Premedication and Chemotherapy Agents Do Not Impair Imgatuzumab

Published OnlineFirst November 18, 2015; DOI: 10.1158/1078-0432.CCR-14-2579

Cancer Therapy: Preclinical Clinical Cancer Research 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, Inja Waldhauer, Marina Bacac, Michaela Roemmele, Esther Bommer, Olivier Freytag, Erwin van Puijenbroek, Pablo Umana,~ and Christian A. Gerdes

Abstract

Purpose: Imgatuzumab (GA201) is a novel anti-EGFR mAb that Results: A majority of premedication and chemotherapy drugs is glycoengineered for enhanced antibody-dependent cellular cyto- investigated had no significant effect on the ADCC activity of toxicity (ADCC). Future treatment schedules for imgatuzumab will imgatuzumab in vitro. Furthermore, enhanced in vivo efficacy was likelyinvolvethe use ofpotentially immunosuppressive drugs,such seen with imgatuzumab combination regimens compared with as premedication therapies, to mitigate infusion reactions charac- single-agent imgatuzumab, single-agent chemotherapy, or cetux- teristic of mAb therapy and chemotherapy combination partners. imab combinations. Because of the strong immunologic component of mode of action Conclusions: These data indicate that medications currently of imgatuzumab, it is important to understand whether these drugs coadministered with anti-EGFR therapies are unlikely to dimin- influence imgatuzumab-mediated ADCC and impact efficacy. ish the ADCC capabilities of imgatuzumab. Further studies Experimental Design: We performed a series of ADCC assays using syngeneic models with functional adaptive T-cell using human peripheral blood mononuclear cells that were first responses are now required to fully understand how chemo- preincubated in physiologically relevant concentrations of com- therapy agents will influence a long-term response to imgatu- monly used premedication drugs and cancer chemotherapies. The zumab therapy. Thus, this study and future ones can provide ability of common chemotherapy agents to enhance the efficacy a framework for designing imgatuzumab combination regi- of imgatuzumab in vivo was then examined using orthotopic mens with enhanced efficacy for investigationinphaseIItrials. xenograft models of human cancer. Clin Cancer Res; 1–9. 2015 AACR.

Introduction body-dependent cellular cytotoxicity (ADCC) activity compared with both the wild-type imgatuzumab antibody and with the The EGFR is activated by mutation or overexpression in a non-glycoengineered mAb cetuximab (3). This enhanced cell variety of epithelial cancers including colorectal cancer, non– killing in vitro translated into superior in vivo efficacy compared small cell lung cancer (NSCLC), and head and neck squamous with cetuximab in a series of orthotopic xenograft models (3). cell carcinoma (1, 2), making anti-EGFR strategies an attractive Early clinical experience with single-agent imgatuzumab demon- therapeutic option for solid tumors. Imgatuzumab (GA201/ strated promising clinical efficacy in heavily pretreated patients RG7160) is a novel humanized anti-EGFR monoclonal antibody with advanced EGFR-positive tumors, including patients with (mAb) with a dual mode of action. Binding of imgatuzumab to KRAS-mutant tumors (4). EGFR inhibits signaling pathways that influence proliferation, Infusion-related reactions (IRR) are common with chimeric survival, and apoptosis in a similar manner to other anti-EGFR and humanized mAbs and chemotherapy agents, such as taxanes mAbs such as cetuximab (3) and panitumumab. However, imga- and platinum analogues, and premedication to reduce the inci- tuzumab is specifically glycoengineered to enhance the binding of dence and severity of these reactions is normal practice (5, 6). IRRs the Fc portion of the antibody to FcgRIIIa receptors on the surface were observed in 76% of patients following the first infusion of of immune effector cells. This results in enhanced in vitro anti- single-agent imgatuzumab in a dose-escalation trial and have been reported with 7.6% to 33% of cetuximab infusions (4, 7). As a fully human antibody, the incidence of infusion reactions with Pharma Research and Early Development, Roche Innovation Center panitumumab is much lower (0%–4%; ref. 7). Premedication Zurich, Schlieren, Switzerland. with corticosteroids and/or antihistamines is mandated for the Corresponding Author: Christian A. Gerdes, Roche Innovation Center Zurich, first infusion of cetuximab and recommended for subsequent Wagistrasse 18, Schlieren CH-8952, Switzerland. Phone: 414-4755-6148; Fax: cycles (8); similar recommendations are made for rituximab, 414-4755-6165; E-mail: [email protected] alemtuzumab, and infliximab (9–11). It is therefore important doi: 10.1158/1078-0432.CCR-14-2579 to establish whether coadministration of premedication drugs 2015 American Association for Cancer Research. might potentially interfere with the mode of action of novel

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Understanding the effect that previously and coadministered Translational Relevance compounds may have on the mode of action of novel mAbs will Therapeutic mAbs are frequently coadministered with che- be important in establishing optimal combinations for clinical motherapy agents and premedication drugs. These drugs are investigation. often immunosuppressive; therefore, it is important to estab- The aim of this study was to investigate the influence of lish whether the activity of novel mAbs is negatively affected by commonly used premedication drugs and potential chemother- these agents. This is particularly important for mAbs which apy partners for imgatuzumab on the in vitro ADCC activity of recruit immune effector cells, such as the anti-EGFR mAb, imgatuzumab and to investigate the in vivo efficacy of imgatuzu- imgatuzumab. We demonstrate that commonly used preme- mab combined with chemotherapy in human orthotopic xeno- dication and cancer drugs have little effect on the degree graft models. of imgatuzumab-mediated antibody-dependent cellular cyto- in vitro toxicity . Furthermore, coadministration of chemother- Materials and Methods apy agents with imgatuzumab achieves greater efficacy than either agent alone in orthotopic xenograft models. These Cell lines, antibodies, and drugs data indicate that imgatuzumab–chemotherapy regimens Human NSCLC (A549) and colorectal (HT29 and LS174T) should not directly diminish the enhanced innate immune adenocarcinoma cell lines were obtained from the ATCC. Cells effector function of the mode of action of imgatuzumab. Our obtained from ATCC were routinely authenticated by karyotyp- findings should facilitate the design of clinical trials of com- ing, short tandem repeat profiling, assessment of cell morphol- bination therapy in patients with solid tumors, which may ogy, and species verification by isoenzymology. Upon receipt, further improve on the efficacy achieved with single-agent cells were expanded and frozen; cells were not passaged for more imgatuzumab. 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 mAbs. Dexamethasone, a potent glucocorticoid frequently used sensitive to ADCC. LS174T cells are also KRAS mutant, whereas to premedicate patients, reduces the efficacy of trastuzumab HT29 cells are KRAS wild-type. Cells were routinely cultured at in vitro by reversing trastuzumab-induced cell-cycle arrest (12). 37 C and 5% CO2 in a humid environment in DMEM (Gibco) Conversely, the opposite effect is seen with rituximab, where supplemented with 10% FCS (PAA laboratories). Commercial- dexamethasone exhibits a synergistic effect on the in vitro com- grade cetuximab was obtained from Merck. plement-dependent cytotoxicity activity of rituximab and has a Premedication drugs investigated in this study included the minimal effect on ADCC (13). immunosuppressive glucocorticoids hydrocortisone (cortisol; Treatment protocols for imgatuzumab will likely involve com- Farmamondo), prednisolone (Farmamondo), and dexametha- bination with conventional chemotherapeutic agents as well as sone (Sigma); the antiemetics granisetron hydrochloride (Kytril; premedication drugs. The efficacy of most mAbs is potentiated serotonin 5-HT3 receptor antagonists) and aprepitant (Emend; when administered with cytotoxic or cytostatic drugs (14). Initial neurokinin 1 receptor antagonist); the antihistamine ranitidine studies with single-agent cetuximab in previously treated hydrochloride (histamine H2-receptor antagonist); the proton advanced colorectal cancer achieved modest response rates pump inhibitor pantoprazole (Pantozol); and vitamin B12 and (8%–11.6%) and time-to-progression/progression-free survival folic acid (all Farmamondo). Chemotherapy agents included (PFS) in the range of 1.4 to 2.6 months (8, 15–19). When gemcitabine (Gemzar, Lilly), cisplatin (Hexal AG), carboplatin combined with first-line irinotecan- or oxaliplatin-based therapy (Teva Pharma AG), irinotecan (Campto, Pfizer), SN-38 (the active in KRAS wild-type patients, response rates of 57.3% and a median metabolite of irinotecan, Farmamondo), doxycycline (Farma- PFS of 9.6 months were achieved (20). Cetuximab has also mondo), oxaliplatin (Sanofi-Aventis), and paclitaxel (Abraxane, demonstrated synergistic effects with platinum compounds Celgene). (21), gemcitabine (22), paclitaxel (23), and topotecan (24). Selection of appropriate chemotherapy partners is particularly ADCC methodology important for mAbs with a strong immunologic component to Peripheral blood mononuclear cells (PBMC) were obtained their mode of action, such as imgatuzumab. Effective induction of from healthy volunteers according to standard methods. All ADCC requires recruitment and activation of host immune effec- PBMCs employed in this study were obtained from individuals tor cells including natural killer (NK) cells, macrophages, and who were homozygous for the low-affinity variant of the human monocytes; therefore, cytotoxic or cytostatic drugs with minimum IgG1 receptor (FcgRIIIa F/F158). PBMCs were pelleted by centri- immunosuppressive effects would be preferable. Paclitaxel is fugation and resuspended in AIM-V media at a concentration of broadly immunosuppressive at standard doses and inhibits cell 2 106 cells per flask. Cells were then incubated for 17 to 23 hours types such as macrophages, effector T cells, and NK cells (25). at 37C with premedication drugs and chemotherapy agents Vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, and diluted in AIM-V media at the following concentrations: bortezomib have been shown to inhibit NK cell–mediated cell 8 mmol/L hydrocortisone, 0.65 mg/mL prednisolone, 0.1 mg/mL killing in vitro, whereas NK cell–mediated killing was unaffected dexamethasone, 1 to 100 mg/mL gemcitabine, 0.1 to 10 mg/mL by therapeutic concentrations of asparaginase, bevacizumab, cisplatin, 28 mg/mL carboplatin, 5 to 2.5 mg/mL paclitaxel, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, 61.6 ng/mL SN-38, 10 mg/mL doxycycline, or 1.5 to 0.3 mg/mL hydroxyurea, streptozocin, and 6-mercaptopurine (26). Differen- oxaliplatin. Drug cocktails consisted of mix 1 (1 mg/mL cisplatin, tial effects of various chemotherapy agents on the cell killing 63.8 ng/mL granisetron hydrochloride, 100 ng/mL dexametha- potential of cytotoxic T cells have also been demonstrated (27). sone, 1.6 mg/mL aprepitant, 10 mg/mL pemetrexed, 1.25 mg/mL

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pantoprazole, 600 pg/mL vitamin B12, and 15 ng/mL folic acid) Results and mix 2 (1 mg/mL cisplatin, 63.8 ng/mL granisetron hydro- Common cancer premedications do not negatively affect chloride, 100 ng/mL dexamethasone, 1.6 mg/mL aprepitant, imgatuzumab-mediated in vitro ADCC or in vivo efficacy in 10 mg/mL gemcitabine, and 94 ng/mL ranitidine hydrochloride). xenograft models After preincubation with investigational drugs, PBMCs were A series of ADCC experiments was performed to investigate washed with AIM-V media, checked for viability with a Vi-cell whether commonly used premedication therapies affected the counter (Beckman Coulter), and used as effector cells in a series of immune effector cells responsible for performing imgatuzumab- ADCC assays. PBMCs (6 105 cells per well) were seeded in mediated cell killing. Incubation of human PBMC effector cells 96-well plates and incubated with A549 target cells (2.4 104 with 8 mmol/L hydrocortisone for 23 hours prior to performing cells per well) at effector:target (E:T) ratios of 25:1 (22:1 for ADCC experiments had no effect on imgatuzumab-mediated carboplatin) and with imgatuzumab or cetuximab concentrations killing of A549 lung adenocarcinoma cells (Fig. 1A). The level ranging from 0.01 ng/mL to 1,000 ng/mL. After 4 hours at 37 C, of imgatuzumab-mediated cell killing achieved by hydrocorti- supernatants were harvested and cell killing was detected using sone-pretreated PBMCs was the same as that of untreated PBMCs the Roche lactose dehydrogenase (LDH) cytotoxicity detection across a range of antibody concentrations. In contrast, ADCC kit. All ADCC assays were performed in triplicate and the per- killing of A549 cells in the presence of cetuximab was decreased centage cell-mediated cytotoxicity was calculated as the average slightly when PBMCs were preincubated with hydrocortisone. No absorbance of the triplicates, from which the absorbance of the significant effect on the cell viability of PBMCs was observed after background controls (untreated PBMCs) was subtracted. Results the incubation period with hydrocortisone, before the ADCC were presented as mean SD. The t test was used to compare cell assay. killing by treated PBMCs with that of untreated control cells. Pretreatment with hydrocortisone also did not affect the in vivo efficacy of imgatuzumab in a human lung adenocarcinoma model Mouse xenograft models (SCID-beige mice inoculated with A549 cells). The median OS Orthotopic xenograft models were used to assess the effect of achieved by imgatuzumab was identical when administered as chemotherapy combinations and the premedication hydrocorti- single agent (25 mg/kg weekly for 3 weeks) to untreated animals sone on the in vivo efficacy of imgatuzumab. The production and or to animals pretreated with 20 mg/kg hydrocortisone 1 hour maintenance of these models has been described previously (3). before the first and second infusion of imgatuzumab (P ¼ 0.36; Briefly, female SCID-beige (Taconics) or SCID-huCD16 transgen- Fig. 1B). Both single-agent imgatuzumab and imgatuzumab ic (tg) mice (Roche-Glycart) were inoculated with 1 or 5 106 administered following hydrocortisone pretreatment achieved A549 cells injected intravenously. For the colorectal liver metas- significantly superior median OS compared with untreated con- tases models, the colorectal cancer cell lines LS174T or HT29 were trol mice (P ¼ 0.0004 and P ¼ 0.00004, respectively). injected into the spleen (3 106 cells). SCID-beige mice contain monocytes and macrophages that express FcgRIV, the murine homolog of human FcgRIIIA. Murine NK cells do not express Preincubation of ADCC effector cells with chemotherapy agents FcgRIV, therefore the SCID/beige mouse model can only dem- at physiologically relevant doses does not affect imgatuzumab- onstrate the antitumoral effect of ADCC (and/or FcgRIV-triggered mediated ADCC antibody-dependent cellular phagocytosis and cytokine release) The effect of chemotherapy agents on the in vitro ADCC activity mediated by monocytes and macrophages as effectors. Mice were and in vivo efficacy of imgatuzumab was investigated using gem- maintained under specific pathogen-free conditions with 12-hour citabine, cisplatin, carboplatin, paclitaxel, and irinotecan. These light/dark cycle daily according to guidelines (GV-SOLAS; compounds are currently used clinically to treat solid tumor types FELASA), and food and water were provided ad libitum. Contin- in which imgatuzumab is likely to be indicated (colorectal cancer, uous health monitoring was carried out and the experimental lung cancer, breast cancer, and head and neck cancer) and are study protocol was reviewed and approved by the Veterinary therefore potential partners for imgatuzumab in future combi- Department of Kanton, Zurich. nation chemotherapy regimens. Mice were randomized into different treatment groups (ten Preincubation of PBMC effector cells in 1 mg/mL gemcitabine mice per group) and therapy started when evidence of tumor had no effect on imgatuzumab- or cetuximab-mediated killing of growth was visible in the target organ of sacrificed scout animals A549 lung cancer cells across the range of mAb concentrations (7 days or 14 days after tumor cell inoculation). All treatments investigated in ADCC assays (Fig. 2A). Cell killing by PBMCs were administered intravenously and nontoxic efficacious doses pretreated with 10 mg/mL gemcitabine was reduced at higher were used as follows: imgatuzumab and cetuximab at a dose of imgatuzumab concentrations compared with controls and ADCC 25 mg/kg, gemcitabine at 150 mg/kg, cisplatin at 2 mg/kg, was significantly reduced for both anti-EGFR mAbs when PBMCs paclitaxel at 20 mg/kg, carboplatin at 60 mg/kg, and irinotecan were preincubated with high concentrations (100 mg/mL) of at 30 mg/kg. Pretreatment with hydrocortisone (20 mg/kg) was gemcitabine (33% relative reduction in maximal cell killing performed 1 hour before the first and second imgatuzumab dose. seen for both mAbs). Preincubation of PBMC effectors cells with All mAbs and drugs were given once weekly for 3 weeks (6 weeks cisplatin (0.1–10 mg/mL; Fig. 2B) and carboplatin (28 mg/mL; Fig. for cisplatin). The termination criteria for sacrificing animals was 2C) had no influence on A549 cell killing mediated by either sickness with locomotion impairment, and median overall sur- antibody, whereas the ADCC capacity of both anti-EGFR mAbs vival (OS) was defined as the experimental day by which 50% of was reduced following preincubation of PBMCs with paclitaxel animals had been sacrificed. Kaplan–Meier survival curves and the (Fig. 2D). Interestingly, SN-38 (the active metabolite of irinote- Pairwise log-rank test were used to compare survival between can) at a single tested concentration of 61.6 ng/mL had no effect animals treated with single-agent imgatuzumab and imgatuzu- on imgatuzumab-mediated ADCC of KRAS wild-type HT29 colo- mab in combination with chemotherapy. rectal cancer cells but reduced the degree of cell killing mediated

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Figure 1. A, pretreatment of PBMC effector cells in vitro with 8 mmol/L 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 follows: before hydrocortisone preincubation, 97.9%; after hydrocortisone preincubation, 96.4%. N ¼ 3. B, in SCID-beige mice (n ¼ 10 per group) inoculated with A549 cells, premedication with hydrocortisone (20 mg/kg) 1 hour before the first and second imgatuzumab infusion did not affect imgatuzumab efficacy; median OS was unchanged (P ¼ 0.36) and significantly longer than with vehicle control.

by cetuximab by approximately 40% (Fig. 2E). No significant gemcitabine plus imgatuzumab compared with 95 days for sin- effect on the cell viability of PBMCs was observed after the gle-agent imgatuzumab (P ¼ 0.05) and 69 days for single-agent incubation period with the different chemotherapeutic agents gemcitabine (P ¼ 0.05; Fig. 4A). Furthermore, OS with imgatu- measured before the respective ADCC assays. zumab plus gemcitabine was significantly longer than for cetux- In all ADCC experiments, the degree of cell killing mediated by imab plus gemcitabine (98 days; P ¼ 0.05). Similar data were seen imgatuzumab was higher than with cetuximab, consistent with when the same xenograft model was treated with imgatuzumab in previous findings (2). combination with cisplatin (Fig. 4B). Median OS was longer with Table 1 summarizes the EC50 and maximal cell killing values for combination therapy (118 days) compared with single-agent all premedication drugs/chemotherapies tested as single agents. imgatuzumab (84 days; P ¼ 0.003) and single-agent cisplatin Significant reductions in the maximal percentage of ADCC were (70 days; P ¼ 0.001), and a trend towards longer survival over the seen following pretreatment of PBMC effector cells with dexa- cetuximab plus cisplatin group (103 days; P ¼ 0.14) was seen. methasone, prednisolone, gemcitabine, and paclitaxel and max- Median OS of SCID/huCD16 tg mice inoculated with A549 cells imal cell killing increased significantly when effector cells were was 102 days when treated with paclitaxel plus imgatuzumab preincubated in oxaliplatin. Pretreatment with prednisolone and compared with 76 days for single-agent imgatuzumab (P ¼ 0.03) paclitaxel also significantly increased the EC50 of imgatuzumab. and 60 days for single-agent paclitaxel (P ¼ 0.0001; Fig. 4C). Similar data were seen when the same xenograft model was The in vitro ADCC potential of PBMCs mediated by treated with imgatuzumab in combination with carboplatin (Fig. imgatuzumab is relatively unaffected by pretreatment with 4D). Median OS was longer with combination therapy (112 days) complex drug mixtures compared with single-agent imgatuzumab (75 days; P ¼ 0.003) or To further investigate the influence of premedication and single-agent carboplatin (48 days; P ¼ 0.001). chemotherapy agents on imgatuzumab activity, ADCC assays Combining imgatuzumab with irinotecan also improved were performed using PBMCs preincubated for 20 hours in the median OS compared with either agent alone in the colorectal presence of two different cocktails (mix 1 and mix 2) of multiple cancer SCID/huCD16 tg xenograft models. In mice inoculated drugs currently used in the clinical setting. At medium to high with KRAS wild-type HT29 cells, median OS was 79 days when concentrations of imgatuzumab (1–1,000 ng/mL), mix 1 had no treated with irinotecan plus imgatuzumab compared with 59 days significant effect on imgatuzumab-mediated ADCC activity (Fig. for single-agent imgatuzumab ( P ¼ 0.03) and 49 days for single- 3). Cell killing was reduced slightly with mix 2 at higher imga- agent irinotecan (P ¼ 0.0001; Fig. 4E). In mice inoculated tuzumab concentrations. In contrast, cetuximab-induced ADCC with KRAS-mutant LS174T cells, median OS was longer with was reduced following preincubation of immune effector cells combination therapy (60 days) compared with single-agent imga- with either mix and at all concentrations of antibody. No signif- tuzumab (45 days; P ¼ 0.04) and single-agent irinotecan (41 days; icant effect on PBMCs cell viability was observed after the incu- P ¼ 0.001; Fig. 4F). bation period with both cocktails (mix 1 and mix 2) measured before the ADCC assays. Discussion Coadministration of imgatuzumab with cytotoxic agents The novel anti-EGFR mAb imgatuzumab is glycoengineered for enhances in vivo efficacy boosted ADCC (3). In this study, we demonstrated that common Combining imgatuzumab with chemotherapy agents signifi- chemotherapy agents and medications used to reduce the risk of cantly improved efficacy compared with both single-agent imga- therapy side effects had no significant negative effect on imgatu- tuzumab and single-agent chemotherapy. This was seen in all zumab-mediated ADCC cell killing in vitro. Moreover, in vivo, combinations tested (Fig. 4). Median OS of SCID-beige mice improved efficacy was observed when chemotherapeutic agents inoculated with A549 cells was 115 days when treated with were combined with imgatuzumab compared with mAb therapy

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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 mg/mL. Pretreatment of PBMCs with 28 mg/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 cetuximab. PBMC viability measured as follows: before gemcitabine preincubation, 98.9%; after 1 mg/mL, 10 mg/mL, and 100 mg/mL gemcitabine preincubation, 96.7%, 96.9%, and 97%, respectively. Before cisplatin preincubation, 99.4%; after 0.1 mg/mL, 1 mg/mL, and 10 mg/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 SN-38 preincubation, 99.8%; after SN-38 preincubation, 97.5%. N ¼ 3.

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

Imgatuzumab EC50 values (ng/mL) Maximal killing (%) Concentration used Cmax reported Untreated Treated P Untreated Treated P Hydrocortisone 8 mmol/L 8 mmol/L 0.28 0.44 0.0994 93.9 87.9 0.0704 Dexamethasone 0.1 mg/mL 0.06 mg/mL 5.46 4.84 0.6066 58.5 49.0 0.0001 Prednisolone 0.65 mg/mL 0.65 mg/mL 0.26 0.68 0.0002 94.9 83.1 <0.0001 Gemcitabine 10 mg/mL 10 mg/mL 0.36 0.38 0.9324 92.4 87.7 0.0058 Cisplatin 1 mg/mL n.c. 0.16 0.16 0.973 73.6 71.2 0.1047 Carboplatin 28 mg/mL 28 mg/mL 0.57 0.60 0.6499 65.6 67.1 0.5488 Paclitaxel 5–2.5 mg/mL 5 mg/mL 1.83 3.0 0.0005 53.5 36.8 <0.0001 SN-38 (metabolite of irinotecan) 0.062 mg/mL 0.062 mg/mL 1.07 1.74 0.1425 66.0 64.0 No difference Doxycycline 10 mg/mL 7 mg/mL 0.81 0.78 0.9392 55.2 55.8 0.5235 Oxaliplatin 1.5–0.3 mg/mL 1.5–0.3 mg/mL 4.01 4.73 0.3495 29.9 31.6 0.0004 NOTE: PBMCs were preincubated at concentrations shown for approximately 24 hours after which ADCC was performed using a range of imgatuzumab concentrations (0.01–1,000 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 ¼ 3. Abbreviation: n.c., not calculated.

alone. This in vivo effect was observed in xenograft models without tent with the known immunosuppressive effects of paclitaxel an acquired immune system but in the presence of the innate (25). However, despite this reduction in ADCC capability, immune effector cells that mediate ADCC. combining paclitaxel with imgatuzumab still resulted in Pretreatment of human PBMCs and murine xenograft models enhanced efficacy in SCID-beige mice models compared with with hydrocortisone at concentrations similar to those used clin- either agent alone. A slight reduction in cell killing at higher ically had no effect on the ADCC activity or efficacy of imgatuzu- concentrations of gemcitabine was also seen with imgatuzumab. These data indicate that strategies to reduce the risk and mab (from 92.4% to 87.7%). Interestingly, the ADCC activity severity of IRRs in the clinic can be potentially achieved with of cetuximab was reduced in the presence of the active metab- hydrocortisone without compromising the efficacy of imgatuzu- olite of irinotecan (SN-38). The ability of cetuximab to both mab-based treatment regimens. Future combination regimens will enhance the efficacy of irinotecan and restore tumor chemo- also necessitate the use of premedications designed to mitigate sensitivity to irinotecan is well established (15, 29–32). How- side-effects, such as nausea and vomiting, commonly observed ever, no such reduction in the ADCC activity of imgatuzumab with the administration of cytotoxic chemotherapies. We demon- inthepresenceofSN-38wasseen,whichmayindicatethat strated that the ADCC activity of imgatuzumab was relatively imgatuzumab–irinotecan combinations can further improve unaffected by the presence of multiple premedication drugs. outcomes in previously treated patients compared with cetux- Two chemotherapy agents reduced the ADCC potential of imab-irinotecan. human effector cells mediated by imgatuzumab. Preincubation Cell killing mediated by imgatuzumab was superior to of PBMCs in paclitaxel reduced the percentage of maximal cell cetuximab-mediated cell killing in all ADCC assays performed. mediated by imgatuzumab from 53.5% to 36.8%. This was also This was expected, as the A549 cell line used in the majority of seen for cetuximab-mediated in vitro cell killing and is consis- ADCC experiments represented a particularly challenging target

Figure 3. Preincubation of PBMC effector cells with a cocktail of cisplatin (1 mg/mL), granisetron hydrochloride (63.8 ng/mL), dexamethasone (100 ng/mL), aprepitant (1.6 mg/mL), pemetrexed (10 mg/mL), pantoprazole (1.25 mg/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 mg/mL), granisetron hydrochloride (63.8 ng/mL), dexamethasone (100 ng/mL), aprepitant (1.6 mg/mL), gemcitabine (10 mg/mL), and ranitidine hydrochloride (94 ng/mL; mix 2). In contrast, both drug cocktails reduced cetuximab- mediated ADCC. PBMC viability measured as follows: before mix 1 and mix 2 preincubation, 99.9%; after mix 1 and mix 2 preincubation, 96.4% and 97.9%, respectively. N ¼ 3.

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Common Cancer Drugs do not Impair Imgatuzumab-Mediated ADCC

Figure 4. SCID-beige mice (A and B) or SCID-huCD16 tg mice (C–F; n ¼ 10 per group) were inoculated with A549 cells (A–D), KRAS wild-type 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 OS with imgatuzumab combined 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. for anti-EGFR mAb-mediated cell killing (low EGFR expression The exact mechanisms by which conventional cytotoxic agents and mutant KRAS), and cetuximab is known to be relatively combine with anti-EGFR mAbs for enhanced efficacy are not fully ineffective against KRAS-mutant tumors (33). The enhanced understood; however, there is accumulating evidence that expo- cell killing and superior efficacy with imgatuzumab compared sure to some chemotherapy agents can upregulate EGFR expres- with cetuximab demonstrated in this study are consistent with sion. Membrane EGFR expression was upregulated following our previous report, which used a variety of human tumor cell exposure of several colorectal cell lines to gemcitabine, irinotecan, line models with different KRAS status and EGFR expression levofolinic acid, and 5-fluorouracil, and this resulted in enhanced level (3). in vitro cetuximab-mediated ADCC and lymphocyte-assisted kill- All chemotherapy agents tested in vivo significantly increased ing (34). Furthermore, levels of membrane EGFR expression OS when combined with imgatuzumab. Imgatuzumab demon- increased in a series of isogenic colorectal cancer cell lines as cell strated additive or synergistic efficacy when coadministered with lines became progressively more oxaliplatin-resistant, and the in gemcitabine and cisplatin in SCID-beige mice and with paclitaxel, vitro sensitivity of the cell lines to cetuximab increased in parallel carboplatin, and irinotecan in SCID-huCD16 tg mice. All imga- (35). The sequence of treatments may also be important; plati- tuzumab-chemotherapy combinations tested to date on xenograft num and taxane chemotherapy followed by anti-EGFR therapy models achieved enhanced efficacy compared with cetuximab (both mAb and small-molecule tyrosine kinase inhibitors) plus chemotherapy. strongly enhanced apoptosis and cell-cycle arrest in vitro, whereas

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Gonzalez-Nicolini et al.

an antagonistic effect was seen when anti-EGFR therapies were Authors' Contributions given before chemotherapy (36). Conception and design: V. Gonzalez-Nicolini, P. Umana,~ C.A. Gerdes One limitation of this study is that the mouse models used lack Development of methodology: V. Gonzalez-Nicolini, C.A. Gerdes a functional adaptive T-cell response; therefore, it was not possible Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): V. Gonzalez-Nicolini, S. Herter, I. Waldhauer, to investigate the imgatuzumab adaptive immune-related mode ﬂ M. Roemmele, E. Bommer, E. van Puijenbroek of action and how the tested agents affect T cells and the in uence Analysis and interpretation of data (e.g., statistical analysis, biostatistics, this has on tumor cell killing. As most chemotherapy agents affect computational analysis): V. Gonzalez-Nicolini, S. Herter, S. Lang, I. Waldhauer, proliferating cells, T-cell response is likely to be affected by some O. Freytag of the drugs if given during previous days or in concomitant Writing, review, and/or revision of the manuscript: V. Gonzalez-Nicolini, ~ administration with the antibody therapy; therefore, further stud- P. Umana, C.A. Gerdes Administrative, technical, or material support (i.e., reporting or organizing ies using syngeneic models with functional adaptive T-cell data, constructing databases): V. Gonzalez-Nicolini, O. Freytag, E. van responses are now required as well as different schedules of Puijenbroek combination therapies. Study supervision: M. Bacac, P. Umana,~ C.A. Gerdes In conclusion, the enhanced innate immune-mediated ADCC activity of imgatuzumab compared with cetuximab appears to be Acknowledgments unaffected by common cancer premedication drugs and chemo- The authors thank Priska Rueegg, Daniela Ackermann, and Michael Mauch therapy agents when tested in models bearing a functional innate for quality administrative support. The authors also appreciate the functional immune system only. Building on these preclinical data, several support of Roche Glycart molecular biology and process biochemistry, Jon Chick, and the rest of the Roche imgatuzumab global team for discussion imgatuzumab combination chemotherapy regimens are under contributions while generating this article. investigation. In NSCLC, the combinations of imgatuzumab plus cisplatin and gemcitabine (for squamous histology) and imga- Grant Support tuzumab plus cisplatin and pemetrexed (for non-squamous his- This study was funded by Roche Glycart AG, Switzerland. Support for third- tology) are investigated as ﬁrst-line treatment (NCT01185847). In party writing assistance for this article was provided by Prism Ideas and funded colorectal cancer, second-line treatment with imgatuzumab plus by F Hoffmann-La Roche. FOLFIRI is also under investigation in patients with KRAS-mutant The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked tumors, and imgatuzumab plus FOLFIRI and cetuximab in advertisement KRAS in accordance with 18 U.S.C. Section 1734 solely to indicate patients with wild-type tumors (NCT01326000; ref. 28). this fact.

Disclosure of Potential Conﬂicts of Interest Received October 9, 2014; revised October 16, 2015; accepted October 20, No potential conﬂicts of interest were disclosed. 2015; published OnlineFirst November 18, 2015.

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Common Cancer Drugs do not Impair Imgatuzumab-Mediated ADCC

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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.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-14-2579

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