The Trifunctional Antibody Ertumaxomab Destroys Tumor Cells That Express Low Levels of Human Epidermal Growth Factor Receptor 2

Published OnlineFirst May 12, 2009; DOI: 10.1158/0008-5472.CAN-08-2861 Published Online First on May 12, 2009 as 10.1158/0008-5472.CAN-08-2861

Research Article

Michael Ja¨ger,1 Alexandra Schoberth,1 Peter Ruf,1 Ju¨rgen Hess,2 and Horst Lindhofer1,2

1TRION Research GmbH, Martinsried, Germany and 2TRION Pharma GmbH, Munich, Germany

Abstract The humanized monoclonal anti-HER2/neu antibody trastuzu- Human epidermal growth factor receptor 2 (HER2/neu) is an mab inhibits growth of tumor cell lines that strongly express the important target for the treatment of the breast cancers in HER2/neu antigen (6). Several clinical studies have shown the which it is overexpressed. However, no approved anti-HER2/ greatest benefit from trastuzumab treatment (7, 8) among women neu therapyis available for the majorityof breast cancer with metastatic breast cancers (scored 3+ or 2+ by immunohis- patients, who express HER2/neu at low levels (with scores of tochemisty), and with HER2 gene amplifications (confirmed by 1+ or 2+/fluorescence in situ hybridization–negative). The fluorescent in situ hybridization; FISH). Based on these results, trifunctional antibodyertumaxomab targets HER2/neu, CD3, assessment of the HER2/neu status is absolutely required for all and activating Fc; receptors. In presence of ertumaxomab, breast cancer patients who may be considered for trastuzumab tri-cell complexes consisting of tumor cells, T cells, and therapy. However, trastuzumab therapy cannot be offered to the majority of breast cancer patients who have low levels of HER2/neu accessorycells form to cause tumor cell lysis.In a phase I trial with metastatic breast cancer patients, ertumaxomab could be expression (scored 1+ and 2+) and negative FISH results. Ertumaxomab is a new member of a family of trifunctional applied safelyand resulted in radiographicallyconfirmed Â clinical responses. In this study, we compare ertumaxomab- bispecific antibodies (anti-HER2/neu anti-CD3). Kiewe and and trastuzumab-mediated killing of cancer cell lines that colleagues (9) presented the first promising clinical data on the express HER2/neu at low and high levels. Under optimal safety and efficacy of ertumaxomab in the treatment of metastatic conditions for trastuzumab-mediated destruction of HER2/ breast cancer patients with different HER2/neu expression levels. neu-overexpressing cells, onlyertumaxomab was able to Such as BiUII and catumaxomab, which target EpCAM instead of mediate the elimination of tumor cell lines that express HER2/neu, ertumaxomab evokes a concerted interaction of HER2/neu at low levels (1+). Ertumaxomab-mediated activity different immune cell types directed against the tumor (10, 11). was accompanied bya Th1-based cytokinerelease, a unique Such as BiUII, ertumaxomab might simultaneously recruit and g g mode of action of trifunctional antibodies. Competitive activate Fc RI and Fc RIII-positive accessory cells (i.e., monocytes, binding studies with trastuzumab and 520C9 mapped the macrophages, natural killer cells, and dendritic cells) through its unique isotype combination (mouse IgG2a and rat IgG2b), leading binding site of ertumaxomab to the extracellular regions II and III of the HER2/neu ectodomain. This site is distinct from to the phagocytosis of the tumor cells (12). The importance of the the binding site of trastuzumab, so that HER2/neu-expressing mouse/rat hybrid Fc region in the process of immunization has tumor cells can be eliminated byertumaxomab in the been shown in immunocompetent mouse tumor models, using the presence of high amounts of trastuzumab. The abilityof trifunctional surrogate antibody BiLu (anti-human EpCAM x anti- ertumaxomab to induce cytotoxicity against various tumor murine CD3; refs. 13, 14). As evidenced by Riesenberg and cell lines, including those with low HER2/neu antigen density, colleagues (15), perforin-mediated cytotoxicity may contribute to mayprovide a novel therapeutic option for breast cancer the antitumor response. Taken together, these data suggest that patients who are not eligible for trastuzumab treatment. ertumaxomab may induce the formation of a tri-cell-complex + + g + [Cancer Res 2009;69(10):4270–6] consisting of HER2/neu tumor cells, CD3 T cells, and Fc R accessory cells, leading to efficient elimination of tumor cells. In this study, we report on the high efficacy of the trifunctional antibody ertumaxomab to specifically eradicate different HER2/ Introduction neu-positive tumor cell lines, accompanied by the activation of a The proto-oncogene HER2 codes for the human epidermal Th1-type cytokine pattern. In contrast to the monospecific growth factor receptor 2 (HER2/neu), which is overexpressed in humanized antibody trastuzumab, ertumaxomab destroys tumor 20% to 30% of breast cancer patients (1–3). HER2/neu over- cell lines with high and also with low HER2/neu expression. In expression is usually based on gene amplification. It is correlated addition, we are able to show that trastuzumab and ertumaxomab with a poor prognosis, reducing progression-free outcomes and recognize different epitopes on Her2/neu. The possible clinical overall survival (4, 5). HER2/neu is an important target for implications of these findings are discussed. antibody-mediated therapy in breast cancer patients. Materials and Methods Antibodies, effector cells, and target cell lines. We used freshly Requests for reprints: Horst Lindhofer, TRION Pharma GmbH, Frankfurter Ring harvested peripheral blood mononuclear cells (PBMC) from healthy donors 193a, 80807 Munich, Germany. Phone: 49-89-324266100; Fax: 49-89-324266199; E-mail: as effector cells. The cells were purified by density centrifugation through [email protected]. Â j I2009 American Association for Cancer Research. Ficoll Histopaque (PAN Biotech) at 897 g, 15 min, 20 C. They were doi:10.1158/0008-5472.CAN-08-2861 washed twice with PBS without Mg2+ or Ca2+ (PAN Biotech), and

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Ertumaxomab Lyses Tumor Cells with Low HER2/neu Levels

Figure 1. HER2/neu-expressing profiles of used cell lines: A, SK-BR-3, (B) HCT-8, (C) BT-20, and (D) SK-LU-1. FACS, fluorescence activated cell sorter histogram, FL1-H = 2502A + secondary detection antibody rat anti-mouse IgG H+L FITC, mouse IgG2a isotype control Me361 (TRION Research) + secondary detection antibody rat anti-mouse IgG H+L FITC; MFI, mean fluorescence intensity; SABC, specific antigen binding capacity as determined by DAKO QIFIKIT. centrifuged at 458 Â g, for 10 min, at 20jC. The supernatant was removed, SPEC HER2/CEN 17 Dual Color Probe is a mixture of an orange and the pellet was resuspended in 20 mL RPMI 1640, supplemented with fluorochrome direct–labeled CEN 17 probe, specific for the a satellite 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 1 mmol/L nonessential centromeric region of chromosome 17 (D17Z1), and a green fluorochrome amino acids, and 10% FCS (PAN Biotech). Cell number and viability direct–labeled SPEC HER2 probe, specific for the HER2 gene at 17q12. After were determined using a Neubauer counting chamber after trypan blue counterstaining with 4¶,6-diamidino-2-phenylindole (Zytovision), slides were staining (Sigma-Aldrich). The breast cancer cell lines SK-BR-3 (ATCC evaluated at Â100 oil magnification, applying a computerized image HTB-30) and BT-20 (ATCC HTB-19), the human ileocaecal adenocarcinoma analysis (MDS; Applied Imaging). cell line HCT-8 (ATCC CCL-244), and the lung cancer cell line SK-LU-1 Fluorescence-activated cell sorter binding competition analysis. (ATCC HTB-57) served as target cells in cytotoxicity assays with PBMC Highly positive HER2/neu SK-BR-3 cells were preincubated with varying effector cells, followed by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]- concentrations of either 520C9 (10,000–100 ng/mL) or trastuzumab (10,000– 2H-tetrazolium-5-carboxanilide inner salt (XTT) cell proliferation assess- 100 ng/mL) for 10 min, followed by addition of a constant concentration of ment (16). The trifunctional antibody ertumaxomab with anti-HER2/neu Â ertumaxomab (1,000 ng/mL), and further incubation for 45 min. After a anti-CD3 antigen specificities was manufactured by TRION Pharma. The washing step, ertumaxomab cell binding was detected by fluorescence- HER2/neu-specific monoclonal mouse antibodies 2502A and 520C9 (ATCC activated cell sorting (FACS) with an anti-rat IgG (H+L) FITC-labeled HB-8696; ref. 17) were produced by TRION Research. 2502A constitutes the secondary detection antibody (Dianova). HER2/neu binding arm of ertumaxomab. The humanized IgG1 antibody Cytotoxicity assay. Effector cells (2 Â 105; PBMC) and target cells trastuzumab (Roche) also recognizes the HER2/neu protein. (SK-BR-3, HCT-8, BT-20, or SK-LU-1) at varying E/T ratios were coincubated Quantitative determination of the cell surface antigen HER2/neu. in flat-bottomed 96-well plates (Greiner) with either trastuzumab or HER2/neu antigen expression on the surface of tumor target cell lines was ertumaxomab and combinations of different antibodies concentrations as quantified using DAKO QIFIKIT (DAKO Cytomation), according to the indicated. PBMCs coincubated with tumor cells alone (allogeneic setting) manufacturer’s protocol. HER2/neu-specific 2502A was used as the primary were used as controls. After 4 d, PBMCs were discarded by washing, and antibody for HER2/neu detection and quantification. HER2/neu antigen proliferation of tumor cells was analyzed using the XTT Cell Proliferation quantity is indicated as specific antibody-binding capacity units after kit II, as described by the manufacturer (Roche). Absorbance was measured subtraction of isotype control (mouse IgG2a) background. in a Versamax microplate reader (Molecular Devices), and raw data were FISH analysis. Target cell lines (e.g., SK-BR-3 or HCT-8) were spun down analyzed in Excel XP (Microsoft). Percent cytotoxicity was calculated as on cytospin slides, and pretreated with pepsin (Sigma-Aldrich) for 5 min follows: [(absorbance allogeneic reaction À absorbance sample)/(absor- at 37jC. Subsequently, cells were fixed with formaldehyde (Sigma-Aldrich) bance allogeneic reaction À absorbance PBMC)] Â 100. All tests were and dehydrated in alcohol (70À100%). Probe hybridization was then performed in duplicate, and experiments were repeated with PBMC from performed with SPEC HER2/CEN 17 (Zytovision), overnight at 37jC. The three different healthy donors. It should be noted that the value of www.aacrjournals.org 4271 Cancer Res 2009; 69: (10). May 15, 2009

Cancer Research allogeneic cytotoxicity in the absence of respective antibodies in each superior ability of ertumaxomab to lyse cells that express high sample was always below 13%. In addition, by using these specific assay levels of HER2/neu under unfavorable E/T ratios. conditions all antibody entities such as trastuzumab or ertumaxomab Cytotoxicity of ertumaxomab and trastuzumab against completely failed to inhibit growth of respective tumor target cells (HCT-8) tumor cells with a low HER2/neu expression profile. The or showed only minimal cytotoxic activity (SK-BR-3 cells) in the absence of efficacious killing of SK-BR-3 tumor cells by ertumaxomab, even at PBMCs (data not shown). The HER2/neu specificity of ertumaxomab was analyzed by preincubation of the PBMC effector and HCT-8 target cells with low effector cell numbers, led to the hypothesis that ertumaxomab excess amounts of the anti-HER2/neu blocking antibody 2502A (200, 500, or might also be able to eliminate tumor cells that express HER2/neu 2,000 ng/mL) for 15 min, followed by addition of different concentrations of at low levels (1+). Based on this hypothesis, a cytotoxicity assay was ertumaxomab (100À0.001 ng/mL). established to analyze the antitumor efficacy of ertumaxomab, Measurement of cytokines. Ertumaxomab- or trastuzumab-induced compared with trastuzumab, on cell lines that express low levels of cytokine release was determined by culturing PBMCs from healthy donors HER2/neu: HCT-8, BT-20, and SK-LU-1. Each of the three cell lines, with target cells (HCT-8) for 24 h in 96flat-bottomed well plates. derived from different carcinomas (colon, breast, and lung), was Ertumaxomab (100-0.001 ng/mL) or trastuzumab (5,000-0.001 ng/mL) were completely killed in the presence of ertumaxomab in a dose range added at different concentrations. After 24 h, the supernatants were of z5 ng/mL. In contrast, trastuzumab entirely failed to exert any collected and frozen at À20jC. Cytokines were measured with the human cytotoxic effects on HCT-8, BT-20, and SK-LU-1 cancer cells, even at Th1/Th2 cytometric bead array kit (BD Biosciences) comprising interleukin (IL)-2, IL-6, IFN-g, and tumor necrosis factor-a (TNF-a; data not shown for high concentrations up to 5,000 ng/mL, and with E/T ratios of TNF-a). Data acquisition and analysis were performed using a FACS-Calibur 20:1 (Fig. 3A, C, and D)—the conditions that are optimal for with the cytometric bead array software (BD Biosciences). trastuzumab induced SK-BR-3 cell lysis (Fig. 2A). Interestingly, the Analysis of cytokine-induced cytotoxicity. Cytokine-induced killing of cytotoxic antitumor potential of ertumaxomab remained compa- the target cell line HCT-8 was analyzed by transferring the supernatants of rable in these experiments, even at an unfavorable E/T ratio of 7:1 cytotoxicity assay samples to 3 Â 104 freshly plated tumor cells (HCT-8). (Fig. 3B). Tumor cell proliferation was analyzed using the XTT Cell Proliferation kit II Cytokines induced by ertumaxomab in the presence of HCT- (Roche). Extinction was measured in a Versamax microplate reader 8 target cells and PBMC. As both CD3+ T cells and accessory (Molecular Devices). Raw data were analyzed in Excel XP (Microsoft). All immune cells are redirected by ertumaxomab to target cells, the experiments were performed in duplicates. interaction of these immune cell types can be assessed by detecting the relevant cytokines secreted into the supernatant of the Results Quantification of HER2/neu expression on target cells. To evaluate the influence of the HER2/neu expression level on ertumaxomab- and trastuzumab-mediated cytotoxicity, the amount of HER2/neu antigen on the surface of different human cancer cell lines (SK-BR-3/breast, BT20/breast, HCT-8/colon, and SK-LU-1/lung) was determined. As expected, SK-BR-3 cells that are scored 3+ (17, 18) stained intensively with anti-HER2/neu monoclonal antibody (mAb) 2502A. Consistent with the intensive staining, the DAKO QIFI test displayed a high specific antigen binding capacity (Fig. 1A). The HCT-8 cells showed a significantly weaker HER2/neu surface density, as did the other cell lines tested (BT-20 and SK-LU-1). Subsequently, the FACS analyses were completed by FISH. Thereby, SK-BR-3 cells revealed amplification of the HER2/neu gene locus, whereas HCT-8, BT-20, and SK-LU-1 cells did not (Fig. 1A, B, C, and D). In summary, the target cell lines HCT-8, BT-20, and SK-LU-1 showed low levels of HER2/neu expression (1+), whereas SK-BR-3 cells scored 3+ for HER2/neu, in agreement with the DAKO HercepTest classification. Cytotoxicity of ertumaxomab and trastuzumab against tumor cells with a high HER2/neu expression profile. Trastuzumab is well-known to promote tumor cell death in HER2/neu-overexpressing cell lines such as SK-BR-3 (17, 18). We compared the ability of ertumaxomab to inhibit SK-BR-3 growth with trastuzumab at effector (PBMC) to target ratios (E/T) of 20:1 and 5:1. Different antibody concentrations of trastuzumab (5,000–0.001 ng/mL) and ertumaxomab (100À0.001 ng/mL) were added to allogeneic cell samples. As shown in Fig. 2A, SK-BR-3 cells were completely lysed by both ertumaxomab and trastuzumab at an E/T ratio of 20:1. However, at suboptimal E/T conditions (5:1), Figure 2. Cytotoxicity against SK-BR-3 cells (HER2 high, HER2 ampl+) trastuzumab-induced killing of SK-BR-3 cells declined to f40%, at E/T ratios of (A) 20:1 and (B) 5:1 mediated by trastuzumab (5,000–0.001 even at high concentrations (5,000–1,000 ng/mL; Fig. 2B). In ng/mL) or ertumaxomab (100–0.001 ng/mL). Cytotoxicity experiments were performed thrice and samples measured in duplicates with 2 Â 105 PBMC contrast, maximal target cell killing remained at ertumaxomab of 3 different healthy donors; sample: PBMC + SK-BR-3 + antibody; allogeneic concentrations as low as 25 ng/mL. These results show the reaction: PBMC + SK-BR-3 cells. Points, mean; bars, SD.

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Ertumaxomab Lyses Tumor Cells with Low HER2/neu Levels

Figure 3. Cytotoxicity against (A) HCT-8 cells (HER2 low, HER2 amplÀ;E/T,20:1);(B) HCT-8 cells (E/T, 7:1); (C) BT-20cells (HER2 low, HER2 ampl À; E/T, 20:1) and (D) SK-LU-1 cells (HER2 low, HER2 amplÀ; E/T, 20:1) mediated by trastuzumab (5,000–0.001 ng/mL) or ertumaxomab (100–0.001 ng/mL). Cytotoxicity experiments were performed thrice and samples measured in duplicates with 2 Â 105 PBMC of 3 different healthy donors; sample: PBMC + HCT-8 + antibody; allogeneic reaction: PBMC + HCT-8 cells. Points, mean; bars, SD. respective cell growth media. Doses of ertumaxomab ranging from on the expression of the HER2/neu antigen, we initiated blocking 1 to 100 ng/mL were able to stimulate high levels of the pro- experiments with an HER2/neu antibody. We preincubated the inflammatory cytokines [i.e., IL-6; IFN-g; TNF-a (data not shown)]- cytotoxicity samples with excess amounts of anti-HER2/neu after 24 hours of incubation time (Fig. 4A and B). Of note, in the antibody 2502A. In three independent experiments with effector presence of accessory cells, T cells, and tumor cells, ertumaxomab cells from different donors, we observed a significant dose- also induced the production of IL-2 (Fig. 4C). A strong Th1 dependent reduction of tumor cell killing at 2502A blocking response is suggested by the high IFN-g and IL-2 levels that were concentrations between 200 and 2,000 ng/mL (Fig. 4D). These present in samples treated with ertumaxomab. Furthermore, IL-6 results show that direct binding of ertumaxomab to HER2/neu is secretion was increased, indicating a proinflammatory response with mandatory for target cell destruction. the contributions of accessory cells (19). In contrast, trastuzumab Inhibition of ertumaxomab binding to HER2/neu by merely stimulated IL-6secretion, and at significantly lower levels antibody520C9. To define the anti-HER2/neu binding region of (Fig. 4B). Interestingly, supernatant transfer experiments showed ertumaxomab, competitive binding analysis of trastuzumab with that all the cytokines induced by ertumaxomab did not have any mAb 520C9 were performed on SK-BR-3 cells. MAb 520C9 was able effect on the growth of HCT-8 cells (Fig. 4A–C). These results strongly to inhibit ertumaxomab binding but trastuzumab was not. This support the view that ertumaxomab-induced killing of HCT-8 cells is result suggests that 520C9 and ertumaxomab recognize similar specific and not mediated by the cytokines per se and depends on epitopes (Fig. 5A). Indeed, the HER2/neu binding site of the mAb physical contact of the re–directed effector cells and HCT-8 cells. 520C9 was previously mapped to the extracellular domain of Our results show that the release of proinflammatory cytokines HER2/neu (amino acid positions 243–370), covering parts of and IL-2, which is stimulated by ertumaxomab, reflects the subdomains II and III (20). This 520C9 site is distinct from the engagement of both Fcg receptor+ accessory immune cells and binding site of trastuzumab, which is located in region IV (21). We CD3+ T-cells. The less pronounced IL-6secretion induced by conclude that trastuzumab and ertumaxomab have different trastuzumab is probably evoked by binding to FcgR on accessory binding epitopes on HER2/neu, which do not interfere with each cells alone. other. Having shown that ertumaxomab and trastuzumab recog- Dependence of ertumaxomab-mediated lysis on HER2/neu nize different HER2/neu epitopes (Fig. 5A), we further asked binding. To address the question of whether the provoked whether ertumaxomab was able to induce the killing of target cytotoxic effects of ertumaxomab on HCT-8 target cells depend cells that express low levels of HER2/neu (e.g., HCT-8) in the www.aacrjournals.org 4273 Cancer Res 2009; 69: (10). May 15, 2009

Cancer Research presence of trastuzumab. Both antibodies, ertumaxomab and istry, showed the most beneficial treatment effects in patients who trastuzumab, were used in combination in competitive cytotoxicity carried FISH-positive tumors. This retrospective analysis indicates assays (Fig. 5B). Effector and HCT-8 target cells were preincubated that breast cancer HER2/neu overexpression most frequently with a constant concentration of trastuzumab (5,000 ng/mL), correlates with gene amplification (22). Unfortunately, overexpres- followed by the addition of ertumaxomab (50 ng/mL). In this sion of the HER2/neu antigen is only detectable in 20% to 30% of setting, trastuzumab alone (5,000 ng/mL) failed again to lyse breast cancer patients (1, 2), leaving the majority with fewer HCT-8 target cells. Ertumaxomab-mediated maximal cell lysis was therapeutic options. present at concentration as low as 50 ng/mL. No difference was This study describes the cytotoxic capacity of the trifunctional observed in the killing efficacy of ertumaxomab between samples antibody ertumaxomab for tumor cell lines that express HER2/neu with (100-fold excess) trastuzumab or without. The observation at high and low levels, in the presence of immune effector cells that excess amounts of trastuzumab do not hamper the lysis of (PBMC) in vitro. Only slight differences could be assessed in the target cells by ertumaxomab confirms that the two antibodies killing efficiency of SK-BR-3 cells between trastuzumab and recognize different HER2/neu epitopes (Fig. 5A). ertumaxomab, at a relatively high E/T ratio of 20:1. However, at more unfavorable E/T ratios (7:1 or lower), probably resembling the situation at the tumor site, only ertumaxomab was able to Discussion efficiently lyse SK-BR-3 cells (Fig. 2). This observed superiority of It is well-established that tumor therapy with the anti-HER2/neu ertumaxomab over trastuzumab is based on the particular mode of antibody trastuzumab achieves significant survival benefits in action that all members of the trifunctional antibody family (e.g., patients with HER2/neu overexpressing metastatic breast cancer, ertumaxomab, catumaxomab, Bi20/FBTA05, BiLu, and BiUII) have whether trastuzumab is used with or without chemotherapy as a in common. These therapeutic antibodies induce a simultaneous first-line therapy until disease progression (7, 8). Moreover, a recruitment and activation of T cells and accessory cells, leading to retrospective analysis of breast cancer patients enrolled during the destruction of targeted tumor cells by different killer clinical trials, with tumors scored 2+ or 3+ by immunohistochem- mechanisms (9, 12, 13, 15, 23).

Figure 4. A-C, influence of ertumaxomab-induced cytokines IFN-g, IL-6, and IL-2 on freshly plated HCT-8 cells (HER2 low, HER2 amplÀ). Cytokine levels of supernatants of ertumaxomab or trastuzumab cytotoxicity experiments were analyzed after 24 h. Supernatants containing ertumaxomab- or trastuzumab-induced cytokines were transferred to freshly plated HCT-8 cells (1 Â 104). HCT-8 growth (o) was measured after a 3-d incubation period with the XTT proliferation assay. As a control was a sample of HCT-8 cells incubated with RPMI1640 medium, which was set as a standard of 100% HCT-8 growth; points, mean. Ab, antibody. D, HER2/neu specificity of ertumaxomab-induced cytotoxicity against HCT-8 cells: Cytotoxicity experiments were done in duplicates with 2 Â 105 PBMC of 3 different healthy donors and an E/T ratio of 6:1. PBMC and target cells were preincubated with 200 ng/mL (E), 500 ng/mL (o), or 2,000 ng/mL (X) of the anti-HER2/neu antibody 2502A followed by addition of ertumaxomab (100–0.001 ng/mL). Ertumaxomab-induced cytotoxicity was compared with samples without preincubation of 2502A (n). Points, mean; bars, SD.

Cancer Res 2009; 69: (10). May 15, 2009 4274 www.aacrjournals.org

Ertumaxomab Lyses Tumor Cells with Low HER2/neu Levels

Figure 5. A, FACS competition binding: inhibition of ertumaxomab (ertu;1Ag/mL) binding to SK-BR-3 cells in the presence of varying ratios of 520C9 or trastuzumab (trastu) compared with ertumaxomab (10:1, 1:1, 1:10); all experiments were performed thrice; as control, the binding capacity of ertumaxomab, 520C9, and trastuzumab to SK-BR-3 cells was analyzed; points, mean; bars, SD. B, cytotoxicity of ertumaxomab (50ng/mL) induced against HCT-8 cells ( HER2 low, HER2 amplÀ)after preincubation with a 100-fold higher concentration of trastuzumab (5,000 ng/mL). As controls were samples with trastuzumab (5,000 ng/mL) and ertumaxomab (50ng/mL) alone. Cytotoxicity experiments were done in duplicate with 2 Â 105 PBMC of 3 different healthy donors and a HCT-8 cell concentration of 1 Â 104 (E/T, 20:1); sample: PBMC + HCT-8 + antibody; allogeneic reaction: PBMC + HCT-8 cells. Points, mean; bars, SD.

The unique mode of action of ertumaxomab led us to the activation. The induction of these cytokines confirms findings hypothesis that tumor cells with low HER2/neu expression profiles, described previously for other trifunctional antibodies (10, 25), and categorized as immunohistochemisty 1+ and FISH negative, might supports the view that their mode of action is mainly independent be potential targets for ertumaxomab therapy. To address this of the targeted antigen. The cytokines IFN-g, TNF-a, and IL-6were question, we chose three tumor cell lines originating from different also released 3 or 6hours after ertumaxomab infusion in a clinical tissues (breast, colon, and lung), each of which has a detectable low trial with metastatic breast cancer patients, confirming the HER2/neu-expressing profile, identified by quantitative FACS relevance of our in vitro results for the clinic (9). measurements and FISH analysis. Cytotoxicity assays revealed that Trastuzumab induced significant amounts of only IL-6, which is ertumaxomab has a strong killing activity against all three cell lines most likely attributable to accessory cells stimulated by Fcg at concentrations above 5 ng/mL, and at E/T ratios of 20:1 or even receptor engagement (Fig. 4B; ref. 26). These results further lower (Fig. 3). Trastuzumab, which acts against tumor cells mainly emphasize the substantial differences in the mechanisms of action by means of antibody-dependent cellular cytotoxicity (17, 24), between ertumaxomab and trastuzumab. without contributions from T cells, completely failed to inhibit Because the transfer of ertumaxomab-treated mixed cell culture growth of these three cell lines, even at high concentrations. supernatants had no growth inhibition effect on freshly plated High quantities of IFN-g, TNF-a(data not shown), and IL-6were HCT-8 cells, the killing of this cell line was not induced by the detected in the supernatants of mixed HCT-8/PBMC samples to detected mediator substances, such as TNF-a or IFN-g. Although which ertumaxomab had been added. IL-2 secretion was stimulat- sufficient at low levels, the necessity of HER2/neu antigen ed as well as IFN-g, a strong indication of a Th1-type T-cell expression for ertumaxomab-provoked tumor cell destruction www.aacrjournals.org 4275 Cancer Res 2009; 69: (10). May 15, 2009

Cancer Research was finally shown by HER2/neu blocking experiments. Preincuba- trastuzumab treatment may benefit from ertumaxomab adminis- tion of HCT-8 target cells with the parental bivalent anti-HER2/neu tration because no interference in binding or killing efficacy was antibody 2502A inhibited ertumaxomab-induced killing up to 60%. observed in coincubation experiments (Fig. 5B). Therefore, a This inhibition shows that physical contact of ertumaxomab with subsequent or even simultaneous application of both antibodies the tumor cell is required for its efficacy (Fig. 4D). This finding has could be reasonable. In conclusion, ertumaxomab may give new been confirmed in vivo for the trifunctional surrogate antibody treatment opportunities for breast cancer patients with HER2/neu BiLu, which has no effect on the growth of target antigen negative expression, independent of the expression profile that is currently tumor cells, whereas it mediates full protection against antigen- under investigation in phase II clinical studies. positive tumor cells in mice (13). An important result, with possible consequences for clinical use, Disclosure of Potential Conflicts of Interest was that ertumaxomab and trastuzumab recognize two different H. Lindhofer: commercial research support, TRION Research GmbH; ownership HER2/neu epitopes. The HER2/neu binding site for trastuzumab is interest and patents, TRION Pharma GmbH. The other authors disclosed no potential located in the cysteine-rich extracellular subdomain IV (amino conflicts of interest. acids 529–627; ref. 21). Ertumaxomab binding is not competitive with trastuzumab. It is competitive with mAb 520C9 (Fig. 5A), Acknowledgments whose HER2/neu binding site was previously mapped to the Received 7/25/08; revised 1/28/09; accepted 3/25/09; published OnlineFirst 5/12/09. extracellular HER2/neu subdomains II and III (amino acid positions The costs of publication of this article were defrayed in part by the payment of page 243–370; ref. 20). The HER2/neu binding epitope of ertumaxomab charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. must be distinct from that of trastuzumab. In consequence, patients We thank Melanie Honcia, Annette Arbter, Melanie Goelden, and Kathrin Gasow that have already received trastuzumab and are refractory to for their expert technical assistance.

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Cancer Res 2009; 69: (10). May 15, 2009 4276 www.aacrjournals.org

The Trifunctional Antibody Ertumaxomab Destroys Tumor Cells That Express Low Levels of Human Epidermal Growth Factor Receptor 2

Michael Jäger, Alexandra Schoberth, Peter Ruf, et al.

Cancer Res Published OnlineFirst May 12, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-08-2861

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