Abstract 5626: Single-chain bispecific BiTE antibody specific for CD3 and melanoma-associated chondroitin sulfate proteoglycan: In vitro and in vivo anti-melanoma activity

Abstract

Abstract Introduction: Bispecific T-cell engager (BiTE) antibodies redirect cytotoxic T cells to malignant cells and are in clinical trials for treatment of various cancers. A recently developed BiTE antibody targets CD3 on T cells and melanoma-associated chondroitin sulfate proteoglycan (MCSP) on human melanoma cells. We examined 1) cytotoxic activity of MCSP-BiTE against a human melanoma cell line cocultured with peripheral blood mononuclear cells (PBMC) from melanoma patients, 2) T cell activation by MCSP-BiTE, and 3) anti-tumor effect of MCSP-BiTE in a human melanoma xenograft model. Methods: 1) Strongly MCSP-positive metastatic human melanoma cell line M27-HI was cultured alone or at a 3:1 effector: target (E: T) ratio with PBMC from 12 melanoma patients. Cocultures were treated for 6-7 days with MCSP-BiTE antibody or a control BiTE antibody that binds CD3 but not MCSP. Cytotoxicity was assessed by a FACS-based assay. 2) PBMC from a healthy donor were incubated with MCSP-BiTE antibody and M27-HI cells, stained with antibody to CD4, CD8, CD69 and granzyme B followed by FACS analysis (Student's t-test). 3) 2 million M27-HI cells, which produce moderately aggressive tumor growth in NOD/SCID mice, were mixed with nonstimulated healthy donor PBMC at an E: T ratio of 1:1 and injected subcutaneously into NOD/SCID mice. MCSP-BiTE antibody was continuously infused intraperitoneally for 4 weeks. Tumor growth in each treatment group of 3 mice was measured in two dimensions twice weekly for 10 weeks; the time for tumors to reach 0.2 cm3 was compared between the treatment and control groups (log-rank test). Results: PBMC of melanoma patients contained 25.93 ± 9.27% CD3+ T cells. 1) The percentage of dead cells was significantly higher when M27-HI cells were cocultured with PBMC and 100 ng/ml MCSP-BiTE (36.07 ± 21.38%; range 2.05-71.17%) versus PBMC and 100 ng/ml control antibody (10.67 ± 6.4%; range 3.05-25.9%; p=0.002) or PBMC alone (12.21 ± 4.18%; range 3.71-19.94%; p=0.003). The percentage of dead cells was lower when PBMC were from patients with clinical evidence of melanoma. 2) A 48-h coculture of healthy donor PBMC, MCSP-BiTE and M27-HI cells showed dose-dependent increases of CD69+ (both on CD4+ and CD8+ cells) and granzyme B (CD8+ cells). 3) Four μg/day MCSP-BITE significantly inhibited tumor growth (p=0.03) compared to a bispecific control antibody. Conclusions: In our previous experiments, CD3+ T cells comprised approximately 60% of healthy donor PBMC and melanoma cell killing was 46-55% (AACR 2009). Here, CD3+ T cells comprised approximately 26% of melanoma patients’ PBMC and melanoma cell killing was 36%; decreased killing could be due to a lower percentage of CD3+ T cells in patients’ PBMC. MCSP-BiTE not only mediated redirected lysis but also T-cell activation as was evident from upregulation of CD69 and granzyme B. MCSP-BiTE shows anti-tumor activity in vivo. 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 5626.