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Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells

Authors


To whom all correspondence should be addressed.
E-mail: syano@staff.kanazawa-u.ac.jp

Abstract

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. (Cancer Sci 2011; 102: 2157–2163)

Lung cancer is the leading cause of malignancy-related deaths worldwide.(1–3) Its high mortality rate has been attributed to its high metastatic potential to multiple organs, including the brain, liver, bones, and lymph nodes.(4,5) Metastasis to these organs frequently causes severe symptoms, such as pain, paresis, and dyspnea, and decreases patients’ quality of life.(6) Approximately 15% of lung tumors are classified as small-cell lung cancer (SCLC); these tumors grow and metastasize to multiple organs much faster than non-small-cell lung cancer (NSCLC).(7) Although initially sensitive to conventional chemotherapy and radiotherapy, SCLC tumors eventually relapse and become refractory to conventional chemotherapy agents.(8) In addition, although several molecularly targeted drugs, such as the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib and the angiogenesis inhibitor bevacizumab, are successful in the treatment of NSCLC, no effective molecularly targeted drugs are currently available for SCLC. Therefore, novel therapeutic methods are essential for improving the poor prognosis of patients with this disease.

Ganglioside GM2 (GM2) is a glycolipid that localizes to plasma membranes and is involved in cell adhesion and signal transduction. GM2 also plays crucial roles in metastasis.(9,10) Highly metastatic tumor cells contain higher amounts of gangliosides than do tumor cells with low metastatic potential.(11,12) Moreover, normal cells such as fibroblasts and epithelial cells express little GM2, indicating that GM2 is an ideal target for antimetastatic therapy.

We recently established a multiple organ metastasis model in natural killer (NK) cell-depleted SCID mice, consisting of the GM2-expressing SCLC cell line SBC-3. We found that the chimeric anti-GM2 antibody, KM966, induced antibody-dependent cellular cytotoxicity (ADCC) and inhibited multiple organ metastasis of SBC-3 cells in vivo.(13,14) Moreover, these SBC-3 cells continued to express GM2 even after becoming resistant to adriamycin.(15,16) These findings provide a therapeutic rationale for the use of anti-GM2 antibody in treating drug-resistant metastatic SCLC. Chimeric antibodies, however, may cause several adverse events, including infusion reactions and the generation of antibodies to these chimeric antibodies,(17–20) because the latter still contain mouse-derived protein sequences.

These problems have been solved by the development of humanized and fully human mAb.(21) Recently, several technologies have been used to further augment the therapeutic potential of humanized antibodies. For example, Potelligent technology (Kyowa Hakko Kirin, Tokyo, Japan) deletes fucose from the antibody, dramatically augmenting ADCC activity,(22) whereas Complegent technology (Kyowa Hakko Kirin, Tokyo, Japan) incorporates IgG3 sequences into IgG1 antibodies, thus augmenting complement-dependent cytotoxicity (CDC) activity. Using these technologies, we have generated two humanized anti-GM2 antibodies. BIW-8962 is a Potelligent antibody with high ADCC activity; KM8927 is a Potelligent and Complegent antibody generated from BIW-8962, with high ADCC and CDC activities. We have assessed the antimetastatic potential of these two humanized anti-GM2 antibodies against GM2-expressing SCLC cells, by evaluating their ADCC and CDC activities in the presence of human mononuclear cells (MNC) and human serum, respectively. We also examined the therapeutic potential of these antibodies in our SCLC multiple organ metastasis model, as well as assaying GM2 expression in clinical specimens obtained from SCLC patients.

Materials and Methods

Cell lines and culture conditions.  The SBC-3 human SCLC cell line was the kind gift of Drs M. Tanimoto and K. Kiura (Okayama University, Okayama, Japan).(13) These cells were cultured in RPMI-1640 medium or DMEM, supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (50 μg/mL), in a humidified CO2 incubator at 37°C.

Reagents.  The anti-mouse interleukin-2 receptor β chain mAb, TM-β1 (IgG2b), was kindly supplied by Drs M. Miyasaka and T. Tanaka (Osaka University, Osaka, Japan).(23) KM966, a chimeric murine–human IgG1 mAb directed against GM2, and BIW-8962 and KM8927, humanized anti-GM2 mAbs, were synthesized by Kyowa Hakko Kirin (Tokyo, Japan).

Flow cytometry.  GM2 expression on SBC-3 cells was examined by flow cytometry.(24) Briefly, cells (5 × 105) were resuspended in PBS, supplemented with 10% pooled AB serum to prevent non-specific binding to the Fc receptor, washed with cold PBS and incubated on ice for 30 min with the humanized anti-GM2 mAb or control IgG. The cells were washed with cold PBS, incubated on ice for an additional 30 min with FITC-conjugated anti-human IgG antibody (Beckman Coulter, Fullerton, CA, USA), washed and resuspended in cold PBS. Cells were analyzed on a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson, San Jose, CA, USA). The mean specific fluorescence intensity was calculated as the ratio of the mean fluorescence intensity of anti-GM2 mAb to that of control mAb.

In vitro effect of anti-GM2 antibody on proliferation of SBC-3 cells.  SBC-3 cells at 80% confluence were harvested, seeded at 2 × 103 cells per well in 96-well plates, and incubated in RPMI-1640 for 24 h. Anti-GM2 antibodies (BIW-8962, KM8927, or KM966) were added at various concentrations, and the cultures were incubated for 72 h at 37°C. A 50 μL aliquot of MTT solution (2 mg/mL; Sigma, St. Louis, MO, USA) was added to each well and the cells were incubated for 2 h at 37°C.(25) The media were removed and the dark blue crystals in each well were dissolved in 100 μL DMSO. Absorbance was measured with an MTP-120 microplate reader (Corona Electric, Ibaraki, Japan) at test and reference wavelengths of 550 nm and 630 nm, respectively. Data shown are representative of three independent experiments.

Isolation of human PBMC.  Peripheral blood MNC were separated from heparinized venous blood samples drawn from healthy donors, diluted twofold with PBS in lymphocyte separation medium (Litton Bionetics, Kensington, MD, USA), and resuspended in medium for use in ADCC and CDC assays.

Antibody-dependent cellular cytotoxicity activity of anti-GM2 antibodies.  SBC-3 cells at 80% confluence were harvested, seeded at 5 × 103 cells per well in 96-well plates, and incubated in the presence of human MNC cells (1 × 105 cells per well) and various concentrations of anti-GM2 antibody (BIW-8962, KM8927, or KM966) in RPMI-1640 for 4 h at 37°C. The ADCC activity was analyzed using the lactate dehydrogenase (LDH) releasing assay (LDH Cytotoxic Test; Wako, Osaka, Japan), in accordance with the manufacturer’s protocols.

Complement-dependent cytotoxicity activity of anti-GM2 antibodies.  SBC-3 cells at 80% confluence were harvested, seeded at 5 × 103 cells per well in 96-well plates, and incubated with diluted human serum (complement) and various concentrations of anti-GM2 antibody (BIW-8962, KM8927, or KM966) in RPMI-1640 for 4 h at 37°C. The CDC activity was assayed using the LDH Cytotoxic Test (Wako), in accordance with the manufacturer’s protocols.

Purification of human peripheral lymphocytes and monocytes.  Leukocytes were obtained from peripheral blood (200 mL) of healthy donors using an RS-6600 rotor of a Kubota KR-400 centrifuge, and PBMC were separated from granulocytes in lymphocyte separation medium (Organon Teknika, Durham, NC, USA). The PBMC were further separated into lymphocytes and monocytes by centrifugal elutriation in a Beckman JE-5.0 elutriation system (Beckman Instruments, Fullerton, CA, USA).(14) Fractions enriched in lymphocytes (>99%) and monocytes (>95%) were obtained at 2000g with flow rates of 26 and 30–36 mL/min, respectively. More than 97% of the cells were viable, as judged by the Trypan blue dye exclusion test. The monocyte fraction was washed twice with PBS and resuspended in RPMI-1640 medium supplemented with 5% FBS. At this point, >90% of the cells were monocytes, as judged by their morphology and non-specific esterase staining, and were used as monocytes for the experiments.

Animals.  Male SCID mice, 5–6 weeks old, were obtained from Nihon Clea (Shizuoka, Japan) and maintained under specific pathogen-free conditions. All animal experiments complied with the Guidelines for the Institute for Experimental Animals, Kanazawa University Advanced Science Research Center (approval no. AP-081088).

SBC-3 cell model of multiple-organ metastasis and antimetastatic effect of anti-GM2 Ab.  To facilitate the metastasis of SBC-3 cells, SCID mice were depleted of NK cells.(26) Briefly, 2 days before tumor cell inoculation, each mouse was injected i.p. with TM-β1 mAb (300 μg/300 μL PBS/mouse). Subconfluent SBC-3 cells were harvested and washed with Ca2+-free and Mg2+-free PBS. Cell viability was determined by the Trypan blue exclusion test, and only single cell suspensions of >90% viability were used. Cells (1 × 106/300 μL) were injected into the lateral tail vein of mice on day 0. To determine the optimum timing and dosage of anti-GM2 antibody, tumor-bearing mice were injected i.p. with control IgG (10 μg/100 μL) on days 7, 14, 21, 28, and 35, or with anti-GM2 antibody (0.1, 1, 10 μg/100 μL) on days 7, 14, 21, 28, and 35. Six weeks after tumor cell inoculation, the mice were anesthetized by i.p. injection of 0.5 mg pentobarbital and killed by cutting the subclavian artery. All major organs were removed, and the number of macroscopic metastatic lesions >0.5 mm in diameter in these organs was counted. Immunofluorescence with anti-NKp46 Ab showed that NK cells were depleted, at least, in the liver longer than 6 weeks (Fig. S1).

Detection of proliferating cells and apoptotic cells. In vivo cell proliferation was quantitated using mouse anti-human Ki-67 mAb (MIB1; Pharmingen, San Diego, CA, USA). Mouse livers were fixed in 10% formalin, followed by processing of 4-μm thick tissue sections. The sections were immersed in 0.01 M citrate buffer (pH 6.0) and boiled in a microwave oven for 10 min for antigen retrieval.

Apoptosis was quantitated in frozen tissue sections using the TdT-mediated TUNEL method, using an Apoptosis Detection System (Promega, Madison, WI, USA). Briefly, frozen tissue sections (9 μm thick) were fixed in PBS containing 4% formalin, washed with PBS and permeabilized with 0.2% Triton X-100. Following equilibration, nucleotide mix and terminal deoxynucleotidyl transferase were added, and DNA strand breaks were labeled with fluorescein-12-dUTP. The reactions were stopped by adding saline sodium citrate, and the localized green fluorescence of apoptotic cells was detected by fluorescence microscopy (×200). All sections were counterstained with H&E for routine histological examinations.

Detection of NK cells in liver metastasis.  Frozen sections (4-μm thick) of liver metastasis produced by SBC-3 cells in NK cell-depleted SCID mice were fixed with cold acetone. After washing, sections were blocked with 5% FBS in PBS for 10 min, and incubated with 10 μg/mL anti-NKp46 Ab conjugated with FITC (1:100, for detecting NK cells; eBioscience, San Diego, CA, USA) at 4°C overnight. After washing with PBS, the section was counterstained using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescence was detected by fluorescence microscopy (×400).

Detection of GM2 expression in tumors.  Five clinical tumor specimens were obtained from five SCLC patients at Kanazawa University Hospital (Kanazawa, Japan). All five patients provided written informed consent, and the study protocol was approved by the Institutional Review Board of Kanazawa University Hospital. Liver metastasis produced by SBC-3 cells in NK cell-depleted SCID mice were also evaluated for GM2 expression. Frozen sections (4-μm thick) of these clinical and preclinical specimens were fixed with cold acetone. After washing, sections were blocked with 5% FBS in PBS for 10 min, and incubated with 10 μg/mL anti-GM2 Ab (BIW-8962) conjugated with Alexa Fluor488 (Invitrogen, Carlsbad, CA, USA) at 4°C overnight. After washing with PBS, sections were counterstained with Vectashield with DAPI (Vector Laboratories).

Quantification of immunohistochemistry and immunofluorescence.  The five areas containing the highest numbers of stained cells within a section were selected for histologic quantitation by light or fluorescent microscopy under 200-fold magnification. All results were independently evaluated by two investigators (T.Y. and H.B.).

Statistical analysis.  All data were expressed as the mean ± SD and were analyzed by one-way anova using GraphPad Prism version 4.01 (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically significant.

Results

Effect of anti-GM2 antibody on GM2-expressing SBC-3 cells in vitro.  Using FACS, we found that the SBC-3 SCLC cell line expressed large amounts of surface GM2 (Fig. 1A). Neither the humanized anti-GM2 antibodies, BIW-8962 and KM8927, nor the chimeric anti-GM2 antibody, KM966, affected the growth of SBC-3 cells in vitro (Fig. S2). In the presence of human MNC, all three anti-GM2 mAbs induced ADCC activity against SBC-3 cells, with the humanized mAbs BIW-8962 and KM8927 having greater ADCC activity than the chimeric KM966 mAb, especially at lower Ab concentrations (0.01–0.10 μg/mL) (Fig. 1B). This phenomenon was confirmed at various effector/target cell ratios (10, 20, and 40) (Fig. 1C). We further purified lymphocytes and monocytes from MNC, and evaluated their potential to induce ADCC. Lymphocytes were more potent than monocytes in our experimental conditions, but both lymphocytes and monocytes induced considerable ADCC activity mediated by KM8927 against SBC-3 cells (Fig. S3).

Figure 1.

 Effect of anti-ganglioside GM2 antibody on GM2-expressing SBC-3 small-cell lung cancer cells in vitro. (A) Assay of GM2 expression on SBC-3 cells by flow cytometry. (B) Effects of three mAbs on antibody-dependent cellular cytotoxicity activity against SBC-3 cells in the presence of human mononuclear cells (MNC), as determined by 4-h LDH releasing assays. Effector (MNC)/target (SBC-3) ratio was 20. (C) Effects of various effector/target (E/T) ratios on antibody-dependent cellular cytotoxicity activity against SBC-3 cells in the presence of human MNC with KM8927 or KM966, as determined by 4-h LDH releasing assays. (D) Effects of three mAbs on complement-dependent cytotoxicity activity against SBC-3 cells in the presence of human serum, as determined by 4-h LDH releasing assays. Results shown are representative of at least three independent experiments. *P < 0.05 compared with KM966.

Moreover, all three mAbs showed CDC activity against SBC-3 cells in the presence of human serum from healthy donors, with KM8927 having greater CDC activity than KM966 at concentrations of 1 μg/mL.

Effect of anti-GM2 antibodies on multiple organ metastasis of SBC-3 cells in NK cell-depleted SCID mice.  To determine the optimal dose of anti-GM2 antibody in vivo, we treated SBC-3 bearing NK cell-depleted SCID mice with various concentrations of KM8927. We found that KM8927 inhibited the production of metastases in the liver, kidneys, and lymph nodes in a dose-dependent manner (Table 1), with 10 μg/dose being the most efficient dose of KM8927.

Table 1.   Dose-dependent effect of KM8927 antibody against multiple organ metastasis by SBC-3 small-cell lung cancer cells in natural killer cell-depleted SCID mice
TreatmentDose (μg)LiverKidneys
No. of metastatic coloniesIncidence median (range)No. of metastatic coloniesIncidence median (range)
  1. SBC-3 (1 × 106) small-cell lung cancer cells were i.v. inoculated into SCID mice depleted of natural killer cells. For control and control IgG, PBS (100 μL) and human IgG (10 μg), respectively, were used. Treatment with KM8927 (0.1 μg, 1.0 μg, 10.0 μg), PBS, or human IgG, was given once a week on days 7, 14, 21, 28, and 35. The mice were killed and metastasis production was valuated. The data shown are representative of two independent experiments. *Significantly different from control group (P < 0.05).

Control 5/525 (8–45)5/55 (3–7)
Control IgG 5/524 (8–45)4/55 (0–8)
KM89270.15/520 (8–48)4/53 (0–8)
1.05/514 (4–39)3/54 (0–9)
10.03/57 (0–9)*2/50 (0–2)*

We next examined the effect of the treatment schedule. We found that i.p. injections of 10 μg KM8927, on days 7, 14, and 21 after inoculation of SBC-3 cells, were all effective in inhibiting the number of multiple organ metastases (Table 2, set 1), although starting KM8927 on day 7 showed the greatest level of inhibition. When we compared the effects of i.p. injections of 10 μg KM8927, BIW-8962, and KM966, beginning on day 7, we found that all three markedly inhibited multiple organ metastases of SBC-3 cells (Table 2, set 2).

Table 2.   Effect of various anti-ganglioside GM2 antibodies against multiple organ metastasis by SBC-3 small-cell lung cancer cells in natural killer cell-depleted SCID mice
TreatmentDay of therapyLiverKidneysLymph nodes
No. of metastatic coloniesIncidence median (range)No. of metastatic coloniesIncidence median (range)No. of metastatic coloniesIncidence median (range)
  1. SBC-3 (1 × 106) cells were i.v. inoculated into SCID mice depleted of natural killer cells. Set 1: PBS for control or KM8927 (10 μg/dose), was given once a week on day 7, 14, or 21. Set 2: PBS or indicated anti-GM2 antibodies (10 μg/dose) given once a week from day 7. The mice were killed on day 42 and metastasis production was valuated. The data shown are representative of two independent experiments. *Significantly different from control group (P < 0.05).

Set 1
 Control75/56 (2–14)2/51 (0–1)5/57 (3–9)
 KM892772/50 (0–1)*0/50 (0–0)3/51 (0–2)*
145/52 (1–3)2/50 (0–5)4/51 (0–3)*
214/52 (0–2)4/51 (0–2)4/53 (0–4)*
Set 2
 Control75/524 (20–32)5/55 (1–7)5/510 (6–12)
 Control IgG75/515 (14–20)5/53 (2–4)5/56 (3–14)
 KM96675/57 (5–9)*3/51 (0–3)*4/52 (0–5)*
 BIW-896275/56 (2–18)*3/52 (0–2)*2/50 (0–3)*
 KM892775/59 (1–14)*3/52 (0–2)*3/51 (0–3)*

To determine the mode of action of these anti-GM2 antibodies, we histologically analyzed liver metastases treated with PBS (control) or anti-GM2 antibodies (Fig. 2A). We found that the numbers of Ki-67-positive proliferating cells did not differ significantly when metastatic lesions were treated with PBS, KM8927, BIW-8962, or KM966. In contrast, the number of TUNEL-positive apoptotic cells was much higher in lesions treated with the anti-GM2 mAbs than with control, although there was no difference among the three types of anti-GM2 antibodies (Fig. 2B). Conversely, the number of GM2-positive cells was much lower in lesions treated with anti-GM2 antibodies than in control lesions. Moreover, the number of GM2-positive cells trended to be fewer in lesions treated with KM8927 and BIW-8962 than with KM966.

Figure 2.

 Histological examination of metastatic lesions treated with anti-ganglioside GM2 antibodies. SBC-3 (1 × 106) small-cell lung cancer cells were inoculated i.v. into natural killer cell-depleted SCID mice. The mice were injected with PBS, control IgG, KM966, BIW-8962, or KM8927, on days 7, 14, 21, 28, and 35 and killed on day 42. Metastatic liver lesions were harvested and stained with H&E, and for Ki-67, an indicator of cell proliferation, TUNEL, an indicator of cell apoptosis, and GM2 expression. (A) Microscopic appearance of lesions. (B) Quantification of the percentage of Ki-67-positive cancer cells and (C) the number of TUNEL-positive apoptotic cells. Data shown are the mean ± SD of five independent areas. Results shown are representative of at least two independent experiments. HPF, high power field. *P < 0.05 compared with control.

In addition, we carried out additional staining to rule out the possibility that decreased GM2 staining of anti-GM2 Ab-treated tumors was due to a masking effect by anti-GM2 Ab used for therapy in vivo. For this purpose, liver metastatic lesions of control and KM8927-treated mice were stained with secondary Ab (anti-human IgG conjugated with Alexa Fluor488) alone or KM8927 and secondary Ab. In the liver lesion of control mice, high GM2 expression was detected when stained with KM8927 and secondary Ab, but it was no detected when stained with secondary Ab alone (Fig. S4). These results indicate the activity and specificity of these Abs. Under these experimental conditions, in the liver lesions of KM8927-treated mice, GM2 expression was considerably lower when stained with KM8927 and secondary Ab (Fig. S4). Importantly, GM2 was not detected when stained with secondary Ab alone, indicating that KM8927 Ab injected for treatment in vivo did not mask GM2 antigen and disturbed GM2 staining in this experiment.

We also assessed the effect of these anti-GM2 mAbs on the survival of SBC-3-bearing mice. KM8927 and BIW-8962 were injected once per week, from day 7, until the mice became moribund. We found that treatment with these mAbs dramatically prolonged survival of the mice compared with control (Fig. 3).

Figure 3.

 Effect of anti-ganglioside GM2 antibodies on survival of SBC-3-bearing mice. SBC-3 (1 × 106) small-cell lung cancer cells were inoculated i.v. into natural killer cell-depleted SCID mice. The mice (10 per group) were treated with PBS, control IgG, KM966, BIW-8962, or KM8927, once a week starting on day 7. Results shown are representative of two independent experiments. *P < 0.001 compared with control.

GM2 expression in clinical specimens obtained from SCLC patients.  We also examined GM2 expression in tumors from SCLC patients by immunofluorescence and immunohistochemistry. We were able to evaluate only five surgical specimens, four primary tumors and a brain metastasis (sample no.1), because SCLC is rarely resected surgically and GM2 can be detected only in frozen sections. GM2-positive SCLC cells were present in three specimens, primary tumors from two patients and a brain metastasis from another patient (Fig. 4).

Figure 4.

 Ganglioside GM2 expression in tumors obtained from patients with small-cell lung cancer. Clinical specimens obtained from the patients were assayed for GM2 expression by immunofluorescence and immunohistochemistry. Of five clinical specimens, three were positive for GM2; representative areas are shown. Results shown are representative of two independent stainings for each specimen.

Discussion

Several mAbs have been successfully introduced into clinical practice to treat cancer patients. For example, the chimeric anti-EGFR mAb cetuximab and the fully human anti-EGFR mAb antibody panitumumab have been approved for the treatment of patients with colorectal cancer. These two antibodies are thought to have similar antitumor activity, but the humanized panitumumab was reported to have better adverse event profiles than the chimeric cetuximab. For example, several patients who were intolerant of cetuximab have been treated with panitumumab without evidence of infusion reactions or allergic responses.(27) We have shown here that the humanized anti-GM2 antibodies BIW-8962 and KM8927 have high therapeutic activities, at least equivalent to that of the chimeric antibody KM966, in our multiple organ metastasis model. Therefore, these humanized anti-GM2 antibodies may retain their therapeutic effect, while being less toxic than the chimeric KM966 antibody. Importantly, we could detect GM2-expressing SCLC cells in clinical specimens. These results collectively suggest a therapeutic rationale for the use of humanized anti-GM2 mAbs in patients with SCLC. In addition, spotty GM2 staining indicates that GM2 expression may be heterogenous among SCLC cells. Therefore, combined use of anti-GM2 Ab with other agents, such as cytotoxic chemotherapeutic drugs, might be necessary for controlling SCLC in patients.

Both ADCC and CDC are important activities of Ab therapy.(28) Potelligent technology was used to develop BIW-8962, with augmented ADCC activity, whereas both Potelligent and Complegent technologies were used to generate KM8927, with augmented ADCC and CDC activities. We found that BIW-8962 had higher ADCC activity using human MNC, and KM8927 had higher ADCC and CDC activities using human serum, than the chimeric anti-GM2 mAb KM966 in vitro. In contrast, the biological effects of these humanized anti-GM2 antibodies were similar to that of KM966 in our mouse metastasis model. This discrepancy may be due to differences in human and mouse biology. Generally, human and rabbit serum, but not mouse serum, has high CDC activity in the presence of antibody.(29) Moreover, although NK cells and monocytes/macrophages are the major effectors of ADCC using anti-GM2 antibody, mouse effector cells are much less potent than human effector cells in inducing ADCC.(14) We confirmed that human peripheral monocytes and lymphocytes induced considerable ADCC mediated by KM8927 against SBC-3 cells (Fig. S3). Furthermore, to facilitate the generation of multiple organ metastases, we depleted NK cells, the major effector cells, from SCID mice.(26) Thus, the humanized anti-GM2 antibodies generated by Potelligent and Complegent technologies may have greater therapeutic potential than chimeric KM966 in humans.

Prolongation of survival is the most reliable and clinically relevant endpoint of preclinical experiments. We found that injection of BIW-8962 and KM8927, beginning on day 7 after inoculation of SBC-3 cells, successfully prolonged the survival of SCID mice bearing multiple organ metastases of SBC-3 cells. In our model, i.v. inoculated SBC-3 cells produced micrometastases by day 7.(13) Small-cell lung cancer generates micrometastases in almost all patients, even when the primary tumor is still small in size. Therefore, these humanized anti-GM2 antibodies may at least prevent the enlargement of systemic micrometastases produced by GM2-expressing SCLC cells.

In conclusion, we have shown that the humanized anti-GM2 mAbs BIW-8962 and KM8927, generated by Potelligent and Complegent technologies, respectively, have therapeutic potential against multiple organ metastases of GM2-expressing SCLC cells. Clinical trials with BIW-8962 are now ongoing in previously treated multiple myeloma (NCT00775502).

Acknowledgments

We thank Drs. Katsuyuki Kiura and Mitsune Tanimoto (Okayama University) for kindly providing SBC-3 cells, and Dr. Masayuki Miyasaka (Osaka University) for providing the TM-β1 hybridoma. We also thank Ms Takako Sano (Kanazawa University), Mr. Takuya Kuramoto, and Yuko Oka (University of Tokushima) for expert technical assistance. This work was supported in part by Scientific Research on Innovative Areas “Integrative Research on Cancer Microenvironment Network” (S. Yano, 22112010A01) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Disclosure Statement

Seiji Yano received honoraria from Chugai Pharma and AstraZeneca. Seiji Yano received research fundings from Chugai Pharma and Kyowa Hakko Kirin. Shiro Akinaga is an employee of Kyowa Hakko Kirin.

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