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Purpose: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti–human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells. Experimental Design: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed. Results: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC. Conclusions: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds.

Background: Endoxifen and 4-hydroxy tamoxifen are both active metabolites of tamoxifen and exert anti-tumor activity for ER+ breast cancer. However, only small numbers of paper reported biological profile of endoxifen. Methods: Cells were incubated with various concentration of endoxifen ( (Z)-4-Hydroxy-N-desmethyl Tamoxifen ( Results: IC50s of endoxifen for MCF-7 were 100 nM in estradiol (E2) deprivation condition and 500 nM in the presence of 1nM E2. These are significantly higher than those of 4-OH tamoxifen, which are 10 nM without E2 and 50 nM with E2, respectively. From the previous reports, plasma endoxifen concentrations in breast cancer patients were ranged from 20 to 200 nM according to CYP2D6 phenotype. Anti-proliferative index of 20 nM endoxifen for MCF-7 was 0.7 (indicating 30% reduction in proliferative activity), and 200 nM endoxifen was 0.5 in the absence of E2. These were 0.95 (20 nM) and 0.75 (200nM), respectively, when incubated with 1 nM E2. 10 nM 4-OH tamoxifen, which is mean concentration in patient plasma, showed about 0.6 anti-proliferative index with no regards to E2 condition. Without E2, endoxifen reduced ERα expression as previously reported, however, endoxifen increased ERα amount in the presence of E2 accompanied with decrease of HER-2 expression. This alternation of ERα and HER2 expression were identical to those observed using 4-OH tamoxifen. There was a weak tendency that ERβ expressing MCF-7 clones showed favorable response to endoxifen compared to parent MCF-7, but this was not significant. Conclusion: Endoxifen is active agent for MCF-7 cell, however, it requires 10-fold higher concentration compared to 4-OH tamoxifen to achieve IC 50. Difference of anti-proliferative effect between 20 nM and 200 nM of endoxifen are relatively small, and it might result in minor difference for clinical response, as expected from recent clinical studies of CYP2D6. This is the first time to show that endoxifen has opposite effect to ERα protein level depending on the presence or absence of E2. ERβ expression did not enhance activity of endoxifen in contrast to previous report. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-20.

Previously, we reported that serum from stage IV breast cancer patients diminished the cytotoxic activity of lymphokine activated killer cells (LAKs) (Abstract #4639 AACR in 2008). Thus, we next tested what the critical role for the inhibitory effect on LAKs was. Based on the study of protein detection in the serum using Cytokine Antibody Array (Ray Biotech), serum from stage IV patients seemed to have higher level of vascular endothelial growth factor (VEGF) as compared to early stage patients. To confirm this by more accurate way, we analyzed the serum level of VEGF by using ELISA in 24 breast cancer patients. When the serum level of VEGF was cut off by median (420 pg/ml), 11 patients with higher level of VEGF out of 12 are included in the patients whose serum inhibited the cyototoxic activity of LAKs. Therefore, we hypothesized that VEGF might be one of the key role for inhibitory effect on LAKs and neutralization of serum VEGF from the patients whose serum inhibited the cyototoxic activity of LAKs could have no inhibitory effect on LAKs. Briefly, PBMCs from a healthy volunteer were used as an effector cell for all patients’ serum incubation. After activation of the PBMCs using IL-2, LAKs were prepared and incubated with the individual patient's serum and washed. The LAKs were incubated with human breast cancer cell line MCF-7 as a target cell and LDH assay was performed to detect cytotoxicity of the LAKs. Fourteen out of 24 patients serum (serum 1) showed inhibitory effect on LAKs and we picked up these patients to test blocking study of serum VEGF. Briefly, serum from these 14 patients was incubated with neutralization antibody of VEGF (R16Y, Santa Cruz Biotechnology) (serum 2) before mix with LAKs. After incubation of serum 1 and serum 2 with LAKs, target cell cytotoxicity of LAKs from incubation with serum 1 showed 30-50% reduced level as compared to control LAKs activity. Interestingly, target cell cytotoxicity of all LAKs from incubation with serum 2 did not show such inhibitory effect (mean cell cytotoxicity of LAKs was 33.48% and 57.27%, serum 1 and serum 2, respectively, control; 55.73%). These data suggest that high level of serum VEGF in breast cancer patients might be a key role of inhibitory effect on LAKs and neutralization of the VEGF could help adoptively transferred LAKs as cancer immunotherapy in the patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5304.