Chimeric and humanized anti-HM1.24 antibodies mediate antibody-dependent cellular cytotoxicity against lung cancer cells

Lung Cancer 63 (2009) 23–31

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Chimeric and humanized anti-HM1.24 antibodies mediate antibody-dependent cellular cytotoxicity against lung cancer cells Wei Wang a , Yasuhiko Nishioka a,∗ , Shuji Ozaki b , Ali Jalili b , Vinod Kumar Verma a , Masaki Hanibuchi a , Shinji Abe c , Kazuo Minakuchi c , Toshio Matsumoto b , Saburo Sone a a Department of Internal Medicine and Molecular Therapeutics, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan b Department of Medicine and Bioregulatory Sciences, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8503, Japan c Department of Pharmacy, Tokushima University Medical and Dental Hospital, Tokushima 770-8503, Japan

a r t i c l e

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Article history: Received 21 December 2007 Received in revised form 2 April 2008 Accepted 21 April 2008 Keywords: Lung cancer Antibody Immunotherapy HM1.24 antigen Cytokine ADCC

a b s t r a c t HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. The aim of this study was to evaluate the antitumor activity of mouse–human chimeric and humanized anti-HM1.24 monoclonal antibodies (mAbs) against lung cancer cells in vitro. Human peripheral blood lymphocytes and monocytes separated from mononuclear cells (PBMCs) were used as effector cells. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of chimeric and humanized anti-HM1.24 mAbs against lung cancer cells were determined by chromium-release assay. In some experiments, target or effector cells were pretreated with various cytokines. Chimeric and humanized anti-HM1.24 mAbs effectively induced ADCC against lung cancer cells mediated more efficiently by lymphocytes than monocytes. The cytotoxic activity correlated with the level of HM1.24 expression on lung cancer cells. Natural killer cells were identified as the major effector cells in ADCC mediated by the anti-HM1.24 mAb. The treatment of lymphocytes or monocytes with IL-2, IL-12, IL-15, M-CSF, or IFN-␥ significantly increased the ADCC activity. Moreover, the culture of lung cancer cells with IFN-␤ or IFN-␥ augmented their susceptibility to ADCC and CDC. PBMCs from patients with lung cancer induced a level of ADCC comparable to that induced by PBMCs from healthy donors. Chimeric or humanized anti-HM1.24 mAbs have potential as a new therapeutic tool in lung cancer, and in combination with interleukins and interferons, could be useful for enhancing ADCC. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of death from cancer worldwide [1,2]. The care rate remains less than 15% despite improvements in surgery, radiotherapy and chemotherapy [1,2]. To prolong the survival of patients with lung cancer, the development of the novel therapeutic modalities is of much interest. Recently, advances in monoclonal antibody (mAb)-based therapies targeting to cell surface antigens and growth factor receptors have lead to success in the treatment of some hematopoietic malignancies as well as solid tumors [3–6]. Recently, administration of anti-vascular endothelial

∗ Corresponding author. Tel.: +81 88 633 7127; fax: +81 88 633 2134. E-mail address: [email protected] (Y. Nishioka). 0169-5002/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2008.04.009

growth factor (VEGF) mAbs (avastin) with chemotherapy (paclitaxel and carboplatin) for NSCLC showed the significant efficacy in the median survival (12.3 months vs. 10.3 months) and the median progression free survival (6.2 months vs. 4.5 months) as compared with the chemotherapy alone group [6]. The phase II trial of the antiEGFR mAb (cetuximab) in patients with previously treated NSCLC showed 4.5% response rate and 30.3% stable disease, which was similar to that of pemetraxed, docetaxel, and erlotinib in similar groups of patients [7]. HM1.24, originally identified as a type II transmembrane protein of 29–33 kDa that is preferentially overexpressed on multiple myeloma (MM) cells, was later found to be identical to bone marrow stromal cell antigen 2 (BST-2) [8,9]. The possibility of the HM1.24 antigen as an immunological target has been extensively studied for MM [10–12]. Recently, using the large scale method to identify the

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target genes such as serial analysis of gene expression (SAGE) and microarray, HM1.24 gene has been reported to be overexpressed in a few of solid tumor cells which exhibited the invasive or drugresistant phenotypes [13,14]. However, little is known about the detail expression status of HM1.24 antigen in solid cancer, particularly at the protein level, and the possibility as an immunological target for therapy with anti-HM1.24 antibody. Recently, we found that 42% of lung cancer cells expressed HM1.24 antigen, and that murine anti-HM1.24 antibody effectively reduced the growth of lung cancer cells expressing HM1.24 antigen via inducing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (submitted for publication). Considering the clinical application of anti-HM1.24 antibody, it is of interest to examine the ADCC activity of chimeric and humanized antibodies using human mononuclear cells. Based on these findings, we examined whether mouse–human chimeric and humanized anti-HM1.24 antibodies can induce ADCC by peripheral blood mononuclear cells not only from healthy subjects, but also from patients with lung cancer. The effects of various cytokines on ADCC by anti-HM1.24 antibodies were also explored. 2. Materials and methods 2.1. Lung cancer cell lines The human lung cancer cell line SBC-5 was provided by Dr. S. Hiraki (Okayama University, Okayama, Japan) [15]. The human adenocarcinoma RERF-LC-OK cells were provided by Dr. M. Akiyama (Radiation Effects Research Foundation, Hiroshima, Japan) [15]. The human lung adenocarcinoma cells ACC-LC-174 and ACC-LC176 were provided by Dr. T. Takahashi (Nagoya University, Nagoya, Japan). The other cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY), 100 U/mL penicillin, and 100 ␮g/mL streptomycin (Meiji Seika Kaisha, Ltd., Tokyo, Japan).

of the cells were viable, as judged with the trypan blue dye exclusion test. The purity of the lymphocytes and monocytes was more than 95%, respectively, as judged from their morphology. Human natural killer (NK) cells were isolated from human peripheral blood mononuclear cells by magnetic cell sorting (MACS) with a NK cell isolation kit (Miltenyi Biotec., Bergisch Gladbach, Germany) [16]. The PBMCs were stained at 4–8 ◦ C for 10 min with NK Cell Biotin-Antibody Cocktail and 15 min with NK Cell MicroBead Cocktail. Cells were washed with MACS buffer (PBS pH 7.2%, 0.5% bovine serum albumin, and 2 mM EDTA) and passed through 30 ␮m nylon mesh to remove cell clumps. The cells were resuspended at up to 108 cells in 500 ␮L of buffer and applied onto the magnetic column. The unlabeled cells that passed through the column were collected as NK cells. The purity of NK cells was more than 95.9% as determined by flow cytometry with anti-CD56 antibody. The magnetically labeled non-NK cells were harvested by flushing the column firmly. 2.4. Flow cytometry The expression of the HM1.24 antigen in lung cancer cells was examined by flow cytometry [17]. Briefly, cells (5 × 105 ) were resuspended in phosphate-buffered saline (PBS) supplemented with 10% pooled AB serum to prevent non-specific binding to the Fc receptor. The cells were washed with cold PBS and stained on ice for 30 min with the murine, chimeric, or humanized anti-HM1.24 mAb or control IgG. After incubation with the primary mAb, cells were washed with cold PBS and then incubated on ice for an additional 30 min with fluorescein isothiocyanate (FITC)-conjugated goat F(ab )2 fragment anti-mouse IgG (H+L) antibody (Beckman Coulter Inc., Fullerton, CA) or a FITC-conjugated goat IgG fraction to human IgG Fc (Beckman Coulter Inc.). The cells were washed again and resuspended in cold PBS. The analysis was performed on a FACS Calibur flow cytometer with CellQuest software (Becton Dickinson, San Jose, CA). The mean specific fluorescence intensity (MSFI) was calculated as the ratio of the MFI of anti-HM1.24 mAb to that of control mAb.

2.2. Reagents Recombinant human interleukin (IL)-2 (specific activity: 107 U/mg), IL-12 (106 U/mg), IL-15 (2 × 106 U/mg) and macrophage colony-stimulating factor (M-CSF) (2 × 105 U/mg) were purchased from PeproTech EC Ltd. (London, UK). Recombinant human IL-23 (3 × 107 U/mg), IL-29 (2 × 105 U/mg), IFN-␤ (8.23 × 107 U/mg), and IFN-␥ (107 U/mg) were obtained from R&D systems (Minneapolis, MN). The mouse anti-HM1.24 monoclonal antibody (mAb) (IgG2a␬), and the mouse–human chimeric and humanized antiHM1.24 mAbs (IgG1␬) were provided by Chugai Pharmaceutical Co., Ltd., (Shizuoka, Japan). The mouse–human chimeric anti-Pglycoprotein mAb MH162 (Human IgG1␬) was provided by Drs. T. Tsuruo and H. Hamada (Japanese Foundation for Cancer Research, Tokyo, Japan). Mouse IgG2a was purchased from BioLegend (San Diego, CA). MH162 and mouse IgG2a were used as control Abs. 2.3. Preparation of effector cells The human study was approved by the ethics committee of the University of Tokushima and written informed consent was obtained from all the subjects. Leukocytes from peripheral blood of healthy donors were collected in an RS-6600 rotor of a Kubota KR-400 centrifuge, and mononuclear cells (PBMCs) were separated from leukocytes in lymphocyte separation medium (Litton Bionetics, Kensington, MD) [16]. Lymphocytes and monocytes were separated from PBMCs by centrifugal elutriation in a Beckman JE5.0 elutriation system as described previously [16]. More than 97%

2.5. Quantification of binding sites of anti-HM1.24 mAb in lung cancer cells The levels of HM1.24 expression in lung cancer cells were quantitatively determined by flow cytometry using a DAKO QIFIKIT (DAKO A/S, Glostrup, Denmark). Briefly, cell samples (5 × 105 ) were washed with PBS and incubated for 30 min at 4 ◦ C in the dark with the murine anti-HM1.24 mAb or control IgG2a at saturating concentrations. Cells were then washed and incubated for 45 min at 4 ◦ C in the dark with 100 ␮L of the F(ab )2 fragment of FITCconjugated goat anti-mouse antibody. Again, cells were washed and resuspended in PBS. A calibration curve linking the intensity of fluorescence and the number of antigenic sites was established using the QIFIKIT. Set-up and calibration beads (100 ␮L) were washed and incubated at 4 ◦ C for 30 min in the dark with 100 ␮L of goat FITClabeled anti-mouse IgG diluted at 1:50. Then, they were washed and suspended in PBS before the flow cytometric analysis. Measurements were performed on a FACS Calibur flow cytometer with CellQuest software. The fluorescence related to specific binding to HM1.24 was obtained by subtracting the MFI of the blank from that of the sample. The number of antigenic sites per cell was calculated from the calibration curve derived from QIFIKIT beads. 2.6. ADCC ADCC was determined by conducting a standard 6 h 51 Cr-release assay as described previously [17]. In some experiments, effector

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Fig. 1. ADCC activity of chimeric and humanized anti-HM1.24 mAbs. (A) The flow cytometric analysis using chimeric and humanized anti-HM1.24 mAbs in SBC-5 cells. SBC-5 cells were stained with control IgG, murine, chimeric, or humanized anti-HM1.24 mAb, and FITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-human IgG, and then analyzed by flow cytometry. The numbers in the right upper corner of each figure represent mean specific fluorescence intensity (MSFI). (B) The dose dependence of chimeric and humanized anti-HM1.24 mAbs in their ADCC against NCI-H196 cells. 51 Cr-labeled NCI-H196 cells were incubated with human lymphocytes or monocytes obtained from healthy donors at an E/T ratio of 40 in the presence of various concentrations of antibodies. (C) Effect of E/T ratio on ADCC mediated by chimeric and humanized anti-HM1.24 mAbs against lung cancer cells. 51 Cr-labeled SBC-5 cells were incubated with human lymphocytes or monocytes in the presence of 1 ␮g/mL of control or antiHM1.24 mAb. (D) NK cells are main effector cells in ADCC mediated by anti-HM1.24 mAb. NK cells were isolated from human peripheral mononuclear cells by miniMACS. 51 Cr-labeled SBC-5 cells were incubated with isolated NK cells () and NK-depleted PBMCs (䊉) in the presence of 1 ␮g/mL of chimeric and humanized anti-HM1.24 mAbs. Data represent the mean ± S.D. of triplicates. Similar results were obtained from three separate experiments.

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cells were cultured in RPMI 1640 medium with or without recombinant human IL-2, IL-12, IL-15, IL-23, IL-29, IFNs or M-CSF for 24 h, then washed and resuspended in the medium prior to use. Target cells were labeled with 0.1 ␮Ci of Na51 CrO4 at 37 ◦ C for 1 h. After three washes with RPMI 1640 medium, 51 Cr-labeled target lung cancer cells (1 × 104 ) were placed in 96-well plates in a triplicate culture, and various concentrations of anti-HM1.24 mAb or control IgG were added to the wells. Effector cells were then added to the plates at various effector to target (E/T) ratios. After a 6-h incubation, supernatants (100 ␮L) were harvested and measured in a gamma counter. Percent cytotoxicity was calculated from the following formula: % specific lysis = (E − S)/(M − S) × 100, where E is the release in the test sample (cpm in the supernatant from target cells incubated with effector cells and test antibody), S is the spontaneous release (cpm in the supernatant from target cells incubated with medium alone), and M is the maximum release (cpm released from target cells lysed with 1% sodium dodecyl sulfate). The spontaneous release observed with different target cells ranged from 5 to 15%. 2.7. CDC Cell lysis with complement was evaluated using a 51 Cr-release assay. Target cells were labeled with 0.1 ␮Ci of 51 Cr-sodium chromate at 37 ◦ C for 1 h. The cells were then washed three times with RPMI 1640 medium. 51 Cr-labeled cells (1 × 104 ) were added into 96-well plates and incubated with serial dilutions of baby rabbit complement (Cedarlane, Ontario, Canada) and the anti-HM1.24 mAb or control IgG for 2 h. After this incubation, the supernatant from each well (100 ␮L) was harvested and 51 Cr-release was measured using a gamma counter. Percent cytotoxicity was calculated as above. 2.8. Patients Ten patients with histologically or cytologically proven lung cancer were enrolled in this study between June and December in 2006 after providing written informed consent. Four of these patients had squamous cell carcinomas, three had adenocarcinomas, two had small cell carcinomas and one had a large cell carcinoma. Clinical stage was defined using the TNM classification (two with IB, four with IIIA, three with IIIB and one with IV) [18]. PBMCs were obtained before starting therapy. 2.9. Statistical analysis A one-way ANOVA with Dunnett’s post test was performed using GraphPad Prism version 4.03 for Windows, GraphPad Software (San Diego, CA). P < 0.05 was considered significant. All experiments were performed at least three times. The correlation between ADCC activity and HM1.24 expression on target cells was evaluated with Pearson’s rank correlation analysis. 3. Results 3.1. Binding ability of chimeric and humanized mAbs In the first set of experiments, to confirm whether the mouse–human chimeric and humanized anti-HM1.24 mAbs had sufficient activity to bind the HM1.24 antigen on lung cancer cells, we performed flow cytometric analysis. The levels of HM1.24 expression in SBC-5 cells determined with mouse–human chimeric or humanized mAb were similar, but slightly lower than the levels obtained with the mouse mAb (Fig. 1A). These results were

confirmed in NCI-H196 and RERF-LC-OK cells, which also express HM1.24 at high levels (data not shown).

3.2. ADCC of anti-HM1.24 mAbs in lung cancer cells Next, we examined the ADCC of these mAbs against in lung cancer cells in a 51 Cr-release assay. As shown in Fig. 1B, the control mAb (MH162) did not induce ADCC mediated by lymphocytes or monocytes isolated from healthy donor against NCI-H196 cells. In contrast, both the mouse–human chimeric and humanized anti-HM1.24 mAbs mediated the ADCC of lymphocytes, but not of monocytes, in a dose-dependent manner (Fig. 1B). The optimal concentration of both antibodies to induce lymphocytemediated ADCC seems to be more than 1 ␮g/mL. The level of ADCC was dependent on the E/T ratio (Fig. 1C). In the case of SBC-5 cells, a weak cytolytic activity by monocytes was detected when a high E/T ratio was used with both antibodies. These results suggested that the ADCC of the anti-HM1.24 mAb was mainly mediated by lymphocytes. In all experiments, the chimeric antibody was more effective than the humanized antibody in inducing ADCC by anti-HM1.24 antibody. Although levels of cytotoxicity comparable with the control mAb were observed in some experiments with SBC-5 (Fig. 1C), these activities were identified as non-specific NK activities, because they were identical to the cytotoxicity without an antibody (lymphocyte alone) (data not shown). To further examine the type of effector cells in lymphocytes, we purified NK cells and examined their ADCC by using antiHM1.24 antibodies against SBC-5 cells. As shown in Fig. 1D, the isolated NK cells from human PBMCs significantly induced the ADCC, but the NK cell-depleted fraction of PBMCs exhibited the marginal level of ADCC, indicating that NK cells are main effector cells of ADCC mediated by the anti-HM1.24 antibody. On the other hand, treatment with the murine, chimeric, or humanized anti-HM1.24 mAb alone did not inhibit the proliferation of SBC-5 even at a concentration of 100 ␮g/mL (data not shown), indicating that the anti-HM1.24 mAbs have no direct anti-tumor activity against lung cancer cells.

3.3. ADCC correlates with HM1.24 levels in lung cancer cells To further analyze the ADCC of chimeric and humanized anti-HM1.24 mAbs, additional human lung cancer cell lines were incubated with human lymphocytes in the presence of the optimal dose of mAb (1 ␮g/mL) and at an E/T ratio of 50:1. As shown in Fig. 2A, both the chimeric and humanized antibodies significantly induced ADCC mediated by lymphocytes, being dependent on the expression level (MSFIs) of HM1.24. To examine the correlation between ADCC activity and HM1.24 levels, we evaluated the ADCC activity in various lung cancer cell lines that express HM1.24 at different levels. The HM1.24 expression levels on the surface of cancer cells were quantitatively determined with the QIFIKIT system. To avoid the variation in NK sensitivity of various lung cancer cell lines, ADCC of the antibody was calculated by subtracting the cytotoxicity that occurred without an antibody from that with anti-HM1.24 antibody. Under this condition, we observed that the level of HM1.24 expression linearly correlated with the ADCC of the chimeric and humanized anti-HM1.24 mAbs (Fig. 2B).

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Fig. 2. Correlation between ADCC activity and the level of HM1.24 expression in lung cancer cells. (A) ADCC against various lung cancer cells. ADCC mediated by human lymphocytes against various lung cancer cells was determined with a 6-h 51 Cr-release assay at an E/T ratio of 50 in the presence of 1 ␮g/mL of control, chimeric or humanized anti-HM1.24 mAb. MFI indicates the mean fluorescence intensity measured by flow cytometry with murine anti-HM1.24 mAb. Data represent the mean ± S.D. of three determinants. The number in parentheses indicates MFI with murine anti-HM1.24 antibody. (B) Correlation between ADCC activity and the expression level of HM1.24 molecules in lung cancer cells. Numbers of HM1.24 molecules per cell were quantitatively determined by flow cytometry using the QIFIKIT system. The ADCC activity against nine lung cancer cell lines (A549, ACC-LC-174, ACC-LC-176, NCI-H196, NCI-H226, NCI-H1437, NCI-H1915, RERF-LC-OK, and SBC-5) was examined with a 6-h 51 Cr-release assay using human lymphocytes at an E/T ratio of 50 in the presence of 1 ␮g/mL of chimeric or humanized anti-HM1.24 mAb. To avoid variation in the sensitivity to NK cells in various lung cancer cell lines, ADCC was calculated by subtracting the cytotoxicity (%) without an antibody from that with anti-HM1.24 mAb. Data represent the mean value of three determinants.

3.4. CDC of chimeric and humanized mAbs The CDC of anti-HM1.24 mAb was examined in the presence of rabbit complement (Table 1). Both chimeric and humanized antiHM1.24 mAbs (1 ␮g/mL) mediated CDC against NCI-H196, SBC-5 and RERF-LC-OK cells. No CDC was induced by control mAb against these lung cancer cells. 3.5. Effect of pretreatment of effector cells with various cytokines on ADCC by anti-HM1.24 antibodies Next, we examined whether pretreatment of effector cells, lymphocytes and monocytes, with various cytokines including IL-2, IL-12, IL-15, IL-23, IL-29, IFN-␤, and IFN-␥ enhanced ADCC of antiHM1.24 antibody against lung cancer cells. Human lymphocytes were incubated for 24 h in medium with or without an opti-

mum concentration of IL-2 (1000 U/mL), IL-12 (20 ng/mL), IL-15 (10 ng/mL), IL-23 (10 ng/mL), IL-29 (10 ng/mL), IFN-␤ (500 U/mL) and IFN-␥ (100 U/mL) and then added to cultures of 51 Cr-labeled lung cancer cells (SBC-5 and RERF-LC-OK) with chimeric or humanized anti-HM1.24 mAb (1 ␮g/mL). As shown in Fig. 3A, non-specific cytolytic activity including lymphokine-activated killer (LAK) activity was found after 24 h culture in the presence of IL-2, IL-12, IL-15 and IFN-␤. Additive effects by chimeric or humanized anti-HM1.24 mAbs were observed in the cytotoxicity mediated by IL-2, IL-12 and IL-15. IL-23, IL-29 and IFN-␥ did not affect lymphocyte-mediated cytotoxicity in this experimental condition (data not shown). We also examined the effect of cytokines on the ADCC of human monocytes. Human monocytes were cultured in medium with or without an optimum concentration of M-CSF (50 ng/mL), IL-29 (10 ng/mL), IFN-␤ (500 U/mL) and IFN-␥ (100 U/mL). M-CSF and IFN-␥ further enhanced the specific ADCC of monocytes with both

Table 1 CDC activity of chimeric and humanized anti-HM1.24 mAbs against lung cancer cells Lung cancer cells

NCI-H196 SBC-5 RERF-LC-OK a

56.7 19.5 17.9

% cytotoxicityb (mean ± S.D.c ) Control

Chimeric

Humanized

0±0 0.3 ± 0.1 0±0

29.5 ± 4.1 31.8 ± 1.3 32.8 ± 1.0

30.5 ± 2.9 19.1 ± 1.2 22.9 ± 1.8

Mean fluorescence intensity analyzed by FACScan using murine anti-HM1.24 mAb (1 ␮g/mL). Cr-labeled cells were incubated for 2 h in medium with mAb (1 ␮g/mL) with rabbit complement at a dilution of 1:4. Mean ± S.D. for triplicate cultures. Data are representative of three separate experiments.

b 51 c

HM1.24 expressiona

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Fig. 3. Effect of pretreatment with various cytokines on ADCC against lung cancer cells mediated by chimeric and humanized anti-HM1.24 mAbs. (A) Effect of pretreatment of effector cells (lymphocytes and monocytes) from healthy donors with various cytokines on ADCC mediated by chimeric and humanized anti-HM1.24 mAbs. Human lymphocytes or monocytes were cultured with various cytokines for 24 h and were used as effector cells. IL-2 (1000 U/mL), IL-12 (20 ng/mL), IL-15 (10 ng/mL), M-CSF (50 ng/mL), IFN-␤ (500 U/mL), and IFN-␥ (100 U/mL) were used at an optimal concentration. ADCC was determined with a 6-h 51 Cr-release assay in the presence of 1 ␮g/mL of control, chimeric or humanized anti-HM1.24 mAb at an E/T ratio of 25 (lymphocytes) or 50 (monocytes) against SBC-5 cells. **P < 0.01 vs. cytotoxicity with control antibody. (B) Effect of pretreatment of lung cancer cells with various cytokines on ADCC mediated by chimeric and humanized anti-HM1.24 mAbs. The lung cancer cell line, ACC-LC-176, was cultured with IFN-␤ or IFN-␥ for 48 h and was used as target cells. ADCC activity was determined with a 6-h 51 Cr-release assay in the presence of 1 ␮g/mL of control, chimeric, or humanized anti-HM1.24 mAb at an E/T ratio of 50. Human lymphocytes or monocytes from healthy donors were used as effector cells. **P < 0.01; *P < 0.05 vs. cytotoxicity for target cells cultured in medium alone. (C) Effect of pretreatment of lung cancer cells with various cytokines on CDC mediated by chimeric and humanized anti-HM1.24 mAbs. The lung cancer cell lines SBC-5 and RERF-LC-OK were cultured with IFN-␤ or IFN-␥ for 48 h and used as target cells. CDC was determined with a 2-h 51 Cr-release assay in the presence of 1 ␮g/mL of control, chimeric, or humanized anti-HM1.24 mAb and rabbit complement at 1:4 dilution. **P < 0.01 vs. cytotoxicity for cells cultured in medium alone. All data represent the mean ± S.D. of three determinants.

chimeric and humanized mAbs (Fig. 3A), whereas IFN-␤ and IL29 improved the non-specific cytotoxicity of monocytes (data not shown). 3.6. Effect of pretreatment of target cells with cytokines on both ADCC and CDC We reported that IFN-␤ and IFN-␥ promoted HM1.24 antigen expression on lung cancer cells [14]. In this study, we further

investigated the effect of IFN-␤ and IFN-␥ on the susceptibility of lung cancer cells to anti-HM1.24 mAbs. ACC-LC-176 cells, whose HM1.24 expression is upregulated by treatment with IFNs, were incubated with IFN-␤ (500 U/mL) or IFN-␥ (100 U/mL) for 48 h and then utilized for ADCC assays with chimeric or humanized anti-HM1.24 mAb at an E/T ratio of 50. Treatment with IFN-␤ efficiently enhanced the cytotoxicity of humanized anti-HM1.24 mAbs (Fig. 3B). We next examined the CDC of these mAbs with SBC-5 and RERF-LC-OK cells pretreated with IFN-␤ and IFN-␥. A more

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Fig. 4. ADCC of PBMCs from patients with lung cancer. (A) PBMCs from healthy donors (open samples, n = 10) and untreated lung cancer patients (close samples, n = 10) were used as effector cells. ADCC was determined against SBC-5 cells at an E/T ratio of 20 in the presence of 1 ␮g/mL of control, chimeric, or humanized anti-HM1.24 mAb. Horizontal bars represent the mean values of each group. (B) PBMCs from lung cancer patients were cultured with IL-15 or IL-12 for 24 h and were used as effector cells. ADCC was determined with a 6-h 51 Cr-release assay against SBC-5 cells at an E/T ratio of 20 in the presence of 1 ␮g/mL of control, chimeric or humanized anti-HM1.24 mAb. Data represent the mean value of three determinants.

efficient cytolysis was also found after treatments with these IFNs (Fig. 3C). 3.7. ADCC of PBMCs from patients with lung cancer Finally, the ADCC of PBMCs from both healthy donors and untreated lung cancer patients was compared using SBC-5 cells as target cells. As shown in Fig. 4A, the lung cancer patients’ PBMCs were as effective as the healthy donors’ PBMCs, although patients with lung cancer showed reduced tendency in NK activity as compared with healthy donors. Next, we examined whether IL-12 and IL-15 could enhance the ADCC against lung cancer cells since these two cytokines had the greatest additive effects on ADCC by anti-

HM1.24 mAbs in Fig. 3A. PBMCs derived from patients with lung cancer were cultured with IL-12 or IL-15 for 24 h before the ADCC assay. An enhancement by IL-15 of ADCC by chimeric mAb was observed in 5 of 9 (56%) and by humanized mAb in 6 of 6 (100%) patients. IL-12 enhanced ADCC by chimeric mAb in 3 of 6 (50%) patients, and by humanized mAb in 3 of 5 (60%) patients (Fig. 4B). 4. Discussion In the present study, we have shown that mouse–human chimeric and humanized anti-HM1.24 mAbs effectively induced ADCC against lung cancer cell lines, which was mainly mediated by NK cells. In our experimental condition, the chimeric anti-HM1.24

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mAb was more effective than the humanized mAb in inducing ADCC. More importantly, the PBMCs from patients with lung cancer also showed a comparable level of ADCC mediated by anti-HM1.24 mAbs to those from healthy donors. These results strongly show the feasibility of therapy with anti-HM1.24 mAbs for patients with lung cancer. We have shown that several antibodies for P-glycoprotein or ganglioside GM2 could induce ADCC against lung cancer cells expressing these molecules using mouse–human chimeric or humanized mAbs [19,20]. Furthermore, the in vivo anti-tumor effects of these antibodies were also demonstrated in mouse models [17,21]. However, one limitation to applying these antibodies for clinical use was the restricted expression of these antigens in lung cancer. In particular, P-glycoprotein was only expressed in some multidrug-resistant lung cancer cells. However, we found the expression of the HM1.24 antigen in more than 40% of lung cancer cells [14]. The high expression rate of HM1.24 seems to be an advantage for this antibody in terms of clinical use. It has been reported that ADCC was enhanced by treatment of effector cells with various cytokines including IL-2, IL-12, IL-15 and M-CSF [12,19,20]. In this study, we also found a significant enhancement by culture of lymphocytes in healthy donors with IL-2, IL-12 and IL-15, and in patients with IL-12 and IL-15. The ADCC of antiHM1.24 mAb by monocytes was weak, but enhanced by treatment with M-CSF and IFN-␥. Furthermore, we found that treatment of tumor cells with IFN-␤ and IFN-␥ enhanced the susceptibility of lung cancer cells to both ADCC and CDC. These results suggest that the combination of these cytokines with anti-HM1.24 mAb might be more useful via not only enhancement of the killing activity of effector cells, but also the susceptibility of cancer cells. Another important issue related to high ADCC could be the level of expression of HM1.24 [22]. We therefore quantified the amount of cell surface HM1.24 by using the QIFIKIT system. The results showed the ADCC of anti-HM1.24 mAbs linearly correlated with the amount of cell surface HM1.24. Anti-tumor activity of the antiHM1.24 mAb is likely to be more profound in patients with high levels of HM1.24. In our previous study, we found that more than 20% of lung cancer cells expressed high levels of HM1.24 which is comparable to that in myeloma cells [14], suggesting that therapy with anti-HM1.24 antibody might be more useful at least for 20% of patients with lung cancer. The precise mechanisms whereby the anti-HM1.24 mAb exerts its anti-tumor activity remain elusive. NK cells and monocytes/macrophages are known to be the major effector cells of ADCC [23,24]. Our study confirmed that NK cells played a major role in the mechanism of anti-tumor activity of anti-HM1.24 mAb. Although NK activity is quite important for host defense against cancers, decreased NK activity [25,26] and monocyte functions [27,28] were reported in lung cancer patients. In our study, NK activity in patients with lung cancer tended to be reduced as compared with that in healthy donors, although the difference was not statistically significant. However, ADCC activity was effectively induced even if we used PBMCs from patients with lung cancer, indicating that the use of an antibody which can induce ADCC can circumvent the tumor-mediated immunosuppression. On the other hand, ADCC is exhibited on ligation of the Fc portion of human IgG to FcR. Although human macrophages constitutively express the high and low affinity Fc receptors specific for IgG (Fc␥RI, Fc␥RIIA, and Fc␥RIIIA), NK cells express only Fc␥RIIIA. Previous study showed that neutrophils expressing Fc␥RI did not exhibit any cytotoxicity with humanized anti-HM1.24 mAb, suggesting that Fc␥R IIIA is the main Fc receptor of humanized anti-HM1.24 mAb. Recently, approaches using peptides of tumor-associated antigens as a cancer vaccine have been tried. We and others reported that dendritic cells (DCs) pulsed with HM1.24–22, 126 and 165

peptides effectively generated HM1.24-specific cytotoxic T lymphocytes (CTLs) which could kill myeloma cells [12,29]. Other approaches using DCs pulsed with HM1.24 protein or transduced with the HM1.24 gene by adeno-associated virus are also effective in generating tumor-reactive CTLs [30,31]. Considering these results, the combination of antibody-based and cellular immunotherapy targeted at HM1.24 would be a good subject for study. In conclusion, the present study demonstrated that the chimeric and humanized anti-HM1.24 mAbs mediated lung cancer-specific cytolysis by ADCC and CDC. Although it is known that administration of mouse mAbs to patients generate human anti-mouse antibody (HAMA) responses, which prevent repeated injections of mAbs and sufficient prolongation of their half-life in the body, chimeric and humanized mAbs could be better to be applied for therapeutic use. Recently, several chimeric or humanized antibodies have been approved and used in the clinic for patients with various malignant diseases [3–6]. Actually, a phase I clinical study of humanized anti-HM1.24 mAb against multiple myeloma has confirmed little toxicity [16]. These results suggest that chimeric or humanized anti-HM1.24 mAbs have the potential as a new therapeutic tool in lung cancer, and in combination with various cytokines including IL-2, IL-12, IL-15, IFN-␤ and IFN-␥ could be useful for enhancing ADCC. Conflict of interest None. Acknowledgments This work is supported by KAKENHI (18590855), the Grantin-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. We thank Ms. Tomoko Oka for technical assistance and Dr. Takashi Takahashi (Nagoya University) for providing ACC-LC-174 and ACC-LC-176 cell lines. References [1] Hoffman PC, Mauer AM, Vokes EE. Lung cancer. Lancet 2000;355:479–85. [2] Spiro SG, Silvestri GA. One hundred years of lung cancer. Am J Respir Crit Care Med 2005;172:523–9. [3] Blackhall F, Ranson M, Thatcher N. Where next for gefitinib in patients with lung cancer? Lancet Oncol 2006;7:499–507. [4] Saijo N. Recent trends in the treatment of advanced lung cancer. Cancer Sci 2006;97:448–52. [5] Egri G, Takats A. Monoclonal antibodies in the treatment of lung cancer. Eur J Surg Oncol 2006;32:385–94. [6] Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paclitaxel–carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006;355:2542–50. [7] Hanna N, Lilenbaum R, Ansari R, Ansari R, Lynch T, Govindam R, et al. Phase II trial of cetuximab in patients with previously treated non-small-cell lung cancer. J Clin Oncol 2006;24:5253–8. [8] Goto T, Kennel SJ, Abe M, Takishita M, Kosaka M, Solomon A, et al. A novel membrane antigen selectively expressed on terminally differentiated human B cells. Blood 1994;84:1922–30. [9] Ohtomo T, Sugamata Y, Ozaki Y, Ono K, Yoshimura Y, Kawai S, et al. Molecular cloning and characterization of a surface antigen preferentially overexpressed on multiple myeloma cells. Biochem Biophys Res Commun 1999;258:583– 91. [10] Ozaki S, Kosaka M, Wakatsuki S, Abe M, Koishihara Y, Matsumoto T. Immunotherapy of multiple myeloma with a monoclonal antibody directed against a plasma cell-specific antigen, HM1.24. Blood 1997;90:3179–86. [11] Ozaki S, Kosaka M, Wakahara Y, Abe M, Koishihara Y, Matsumoto T. Humanized anti-HM1.24 antibody mediates myeloma cell cytotoxicity that is enhanced by cytokine stimulation of effector cells. Blood 1999;93:3922–30. [12] Jalili A, Ozaki S, Hara T, Shibata H, Hashimoto T, Abe M, et al. Induction of HM1.24 peptide-specific cytotoxic T lymphocytes by using peripheral-blood stem-cell harvests in patients with multiple myeloma. Blood 2005;106:3538–45.

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