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Identification of novel tumor antigens with patient-derived immune-selected antibodies

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Abstract

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions.

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Acknowledgments

This work was supported by NIH:NCI IMAT grant, CA114489 (KPC) and The Patrick and Catherine Weldon Donaghue Foundation (KPC). Many thanks to Nancy Ryan for her technical assistance in histology and immunohistochemistry.

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Authors and Affiliations

  1. Center for Vascular Biology, EM028, Department of Cell Biology-MC3501, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT, 06030-3501, USA

    Daniel Rodriguez-Pinto, Martin P. Keough, Kathryn N. Phoenix, Frank Vumbaca, David K. Han & Kevin P. Claffey

  2. The Feinstein Institute for Medical Research, Manhasset, NY, 11030, USA

    Daniel Rodriguez-Pinto

  3. Merck & Co, Inc., Upper Gwynedd, PA, 19454, USA

    Jason Sparkowski

  4. Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Martin P. Keough

  5. Leibniz Institute for Neurobiology, Magdeburg, Germany

    Eckart D. Gundelfinger

  6. School of Biological Sciences, Royal Holloway University of London, Surrey, UK

    Philip Beesley

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  1. Daniel Rodriguez-Pinto
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  2. Jason Sparkowski
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  9. Kevin P. Claffey
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Correspondence to Kevin P. Claffey.

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Rodriguez-Pinto, D., Sparkowski, J., Keough, M.P. et al. Identification of novel tumor antigens with patient-derived immune-selected antibodies. Cancer Immunol Immunother 58, 221–234 (2009). https://doi.org/10.1007/s00262-008-0543-0

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  • DOI: https://doi.org/10.1007/s00262-008-0543-0

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