Identification of prostate specific membrane antigen (PSMA) as the target of monoclonal antibody 107-1A4 by proteinchip�; array, surface-enhanced laser desorption/ionization (seldi) technology

Abstract

Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in men in the United States. In spite of increasing attention and accumulating knowledge, advances in treatment that improve survival remain elusive. Prostate specific antigen (PSA) is the most widely used marker of prostate cancer. Immunoassays for PSA using monoclonal (MAb) or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA is not a perfect tumor marker because serum levels often are elevated in men with benign prostatic hyperplasia, prostatitis and other nonmalignant disorders and also because levels are not always elevated in individuals with early prostate cancer. 1 In an effort to identify additional proteins that could be of value for the diagnosis and treatment of prostate cancer, we have recently described the generation of a new prostate cancer-reactive MAb, designated 107-1A4, using a two-phase immunization protocol involving the prostate cancer cell line, LNCaP. 2 This MAb recognizes an antigen, which appeared to be distinct from those previously described and shows specific tumor targeting in preliminary in vivo studies. In part because MAb 107-1A4 recognized a conformational epitope and, thus, could not be used in Western blots, we were unable to characterize its antigen using conventional approaches. 2 In this report we describe the identification of the target antigen of MAb 107-1A4 using ProteinChip array, surface-enhanced laser desorption/ionization (SELDI) technology from Ciphergen Biosystems (Fremont, CA). SELDI, a concept introduced by Hutchens and Yip 3 combines ProteinChip technology with timeof-flight mass spectrometry and offers the advantages of speed, simplicity, sensitivity and accuracy. Briefly, each ProteinChip array has a number of spots that contain functional groups, chemical or biological "docking sites," for the selective binding and washing of proteins/peptides from complex mixtures. After the sample is applied to the surface, unbound proteins and interfering substances are washed away. A solution containing laser energyabsorbing molecules, often referred to as a matrix, is then added and allowed to dry. In this fashion, the laser energy absorbing matrix molecules co-crystallize with the adsorbed proteins. The captured proteins are then detected using laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS). A more comprehensive description of the overall history and recent advances in SELDI technology and comparisons with other laserbased mass spectrometry...

Further investigation of the epitope recognized by the new monoclonal antibody 2C9

“…Several kinds of anti‐PSMA antibodies have been established and characterized for reactivity with normal and cancer tissues 5,16–18 . Differences in epitopes of the PSMA antigen recognized resulted in unique binding patterns.…”

Principles of Molecular Oncology

“…Early clinical studies are already demonstrating the value of profiling proteins in a complex mixture of tumor cells for monitoring drug responsiveness (425,426), for identifying new drug targets for prevention (427,428) and treatment (429,430), and for the detection and diagnosis of cancers (431)(432)(433)(434).…”

“…In addition to an immediate impact in therapeutic monitoring and drug development, proteomic applications are showing similar clinical promise in the application areas of early detection and cancer diagnosis (418,427,428,431,432,434). For example, serum protein profiles generated using surface-enhanced laser desorption ionization-mass spectrography (SELDI-MS) technology were shown to reliably identify women who harbored all stages of ovarian tumors, including women with early stage I disease (433).…”

“…This tumor, and its juvenile form, giant cell fibroblastoma, are cytogenetically characterized by the presence of supernumerary ring(s) derived from the t(17;22) (427). The breakpoints from translocations and rings contain the fusion of PDGF chain and the COL1A1 gene(428).…”

Treatment of Carcinogenesis

“…In addition to an immediate impact in therapeutic monitoring and drug development, proteomic applications are showing similar clinical promise in the application areas of early detection and cancer diagnosis (418,427,428,431,432,434). For example, serum protein profiles generated using surface-enhanced laser desorption ionization-mass spectrography (SELDI-MS) technology were shown to reliably identify women who harbored all stages of ovarian tumors, including women with early stage I disease (433).…”