Pegfilgrastim Enhances the Antitumor Effect of Therapeutic Monoclonal Antibodies

Published OnlineFirst March 17, 2016; DOI: 10.1158/1535-7163.MCT-15-0759

Small Molecule Therapeutics Molecular Cancer Therapeutics Pegﬁlgrastim Enhances the Antitumor Effect of Therapeutic Monoclonal Antibodies Sebastien Cornet1,2,3,4, Doriane Mathe2,3,4, Kamel Chettab1,2,3,4, Anne Evesque2,3,4, Eva-Laure Matera2,3,4, Olivier Tredan 5, and Charles Dumontet1,2,3,4

Abstract

Therapeutic mAbs exert antitumor activity through various filgrastim in murine xenograft models treated with mAbs. This þ mechanisms, including apoptotic signalization, complement- was observed with rituximab in CD20 models and with trastu- þ dependent cytotoxicity, and antibody-dependent cellular cyto- zumab in HER2 models. Stimulation with pegfilgrastim was toxicity (ADCC) or phagocytosis (ADCP). G-CSF and GM-CSF associated with significant enhancement of leukocyte content have been reported to increase the activity of antibodies in in spleen as well as mobilization of activated monocytes/ preclinical models and in clinical trials. To determine the poten- granulocytes from the spleen to the tumor bed. These results tial role of pegfilgrastim as an enhancer of anticancer antibodies, suggest that pegfilgrastim could constitute a potent adjuvant for we performed a comparative study of filgrastim and pegfilgrastim. immunotherapy with mAbs possessing ADCC/ADCP properties. We found that pegfilgrastim was significantly more potent than Mol Cancer Ther; 15(6); 1238–47. 2016 AACR.

Introduction Most clinically used mAbs belong to the IgG subclass and their Fc domain interacts with IgG Fc receptors (Fcg receptors). The mAbs are increasingly used for the treatment of cancer patients. human IgG receptor family includes several members, most of Their mechanisms of action are complex as antibodies can either which are activating (CD64 or FcgRI, CD32a or FcgRIIa, CD16a or target the tumor cells themselves or the microenvironment, FcgRIIIa, CD16b or FcgRIIIb) and one which is inhibitory including tumor vasculature or the surrounding immune cells. (CD32b or FcgRIIb) with various affinities for the IgG subclasses. Among antibodies targeting tumor cells directly, various mechan- Several of the mAbs currently used in the clinic rely at least partly isms of action are likely to be involved including direct apoptotic on Fcgreceptor–mediated mechanisms and novel antibodies, signalization after recognition of the cognate antigen and extra- such as obinutuzumab, have been specifically engineered to cellular effector mechanisms such as complement-dependent possess enhanced ADCC properties (3). Fc receptor polymorph- cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity isms that affect affinity for IgG (FcgRIIa-131H/R and FcgRIIIa- (ADCC) or its closely related phenomenon antibody-dependent 158V/F) have been found to influence the clinical response to cellular phagocytosis (ADCP; ref. 1). Despite this diversity of rituximab (anti-CD20; ref. 4), cetuximab (anti-EGFR; ref. 5), or mechanisms of antitumor activity, treatment by mAbs has not trastuzumab (anti-HER2; ref. 5) therapy in lymphoma, colorectal, allowed to obtain cure in most of the approved indications, either and breast cancer patients, respectively, suggesting that Fcg recep- as a single agent or in combination with other agents. The case of tor–mediated effector functions play an important role in cancer follicular non-Hodgkin lymphoma (NHL) is exemplary as certain treatment. patients can benefit from single-agent therapy but resistance to The nature of the Fcg receptor–bearing cells involved in cyto- treatment will eventually develop. In addition, a significant pro- toxic activity is not completely elucidated. Classically, natural portion of responding patients will no longer benefit of retreat- killer (NK) cells have been considered as the main effector cells for ment with this mAb after relapse (2). ADCC (6). NK cells express the high-affinity receptor FcgRIIIa, and the association between Fc receptor alleles and clinical success of mAb therapy has been attributed to NK cells (7). However, 1 2 Hospices Civils de Lyon, Pierre Benite, France. Universite de Lyon, other cell populations can act as potential effector cells for mAb- Lyon, France. 3INSERM U1052, Centre de Recherche en Cancerologie de Lyon, Lyon, France. 4CNRS UMR 5286, Centre de Recherche mediated tumor regression, including myeloid effector cells such en Cancerologie de Lyon, Lyon, France. 5Departement d'Oncologie as monocytes/macrophages and neutrophils (8). ^ Medicale,Centre de Lutte Contre le Cancer Lyon et Rhone-Alpes, Lyon, Neutrophils are the most abundant circulating leukocyte sub- France. population in peripheral blood. In addition, this population can Note: Supplementary data for this article are available at Molecular Cancer be amplified and/or mobilized by the administration of granu- Therapeutics Online (http://mct.aacrjournals.org/). locyte (macrophage)-colony stimulating factors (G-CSF and GM- Corresponding Author: Charles Dumontet, Centre de Recherche en CSF; refs. 9–11). Neutrophils have been described as potent Cancerologie de Lyon, 8 avenue Rockefeller, Lyon 69008, France. Phone: cytotoxic effectors, able to produce many cytotoxic molecules 334-7877-7123; Fax: 334-7877-7088; E-mail: [email protected] (12) and exert direct tumoricidal activity (13). Their ability to doi: 10.1158/1535-7163.MCT-15-0759 directly recognize tumor cells is limited but is highly enhanced in 2016 American Association for Cancer Research. the presence of mAbs (14).

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Pegﬁlgrastim and Anticancer Antibodies

Fcg receptors expressed on neutrophils are FcgRIIa, FcgRIIIb, cinoma epithelial cell line A549 (ATCC: CCL-185), and the breast and FcaRI. FcgRIIIb is the most abundant Fcg receptor on neu- cancer cell line MDA-MB361 (ATCC: HB-27), were purchased trophils, but evidence suggests that it is not involved in efficient directly from the ATCC. These cells were authenticated by ATCC tumor cell cytotoxicity (15). Antibody-dependent killing of tumor by generating human short tandem repeat profiles by simulta- cells by neutrophils in the absence of G-CSF is described to be neously amplifying multiple STR loci and amelogenin (for gender nonexistent or weak (16, 17) and is likely mediated through determination) using the Promega PowerPlex Systems. These cells FcgRIIa (18). Therefore, specific targeting of FcgRI has been were cultured in the laboratory for less than 6 months in culture proposed, as this may overcome potential inhibitory signals medium consisting of RPMI1640 (Life Technologies), 10% FCS through FcgRIIb when IgG mAbs are employed (19). (Integro), 100 U/mL of penicillin, and 100 mg/mL of strepto- To improve antibody-dependent cytotoxicity of leukocytes, mycin (Life Technologies). NFS-60 cells, kindly provided by coadministration of mAbs and different cytokines has been per- Dr.J.Ihle(St.JudeChildren'sResearchHospital,Memphis,TN) formed, in particular, with GM-CSF and G-CSF. Cartron and were maintained in RPMI1640 with 10% of FCS, 1% L-gluta- colleagues (20) performed a phase II clinical study combining mine (2 mmol/L), 1% b-mercaptoethanol (Sigma Aldrich), and GM-CSF and rituximab in patients with relapsed follicular lym- 20 U/mL of IL3 (Sigma-Aldrich). All cells were cultured at 37C phoma. This study reported an improvement in the expected in a 5% CO2 atmosphere. complete response rate with the combination compared with During the course of our experiments, cell culture was system- monotherapy (39% vs. 6%, respectively; ref. 21). The authors atically performed in compliance with good laboratory practice explain this result by the recruitment and activation of granulocytes and the specific culture conditions for each cell line used. Cell line and monocytes, leading to an increased antitumor potential, morphology was checked and the expressions of cell surface involving both ADCC and ADCP (22). While increasing the dose markers CD20 and HER2 were assessed by flow cytometry on of GM-CSF appears to decrease the ADCC exerted by peripheral RL, A549, and MDA-MB361 cell lines, respectively. Using MycoA- mononuclear cells (23), G-CSF or IFNg-primed neutrophils have lert Kit (Lonza) all cell lines used in this study tested mycoplasma- an increased potential for mAb-mediated cytotoxic activity through negative. upregulated expression of FcgRI both in vitro and in vivo (24). Van der Kolk and colleagues showed in a phase I/II clinical trial that, Cell assays when G-CSF was combined with rituximab in CLL, the median NFS-60, a murine myeloid leukemia cell line which prolif- time to progression observed for the combination was 24 months, erates in response to G-CSF, was used for cell-cycle experiments and only 13 months for rituximab as a single agent although the (26, 27). Cells were starved for 18 hours (no IL3 nor G-CSF) to – overall response rate was comparable in both groups (25). block NFS-60 cells in G0 G1 phase, then various concentrations Recombinant G-CSF (filgrastim) used to treat patients has a of filgrastim or pegfilgrastim (from 5 pmol/L to 1 nmol/L) were short half-life of in the blood stream (3–4 hours). Pegfilgrastim, a added to the cells for 18 hours. Cell-cycle distribution was filgrastim analogue carrying a 20 kDa polyethylene glycol (PEG) estimated by flow cytometry using propidium iodide as molecule on its N-terminus has a half-life of 15 to 80 hours in described previously (28). blood. Pegylation increases the molecular weight of filgrastim above the threshold for renal clearance. Consequently, the drug is Neutrophil preparation believed to be mostly cleared by neutrophils. Neutrophil-medi- Blood samples from healthy donors were provided by the Lyon ated clearance takes longer than renal clearance, thereby increas- Blood Bank. Neutrophils were obtained by performing a density ing the half-life of the drug. We show that filgrastim and pegfil- gradient centrifugation (Pancol, Pan-Biotech) followed by a dex- grastim display similar effects on normal human polymorpho- tran separation (3% Dextran, Sigma Aldrich). Remaining red nuclear cells in vitro. However, in vivo, pegfilgrastim proved to be blood cells were removed using a lysis solution (BD Pharm Lyse, significantly more potent than filgrastim when combined with BD Biosciences) and purity of neutrophils was evaluated by flow therapeutic mAbs such as rituximab and trastuzumab. This in vivo cytometry using CD45/SSC gating. effect was associated with a strong increase in spleen size as well as recruitment of neutrophils and activated monocytes in various Leukocyte subpopulations in mice compartments including the tumor site. The phenotype of leukocyte subpopulations in the blood, spleen, bone marrow, and tumor were studied after four injec- Materials and Methods tions with rituximab combined or not with filgrastim or pegfil- fl Reagents grastim. Brie y, spleen and tumor were cut in small pieces and Rituximab (MabThera, Roche) a chimeric mAb directed against incubated in PBS containing 0.5% FCS and 1 mg/mL collagenase A (Roche Diagnostics GmbH), 0.1 mg/mL hyaluronidase and 10 CD20, and trastuzumab (Herceptin, Genentech) a chimeric mAb directed against HER2/neu receptors were purchased as their mg/mL DNase (Sigma) at 37 C for 45 to 60 minutes. Digested fi commercial formulations. Filgrastim (Granocyte 34M, Chugai fragments were ltered through a stainless steel sieve and cell þ Pharmaceuticals), a recombinant G-CSF synthesized in Chinese suspensions washed in PBS 0.5% FCS. Blood and bone fi fi marrow samples were processed directly with no prior treat- hamster ovary cells (CHO cells), and peg lgrastim (Peg lgrastim, Amgen) a recombinant human factor-CSF (filgrastim) coupled ment. Cells were stained in 100 mL PBS for 30 minutes at 4 C with a 20 kDa PEG molecule to the N-terminus were also used as with anti-CD11b Alexa Fluor 488 (clone M1/70), anti-CD16/ their commercial formulations. CD32 PerCP-Cy5.5 (clone 2.4G2), anti-CD49b PE-CF594 (clone DX5), anti-Ly-6C APC-Cy7 (clone AL-21), anti-Ly-6G Cell lines and culture (clone 1A8), purchased from BD Pharmingen, and anti-f4/80 RL (ATCC: CRL-226), a human NHL B-cell line expressing Pe-Cy7 (clone BM8) purchased from eBioscience. For intratu- CD20 and two HER2-positive lines, the human lung adenocar- moral leukocytes, anti-human CD20 V450 (clone L27, BD

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Pharmingen) was used to distinguish human cells and murine twice a week. Treatments were administered once a week: mAbs leukocytes. The cells were washed once in PBS supplemented were injected intraperitoneally and G-CSF formulations intrave- with 2% FCS and immediately analyzed in a LSR II flow nously on the same day. cytometer (Becton Dickinson). Data were analyzed with BD To define optimal doses of filgrastim and pegfilgrastim for FACSDiva7 software. tumor growth inhibition experiments, a dose–response experi- ment was performed using various quantities of filgrastim and Human leukocytes pegfilgrastim (3 to 60 mg per injection per mouse) combined with Leukocyte populations of healthy donors used for in vitro rituximab. Optimal doses were then used for in vivo experiments experiments were assessed by flow cytometry using anti-CD13 to evaluate their effect when coinjected with different doses of VioBlue (clone BW264/56), anti-CD16 (VEP13), anti-CD14 mAb (data not shown). APC-Vio770 (clone TUK4), anti-CD45 APC-vio700 (clone 5B1), anti-CD19 Pe-Vio770 (LT19), anti-CD15 APC (clone Statistical analysis VIMC6), and anti-CD56 PE (AF12-7H3) obtained from Milte- In vitro data were analyzed using Student t test. For tumor nyi Biotec. growth inhibition assays, one-way ANOVA was used to compare treatment groups. Analyses were performed with Statistica 8.0 Human granulocyte studies (StatSoft, Inc.). Effect of G-CSF formulations on the phagocytic activity of normal human leukocytes was assessed with two different Results assays. Phagotest (Glycotope Biotechnology), which measures fi fi the ingestion of one or more labeled and opsonized bacteria, Effect of lgrastim and peg lgrastim on the antitumor activity in vivo E. coli–FITCpercell,wasusedtodeterminethephagocytic of therapeutic mAbs fi fi fi properties of granulocytes, lymphocytes, and monocytes. The ef cacy of the combination of peg lgrastim, lgrastim, and FagoFlowEx Kit (Exbio Diagnostics) was used to measure the therapeutic antibodies was compared in the RL, MDA-MB431, respiratory (oxidative) burst after their stimulation with E. coli and A549 models. For the evaluation of tumor growth, calcula- fi fi bacteria in human heparinized whole blood using flow tions were started on the rst day of treatment until sacri ce of the cytometry. control group. Values were documented as medians and SDs. The tumor growth inhibition (TGI) for volume (T/C) was calculated ½TV ðday ddxxÞ100 according to the NCI formula: 1 ð treated Þ. ADCP assay ½TVcontrolðday ddxxÞ100 ADCP mediated by neutrophils was evaluated by fluores- In the first study, SCID mice bearing RL tumors were random- cence transfer of target cells to effector cells. The assay was ized into six groups, with 5 mice per group: controls, filgrastim, adapted from McEarchern and colleagues (29). Tumor cells pegfilgrastim, rituximab, filgrastim þ rituximab, pegfilgrastim þ were labeled with PKH26, a fluorescent cell membrane dye, rituximab. As shown in Fig. 1A, at day 42, TGI values calculated as according to the manufacturer's instructions (Sigma). Neutro- indicated previously were 95%, 78%, and 80% for pegfilgrastim/ phils stimulated or not with filgrastim or pegfilgrastim (at a rituximab, filgrastim/rituximab, and rituximab alone, respective- concentration of 10 ng/mL during 2 hours at 37 C) were used ly. In addition, a one-way ANOVA showed that pegfilgrastim/ as effector cells. Effector and target cells were coincubated at an rituximab treatment significantly decreased tumor size when effector:target (E:T) ratio of 2:1 overnight at 37 C5%CO2,in compared with filgrastim/rituximab and rituximab alone, where- the presence or absence of mAbs. After incubation, cells were as no difference could be measured between filgrastim/rituximab washed once in PBS 2% FCS stained with 3 mL of anti-CD15- and rituximab alone. In this model, pegfilgrastim effect on tumor APC (clone HI98, BD Biosciences) for 30 minutes at 4 Cand size was equivalent to that observed with rituximab treatment. analyzed by flow cytometry. This may be the consequence of the significant changes in immune cell number and function observed after pegfilgrastim In vivo experiments inoculation (30). Six-week-old female CB17 severe combined immunodeficient In the MDA-MB361 model, with 3 mice per group (Fig. 1B), TGI (SCID) mice purchased from Charles River Laboratories were bred values were 17%, 94%, and 112% for pegfilgrastim alone, tras- under pathogen-free conditions at the animal facility of our tuzumab alone, and pegfilgrastim/trastuzumab, respectively. In institute. Animals were treated in accordance with the European addition to the significant differences between the treatment Union guidelines and French laws for the laboratory animal care groups, a complete tumor remission was observed in the pegfil- and use. The animals were kept in conventional housing. Access to grastim/trastuzumab group but not in the other groups. Similar food and water was not restricted. This study was approved by the observations were obtained with a third model (A549 cell line, University of Lyon Animal Ethics committee. Continuous health Supplementary Fig. S1). Furthermore, in the RL model, an monitoring was carried out on a regular basis, with daily mon- increased mice survival was observed in the pegfilgrastim/ritux- itoring of clinical symptoms and adverse effects. Animals of an imab–treated group (Supplementary Fig. S2). average weight of 20 g were inoculated subcutaneously with tumor cells (3–5 106 cells) into the right flank. When subcuta- Effect of filgrastim and pegfilgrastim on cell-cycle distribution neous tumors were established (median volume, 100–150 mm3), of NFS-60 cells animals were randomized and treatments were initiated. Primary Cell-cycle experiments comparing variable concentrations of tumor volume (TV) was calculated according to the National filgrastim and pegfilgrastim showed that both molecules induce Cancer Institute (NCI, Bethesda, MD) protocol [TV ¼ (length cell-cycle entry of starved NFS-60 cells, with a decrease in the 2 width )/2, where "length" and "width" are the long and short percentage of cells in G0–G1 and a corresponding increase of the diameters of the tumor mass]. Measurements were performed cells in G2–M phase. In this assay, filgrastim was more potent than

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Pegﬁlgrastim and Anticancer Antibodies

AB 1,600 900 Untreated Untreated ) 3 ) 1,400 Ritux 800 Trast 3 1,200 Filgrast 700 Pegfilgrast Filgrast + Ritux Trast + Pegfilgrast 1,000 Pegfilgrast 600 Pegfilgrast + Ritux 500 800 400 600 300 400 Tumor volume (mm Tumor 200 Tumor volume (mm Tumor 200 100 0 0 22 29 36 43 20 27 34 41 48 55 62 Days after tumor engraftment Days after tumor engraftment

Figure 1. Effect of filgrastim and pegfilgrastim on the antitumor activity of therapeutic mAbs in vivo.A,efficacy of combination of pegfilgrastim and rituximab (Ritux) on RL xenograft growth in mice. The TGI calculated on day 42 confirmed the potentiation of rituximab antitumor activity when coinjected with pegfilgrastim. Cotreatment with pegfilgrastim (Pegfilgrast) and rituximab significantly decreased RL growth when compared with untreated, rituximab alone, and filgrastim with rituximab groups according to one-way ANOVA (P < 0.001). No significant differences were observed between rituximab and filgrastim (Filgrast) with rituximab. B, efficacy of combination of pegfilgrastim and trastuzumab (Trast) on MDA-MB 361 xenograft growth in mice. The TGI calculated on day 62 showed the potentiation of trastuzumab efficacy when coinjected with pegfilgrastim. With a complete relapse in the MDA-MB 361 model, cotreatment with pegfilgrastim and trastuzumab significantly decreased tumor growth when compared with untreated and trastuzumab alone according to one-way ANOVA (P < 0.001).

pegfilgrastim over the range of concentrations tested (0.1–2 in 2 of 3 donors, pegfilgrastim was more potent than filgrastim in ng/mL) with no dose–response effect for either molecule. Similar inducing an increased phagocytosis by neutrophils (, P < 0.05). results were observed at concentrations of 5 ng/mL or greater (Fig. 2A and B). Effect of filgrastim and pegfilgrastim on leukocyte populations in vivo Effect of filgrastim and pegfilgrastim on phagocytic activity of Mice received repeated injections of rituximab combined or not normal human neutrophils with filgrastim or pegfilgrastim (n ¼ 5 mice per group). The Phagocytic activity of normal human neutrophils was explored cellularity of spleens was found to strikingly increase in mice in samples obtained from three healthy donors (Fig. 3A). Filgras- treated with pegfilgrastim without rituximab in comparison with tim induced a significant increase of the percentage of neutrophils controls, with cell numbers increased 5- to 6-fold (Fig. 4A). There performing phagocytosis in 2 of 3 healthy donors (, P < 0.005), was also a significant increase of spleen size of mice treated with whereas pegfilgrastim induced a significant increase of the per- pegfilgrastim (with or without rituximab) compared with the centage of neutrophils performing phagocytosis in 3 of 3 healthy other groups (P < 0.05; Fig. 4B and C). The ratio of spleen donors (, P < 0.05). However, no significant difference could be leukocyte subpopulations was also analyzed. For each subpop- measured between samples stimulated with filgrastim or pegfil- ulation assessed, association of pegfilgrastim and rituximab grastim. Evaluation of ROS production by neutrophils using induced an increase in the number of cells when compared with Fagoflow showed similar results. As shown in Fig. 3B, filgrastim rituximab alone. The activated monocyte population was multi- and pegfilgrastim induced a significant increase of the percentage plied 19-fold and the granulocyte population 18-fold (Supple- of neutrophils producing ROS (, P < 0.05), whereas no significant mentary Fig. S3). difference of ROS production could be measured between leu- We analyzed leukocyte subpopulations to identify those kocytes stimulated with filgrastim or pegfilgrastim. These two leukocytes which were preferentially increased. As shown functional assays confirmed the ability of filgrastim and pegfil- in Fig. 5A, four leukocyte subpopulations were evaluated, grastim to enhance phagocytic and ROS-producing properties of including NK cells, resting monocytes, activated monocytes, neutrophils, with no significant difference observed between the and granulocytes. None of the treatments induced variation of þ two molecules. the percentage of NK cells, identified with CD49b ,among CD11b cells. Among the monocytic population (Ly6G Effect of filgrastim and pegfilgrastim on ADCP properties of Ly6Chighorlow),thepercentageofactivated monocytes (Ly6G normal human neutrophils Ly6Chigh) was slightly but significantly increased after pegfil- Phagocytosis of target cells was measured by incorporation of grastim/rituximab injections (P < 0.05), whereas resting mono- the fluorescent lipophilic molecule PKH67 used to label target cytes (Ly6G Ly6Clow)weresignificantly decreased (P < 0.05). þ cells into neutrophils. While trogocytosis may occur, this assay is The percentage of the Ly6G Ly6Clow population, usually commonly used to evaluate ADCP in vitro (31). As presented described as granulocytes, was increased in the spleen of mice in Fig. 3C, the presence of mAb induced a significant increase of treated with filgrastim and pegfilgrastim compared with both phagocytosis of tumor cells by neutrophils. In addition, preincu- untreated and rituximab-treated mice, but no significant dif- bation of neutrophils with filgrastim and pegfilgrastim signifi- ference was observed between filgrastim and pegfilgrastim cantly enhanced their capacity to perform phagocytosis. Moreover treatment groups.

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A 45 B Filgrastim 80 Filgrastim Pegfilgrastim Pegfilgrastim 40 75 phase phase 2 1 - G 0 35 70

30 65

% of NFS-60 cells in S - G 60 % of NFS-60 cells in G

20 55

5 pmol/L 5 pmol/L 25 pmol/L 50 pmol/L 100 pmol/L 25 pmol/L 50 pmol/L 100 pmol/L Unstimulated Unstimulated Stimulation Stimulation

Figure 2. Effect of filgrastim or pegfilgrastim on cell cycle of NFS-60 cells. After 18 hours of G-CSF starvation of NFS-60 cells to stop their proliferation, NFS-60 cells were stimulated with different concentrations of filgrastim or pegfilgrastim. Both filgrastim and pegfilgrastim induced a re-entry into the cell cycle of NFS-60 cells (P < 0.05) compared with unstimulated NFS-60 (P < 0.005 for filgrastim and P < 0.05 for pegfilgrastim). A and B, percentage of NFS-60 re-entry into the cycle phases S–G2 and G0–G1, respectively. In addition, filgrastim was significantly more potent to induce re-entry of NFS-60 cells compared with pegfilgrastim (P < 0.05) except at the highest concentrations of pegfilgrastim [no significant difference at 100 pmol/L and above (data not shown)].

We also assessed absolute number of leukocyte subpopula- be more prolonged in the case of pegfilgrastim or enhanced tions within the spleen (Fig. 5B). In parallel with the weight uptake of pegfilgrastim by accessory cells. increase observed with pegfilgrastim treatment (with or with- Some attempts have been made to potentiate the effect of mAbs out rituximab), a systematic and significant increase of the in the clinic with growth factors targeting potential effector cells. number of cells of all leukocyte subpopulations was observed An immunocytokine fusion protein combining IL2 with an anti- (P < 0.05). In blood (Fig. 5C), significant differences were only fibronectin antibody in combination with rituximab was found to observed for the pegfilgrastim groups (with or without ritux- eradicate B-cell lymphoma xenografts (32). Antibodies directed þ imab) with a significant increase of the Ly6G Ly6Clow popu- against the inhibitory Kir molecules expressed by NK cells have lation and a significant decrease of the monocytic populations been evaluated in combination with rituximab (33). IL15 has (both Ly6G Ly6Chigh and Ly6G Ly6Clow). Within the tumor, been shown to induce NK-mediated CLL depletion in vitro, with a no significant variation of NK cells or monocytes (both potentiation of rituximab (34). IL21 has been shown to enhance Ly6Chigh and Ly6Clow) could be observed, but a significant the activity of rituximab in Cynomolgus monkeys (35). IFNg has þ increase of the percentage of neutrophils within CD11b cells been reported to enhance antibody-mediated toxicity of neutro- up to 18% (vs. 2.85% in controls) was detected in mice phils against a colorectal model (36). Currently, none of these receiving pegfilgrastim (, P < 0.05; Fig. 5D). approaches have yet been validated in the clinic. Some investigators have used G-CSF or GM-CSF in an attempt Discussion to enhance mAb potency in preclinical models and patients. Hernandez-Ilizaliturri and colleagues combined murine G- or Our results show that pegfilgrastim enhances the antitumor GM-CSF with rituximab in a murine xenograft model in SCID activity of ADCC/ADCP-performing antibodies such as rituximab mice and found that these molecules significantly increased the and trastuzumab in preclinical models and that this effect was antitumor effect of the antibody in this setting (37). These authors greater than that observed with filgrastim. This immuno-adjuvant also showed that neutrophil depletion in a murine model was effect was associated with the amplification and recruitment of associated with a loss of antitumor activity of rituximab (38). Van neutrophils, including in the tumor site. Clinical studies, such as der Kolk and colleagues administered G-CSF 5 mg/kg/day for 3 the one currently being conducted combining rituximab and days in their study with rituximab infusion on day 2 in 26 patients pegfilgrastim in patients with NHL (NCT01682044) should with relapsing low-grade lymphoma (25). Tolerance was satis- determine whether this combination is beneficial in the clinic. factory and time to progression was considered to be unexpect- Possible differences between filgrastim and pegfilgrastim may be edly long in some patients. Cartron and colleagues treated the kinetic of stimulation of accessory cells, which is expected to 33 patients with relapsed follicular lymphoma with GM-CSF

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Pegﬁlgrastim and Anticancer Antibodies

AB 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 % Neutrophils producing ROS

% Granulocytes phagocyting E. coli 0 E. coli E. coli E. coli None None None PMA None None None Filgrast Filgrast Filgrast Unstimulated Filgrast Pegfilgrast Pegfilgrast Pegfilgrast Pegfilgrast

Donor 1 Donor 2 Donor 3

C 45 40 35 30

25 20 15

% Neutrophils PKH67+ 10 5

0 None Ritux None Ritux None Ritux None Ritux None Ritux None Ritux None Ritux None Ritux None Ritux

None Filgrast Pegfilgrast None Filgrast Pegfilgrast None Filgrast Pegfilgrast

Donor 1 Donor 2 Donor 3

Figure 3. Effect of filgrastim (filgrast) and pegfilgrastim (pegfilgrast) on phagocytic and ADCP activity of normal human neutrophils. A, effect of filgrastim or pegfilgrastim on phagocytic activity of neutrophils using FITC E. coli from PhagoTest. Compared with unstimulated samples, filgrastim induced a significant increase of the percentage of neutrophils performing phagocytosis in 2 of 3 healthy donors (, P < 0.005), whereas pegfilgrastim induced a significant increase of the percentage of neutrophils performing phagocytosis in 3 of 3 healthy donors (, P < 0.05; , P < 0.005). B, effect of filgrastim or pegfilgrastim on the production of reactive oxygen species (ROS) by granulocytes in presence or absence of E. coli using FagoflowEx Kit. Results shown are median values obtained with 4 different healthy donors. Filgrastim and pegfilgrastim induced a significant increase of percentage of neutrophils producing ROS (P < 0.05). C, effect of filgrastim or pegfilgrastim on ADCP activity of fresh human neutrophils. Compared with unstimulated samples, filgrastim and pegfilgrastim induced a significant increase of the percentage of neutrophils performing phagocytosis (P < 0.005). A significant difference was measured for some of the healthy donors (2/3) between samples stimulated with filgrastim and pegfilgrastim (P < 0.005; comparison between samples with or without rituximab).

5 mg/kg/day on days 1 to 8 and rituximab 375 mg/m2 on day 5 of the sequence of administration. In our study, both molecules each 21-day cycle for four cycles (20). They observed a 70% overall were administered simultaneously. This may prove to be an response rate and tolerance was satisfactory with 39% complete issue if patients receive simultaneous cytotoxic chemotherapy responses, which appears to be greater than what is expected with as it may sensitize hematopoietic cells to myeloid toxicity. A rituximab as single-agent therapy. small yet provocative study by Gruber and colleagues on 32 Several issues remain to be addressed concerning the poten- CLL patients receiving the ﬂudarabine/cyclophosphamide/ tial clinical beneﬁt of combining a leukopoietic growth factor rituximab combination with or without G-CSF, administered with ADCC/ADCP-performing mAbs. One question deals with in case of profound neutropenia, reported much better

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AB 200 450 400 350 300 ) per spleen 6 250 20 200 150

Spleen weight (mg) 100

Cell number ( ¥ 10 50 2 0

Ritux Filgrast Ritux Untreated Pegfilgrast Filgrast Untreated Pegfilgrast Filgrast + Ritux Pegfilgrast + Ritux Filgrast + Ritux Pegfilgrast-Ritux C Ritux Filgrast + Ritux Pegfilgrast + Ritux

1 cm

Figure 4. Effect of filgrastim and pegfilgrastim on spleen cellularity, spleen size, and leukocyte subpopulations. A, effect of filgrastim (filgrast) and pegfilgrastim (pegfilgrast) on spleen cellularity. Spleens were obtained 2 days after the last treatment from 5 mice that were either not treated or treated weekly with rituximab (Ritux; 30 mg/kg), filgrastim (375 mg/kg), with or without rituximab (30 mg/kg) and pegfilgrastim (750 mg/kg) with or without rituximab (30 mg/kg), during 4 weeks. Spleens were dissociated into single-cell suspensions and leukocytes counted by Trypan blue exclusion. Results are the mean SEM of 5 mice per group (, P < 0.005). B, effect of filgrastim and pegfilgrastim on spleen size and leukocyte subpopulations. Spleens were obtained 2 days after the last treatment from 5 mice that were either not treated or treated weekly with rituximab (30 mg/kg), filgrastim (375 mg/kg) with or without rituximab (30 mg/kg) and pegfilgrastim (750 mg/kg) with or without rituximab (30 mg/kg), during 4 weeks. Spleen size was not increased in mice exposed to filgrastim þ rituximab in comparison with mice exposed to rituximab alone but was significantly increased in mice exposed to pegfilgrastim þ rituximab (up to 6-fold when compared with rituximab alone (, P < 0.005), and up to 4-fold (, P < 0.005) when compared with filgrastim þ rituximab. C, pictures of spleens of mice treated with rituximab, filgrastim þ rituximab, or pegfilgrastim þ rituximab after 4 weeks of treatments.

outcomes in the G-CSF group (39). One possible explanation neutrophils by TGFb, with the induction of a protumor phe- is that G-CSF potentiated rituximab in this setting, as the half- notype by this cytokine (40). More recently, colon epithelial life of rituximab in patients is in the order of 15 days. Another cells exposed to mutagenic agents have been shown to produce potential issue is that of the effective dose. It is possible that GM-CSF, promoting an inﬂammatory procarcinogenic environ- the dose required to obtain an immunoadjuvant effect in ment (41). combination with mAbs may be different from that classically In conclusion, pegﬁlgrastim appears to be an attractive used to prevent or treat neutropenia. candidatetopotentiatetheantitumoractivityofmAbswith Another important aspect to consider when administering ADCC or ADCP properties such as rituximab and trastuzumab. neutrophil growth factors is the existence of tumor-associated Further studies are required to determine whether these obser- neutrophils with potential tumor-promoting properties. Fridle- vations can be generalized to other target antigens and nder and colleagues described the polarization of intratumoral other mAbs.

1244 Mol Cancer Ther; 15(6) June 2016 Molecular Cancer Therapeutics

Pegﬁlgrastim and Anticancer Antibodies

Untreated Untreated Rituximab Rituximab Filgrastim alone Filgrastim alone ABFilgrastim rituximab Filgrastim rituximab Pegfilgrastim alone 1,E+08 Pegfilgrastim alone Pegfilgrastim rituximab Pegfilgrastim rituximab 70

60 1,E+07 cells + 60

40 1,E+06

20 1,E+05

% Cells within CD11b 10 Absolute number of cells within spleen 0 1,E+04 + − − − CD49d Ly6G+Ly6C Ly6G Ly6G CD49d+ Ly6G+ Ly6G− Ly6G− high low Ly6C Ly6C cells LyClow Ly6Chigh Ly6Clow Leukocyte subpopulation Leukocyte subpopulation

CD100 Untreated 45 Untreated Rituximab Rituximab 90 Filgrastim alone 40 Filgrastim alone Filgrastim rituximab Filgrastim rituximab 80 Pegfilgrastim alone cells Pegfilgrastim alone 35 + Pegfilgrastim rituximab Pegfilgrastim rituximab 70 30 60 25 50 20 40 15 30 10 20 leukocytes % Tumor-infiltrated % Cell population within CD11b 10 5

0 0 − − Ly6G+ Ly6Clow Ly6G LyChigh Ly6G Ly6Clow CD49d+ Ly6G+ Ly6G− Ly6G− Ly6Clow Ly6Chigh Ly6Clow Leukocyte subpopulation Leukocyte subpopulation

Figure 5. Effect of treatments on leukocyte subpopulations. A, spleen leukocyte subpopulations. After 4 weeks of treatment, the percentage of activated monocytes (Ly6G Ly6Chigh) was significantly increased after pegfilgrastim/rituximab injections (P < 0.05), whereas resting monocytes (Ly6G Ly6Clow) were significantly þ decreased (P < 0.005). The percentage of the granulocytes (Ly6G Ly6Clow) was increased in the spleen of mice treated with filgrastim and pegfilgrastim compared with both untreated and rituximab-treated mice, but no significant difference was observed between filgrastim and pegfilgrastim treatment groups. B, absolute number of leukocyte subpopulations in spleens. Considering the important weight variation of spleens depending on treatment, we assessed absolute cell number in the different treatment groups. For each studied subpopulation, pegfilgrastim with rituximab treatment induced an increased number of cells (P < 0.05 when compared with rituximab filgrastim). C, leukocyte subpopulations within blood. After 4 weeks of treatments with pegfilgrastim with rituximab, the percentage of granulocytes within myeloid cells significantly increased compared with mice treated with rituximab alone or filgrastim with rituximab (P < 0.05). In parallel, pegfilgrastim/rituximab–treated mice presented a significant decrease of monocytic populations (both activated Ly6Chigh and resting Ly6Clow; P < 0.05). D, leukocyte subpopulations within tumors. After 4 weeks of treatments with pegfilgrastim with rituximab, the percentage of granulocytes þ within CD11b cells significantly increased in comparison with mice treated with rituximab alone or filgrastim with or without rituximab (P < 0.05). In parallel, pegfilgrastim/rituximab–treated mice presented a significant decrease of activated monocytes (Ly6G Ly6Chigh) in comparison with rituximab-treated mice (P < 0.05).

www.aacrjournals.org Mol Cancer Ther; 15(6) June 2016 1245

Cornet et al.

Disclosure of Potential Conﬂicts of Interest Administrative, technical, or material support (i.e., reporting or organizing C. Dumontet reports receiving a commercial research grant from Roche data, constructing databases): S. Cornet, D. Mathe, K. Chettab, C. Dumontet Glycart. No potential conﬂicts of interest were disclosed by the other authors. Study supervision: S. Cornet, C. Dumontet

Authors' Contributions Grant Support Conception and design: S. Cornet, C. Dumontet This work was supported by the Lyric Grant INCa-DGOS-4664 (to C. Development of methodology: S. Cornet, D. Mathe, K. Chettab, A. Evesque, Dumontet). E.-L. Matera, C. Dumontet The costs of publication of this article were defrayed in part by the Acquisition of data (provided animals, acquired and managed patients, payment of page charges. This article must therefore be hereby marked provided facilities, etc.): S. Cornet, D. Mathe, A. Evesque, E.-L. Matera, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate O. Tredan, C. Dumontet this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Cornet, D. Mathe, C. Dumontet Writing, review, and/or revision of the manuscript: S. Cornet, K. Chettab, Received September 11, 2015; revised March 8, 2016; accepted March 8, 2016; O. Tredan, C. Dumontet published OnlineFirst March 17, 2016.

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www.aacrjournals.org Mol Cancer Ther; 15(6) June 2016 1247

Pegfilgrastim Enhances the Antitumor Effect of Therapeutic Monoclonal Antibodies

Sébastien Cornet, Doriane Mathé, Kamel Chettab, et al.

Mol Cancer Ther 2016;15:1238-1247. Published OnlineFirst March 17, 2016.

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