs preincubated for 17–23 hours with increasing concentration of gemcitabine (A) or cisplatin (B) or a single concentration of , carboplatin (C), paclitaxel (D) or SN 38, the active metabolite of irinotecan (E). ADCC (4-hour LDH release assay) mediated by either imgatuzumab or cetuximab was not affected by preincubation of effectors cells with gemcitabine at concentrations up to

10 µg/mL. Pretreatment of PBMCs with 28 µg/mL paclitaxel significantly reduced the maximal killing and EC50 for both mAbs compared with untreated PBMCs; whereas cisplatin and carboplatin had no influence on ADCC for both mAbs at all tested concentrations. SN 38 did not affect imgatuzumab-induced ADCC of HT29 cells but reduced the level of cell killing with

Gonzalez-Nicolini et al. cetuximab. PBMC viability measured as follow: before gemcitabine preincubation 98.9%; after 1

µg/ml, 10 µg/ml and 100 µg/ml gemcitabine preincubation 96.7%, 96.9% and 97 % respectively.

Before cisplatin preincubation 99.4%; after 0.1 µg/ml, 1 µg/ml and 10 µg/ml cisplatin preincubation 99.8%, 99.6% and 99.5 % respectively. Before carboplatin preincubation 98.4%; after carboplatin preincubation 98.1%. Before paclitaxel preincubation 99.8%; after paclitaxel preincubation 99.7%. Before SN38 preincubation 99.8%; after SN38 preincubation 97.5%. N= 3.

Figure 3. Preincubation of PBMC effector cells with a cocktail of cisplatin (1 µg/mL), granisetron hydrochloride (63.8 ng/mL), dexamethasone (100 ng/mL), aprepitant (1.6 µg/mL), pemetrexed

(10 µg/mL), pantoprazole (1.25 µg/mL), vitamin B12 (600 pg/mL), and folic acid (15 ng/mL; Mix

1) had no effect on imgatuzumab-mediated antibody-dependent killing (4-hour LDH release assay using A549 target cells at an E:T ratio of 25:1). A slight reduction in ADCC was seen at higher imgatuzumab concentrations after preincubation of PBMCs in cisplatin (1 µg/mL), granisetron hydrochloride (63.8 ng/mL), dexamethasone (100 ng/mL), aprepitant (1.6 µg/mL), gemcitabine (10 µg/mL), and ranitidine hydrochloride (94 ng/mL; Mix 2). In contrast, both drug cocktails reduced cetuximab-mediated ADCC. PBMC viability measured as follow: before Mix 1 and Mix 2 preincubation 99.9%; after Mix 1 and Mix 2 preincubation 96.4% and 97.9 % respectively. N= 3.

Figure 4. SCID-beige mice (A&B) or SCID-huCD16 tg mice (C–F; n=10 per group) were inoculated with A549 cells (A–D), KRAS-wildtype HT29 cells (E) or KRAS-mutant LS174T cells

(F) on Day 0. Animals were treated with either saline (vehicle control), single-agent imgatuzumab

(25 mg/kg), single-agent chemotherapy (gemcitabine 150 mg/kg; cisplatin 2 mg/kg; paclitaxel

20 mg/kg, carboplatin 60 mg/kg or irinotecan 30 mg/kg), imgatuzumab plus chemotherapy or cetuximab (25 mg/kg) plus chemotherapy. All therapies began on Day 7 and were given once weekly for 3 weeks (6 weeks for cisplatin). Median overall survival with imgatuzumab combined

Gonzalez-Nicolini et al. with chemotherapy agents was superior to both single agent imgatuzumab and single agent chemotherapy in all models investigated. Imgatuzumab combined with chemotherapy was also superior to cetuximab combined with chemotherapy.

Premedication and chemotherapy agents do not impair imgatuzumab- (GA201) mediated antibody-dependent cellular cytotoxicity and combination therapies enhance efficacy

Valeria Gonzalez-Nicolini, Sylvia Herter, Sabine Lang, et al.

Clin Cancer Res Published OnlineFirst November 18, 2015.

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