Identification of novel tumor antigens with patient-derived immune-selected antibodies.
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.
Center for Vascular Biology, EM028, Department of Cell Biology-MC3501, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3501, USA.
Daniel
D
Rodriguez-Pinto
Jason
J
Sparkowski
Martin P
MP
Keough
Kathryn N
KN
Phoenix
Frank
F
Vumbaca
David K
DK
Han
Eckart D
ED
Gundelfinger
Philip
P
Beesley
Kevin P
KP
Claffey
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008198:Lymph Nodes / Q000276:immunology* / Q000473:pathology; D008213:Lymphocyte Activation / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D008562:Membrane Glycoproteins; C518360:NPTN protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins