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TYRP1 (tyrosinase-related protein 1) is a melanoma antigen expressed in melanosomes and on the surface of melanoma cells. Previous studies have shown that mouse antibodies to TYRP1 localized to melanomas in vivo and inhibited tumor growth and metastasis. Here, we describe the characterization of a novel fully human anti-TYRP1 MAb (20D7) generated by immunizing HuMAb mice (Medarex). 20D7 recognized recombinant and native human TYRP1 by Western blotting and ELISA, and native TYRP1 in melanoma cells as determined by flow cytometry analysis. 20D7 cross-reacted with mouse TYRP1. The binding affinity to human TYRP1 for the human MAb was in the low nM range as determined by surface plasmon resonance kinetics. 20D7 can bind to human and mouse Fc receptor and induce a strong ADCC response against human melanoma cells in vitro. The antitumor activity of 20D7 was tested in human melanoma xenografts and mouse metastatic melanoma models in athymic nude mice. Growth of s.c. human melanoma tumors and metastatic nodules of murine B16 tumor were significantly suppressed by 20D7 compared to human IgG control. These results suggest that human anti-TYRP1 MAb may be a potent therapeutic for the treatment of malignant melanoma. [ABSTRACT FROM AUTHOR]

Human carcinomas frequently express one or more members of the epidermal growth factor receptor family. Two family members, epidermal growth factor receptor (EGFR) and c-erbB2/neu (HER2), homodimerize or heterodimerize upon activation with ligand and trigger potent mechanisms of cellular proliferation, differentiation and migration. In this study, we examined the effect of the anti-EGFR monoclonal antibody Erbitux (cetuximab) on human tumor cells expressing both EGFR and HER2. Investigation of the effect of cetuximab on the activation of EGFR-EGFR, EGFR-HER2 and HER2-HER2 homodimers and heterodimers was conducted using the NCI-N87 human gastric carcinoma cell line. Treatment of NCI-N87 cells with cetuximab completely inhibited formation of EGFR-EGFR homodimers and EGFR-HER2 heterodimers. Activation of HER2-HER2 homodimers was not appreciably stimulated by exogenous ligand and was not inhibited by cetuximab treatment. Furthermore, cetuximab inhibited EGF-induced EGFR and HER2 phosphorylation in CAL27, NCI-H226 and NCI-N87 cells. The activation of downstream signaling molecules such as AKT, MAPK and STAT-3 were also inhibited by cetuximab in these cells. To examine the effect of cetuximab on the growth of tumors in vivo, athymic mice bearing established NCI-N87 or CAL27 xenografts were treated with cetuximab (1 mg, i.p., q3d). The growth of NCI-N87 and CAL27 tumors was significantly inhibited with cetuximab therapy compared to the control groups (p<0.0001 in both cases). In the CAL27 xenograft model, tumor growth inhibition by cetuximab treatment was similar to that by cetuximab and trastuzumab combination treatment. Immunohistological analysis of cetuximab-treated tumors showed a decrease in EGFR-HER2 signaling and reduced tumor cell proliferation. These results suggest that cetuximab may be useful in the treatment of carcinomas co-expressing EGFR and HER2.

Targeted immunotherapy against tumors or angiogenesis has shown promise as an alternative approach for the treatment of malignant disease. Whether or not combining these two treatment modalities would enhance the antitumor effect was tested in mouse models of malignant melanoma. C57BL/6 mice bearing established subcutaneous B16 tumors were treated with anti-vascular endothelial growth factor receptor (anti-VEGFR) fetal liver kinase-1 (Flk-1) monoclonal antibody (mAb) DC101 and/or anti-TYRP-1/gp75 (tyrosinase-related protein-1) mAb TA99. The growth of subcutaneous B16 tumors was significantly suppressed by the mAb DC101 (63%, p<0.001) and by mAb TA99 (75%, p<0.001) treatment alone. The combined antibody (TA99+DC101) treatment resulted in a significant enhancement (93%, p<0.001) of tumor growth suppression. In a B16 pulmonary metastasis model, combined therapy with mAb DC101 and mAb TA99 resulted in a significant reduction of lung metastases compared to the control (p<0.001) and the single agent treatment groups (p<0.05). A combined modality approach that provides passive immunity to melanoma differentiation antigens as well as inhibiting tumor neovascularization may be valuable for the treatment of malignant melanoma.

Background: The epidermal growth factor receptor (EGFR) plays an important role in the growth and survival of many human tumors of epithelial origin. EGFR variant III (EGFRvIII) is a truncated form of EGFR that does not bind ligand, is constitutively active, and is reported to be coexpressed with EGFR in some human tumors including breast, glioblastoma, lung, and prostate., Materials and Methods: Here we have tested the anti-EGFR monoclonal antibody cetuximab for its interaction with EGFRvIII. Chinese hamster ovary (CHO), 32D (non-tumorigenic murine hematopoetic cells), and U87-MG stable transfectants were generated to express EGFRvIII., Results: Analysis of receptor phosphorylation showed that the EGFRvIII was constitutively phosphorylated in transfected cells. Flow cytometry, direct binding, and immunoprecipitation analysis of EGFRvIII transfectants showed specific binding of cetuximab to EGFRvIII. Cetuximab bound to EGFRvIII with a KD of 0.38 nM determined by Scatchard analysis and 1.1 nM determined by Biacore analysis respectively. In internalization studies, binding of cetuximab to the EGFRvIII on the cell surface led to at least 50% of the cetuximab-EGFRvIII complex internalized from cell surface of CHO-EGFRvIII after 3 hours. This internalization led to a reduction in phosphorylated EGFRvIII in transfected cells. Furthermore, incubation of cells expressing EGFRvIII with cetuximab resulted in 40-50% inhibition of cell proliferation., Conclusion: These data suggest that cetuximab may be a potential candidate for the treatment of tumors that also express EGFRvIII.

CD4(+) T cells can recognize "self" tumor antigens, but the impact of tumor cell expression of self-antigens on CD4(+) T-cell function in humans is unknown. Here, we identify a new epitope (ISPNSVFSQWRVVCDSLEDYD) derived from tyrosinase-related protein-1 (TRP-1) using a predictive algorithm and mice transgenic for a chimeric HLA-DRB1*0401 molecule. We then compared the functions of TRP-1-epitope-specific, CD4(+) T-cell responses in normal healthy individuals to those found in patients with metastatic malignant melanoma. Surprisingly, we found that tumor-bearing patients had significantly higher levels of TRP-1-specific, CD4(+) T-cell function than healthy volunteers as measured ex vivo. Thus, the net effect of "self" antigen expression by tumor cells was the enhancement of tumor antigen-specific CD4(+) T-cell function, rather than immunosuppression. These findings indicate that antigens expressed by malignant melanoma cells can partially activate CD4(+) T lymphocytes.