Identification of prostate cancer antigens by automated high-throughput filter immunoscreening.

There is a need for earlier and more accurate cancer diagnostics as well as new targets for cancer immunotherapy. To this end, it is important to identify sets of tumour antigens specific for different cancer forms. Several methods that identify potential tumour antigens in an arrayed and high-throughput format have been developed during the last years of SEREX (serological identification of antigens by recombinant expression cloning) related research. Such techniques may hold the potential to describe the complete immunogenic part of the cancer proteome, also called the cancer immunoproteome. We have developed a powerful platform for automated serological high-throughput filter screening of tumour cDNA libraries. The screening format of this method is 18,000 single cDNAs clones, which is superior to other high-throughput methods described. The output is antigens, which are potential diagnostic cancer markers and vaccine targets. We present here the results from the screening of a prostate tumour cDNA library with autologous patient antibodies. We first demonstrated the feasibility of the automated high-throughput filter immunoscreening method by use of the NY-ESO-1sv (NY-ESO-1 splice variant) antigen. We then screened 18,000 cDNA clones from a phage display selected prostate tumour cDNA library with autologous patient antibodies and identified several relevant antigens such as NY-ESO-1, XAGE-1, DJ-1 and transcription factor 25 (TCF25). The present high-throughput immunoscreening method has the potential to identify both patient-specific and disease-specific antigens for use in diagnostics and therapy.

Direct isolation of recombinant human antibodies against group B Neisseria meningitidis from scFv expression libraries.

The successful generation of human antibodies from large nai;ve antibody libraries requires iterative selection steps. Here, we describe a new and fast method for the isolation of high affinity antibodies directly from human single chain Fv antibody (scFv) expression libraries. Escherichia coli scFv expression libraries were made from peripheral blood lymphocytes from four individuals vaccinated with group B Neisseria meningitidis outer membrane vesicle (OMV) vaccine. Forty thousand clones were directly screened for antibodies binding N. meningitidis strain 44/76 (B:15:P1.7,16). Of 430 specific clones detected, 225 candidates were isolated and re-screened against the N. meningitidis strains NZ-98/254 (B:4:P1.7b,4) giving 4% cross-reactive clones. Antibodies were further characterized by DNA sequencing, ELISA and surface plasmon resonance (SPR) analysis, showing broad V-gene diversity and nanomolar scFv affinities. Antibodies derived by this method may assist in the discovery and development of new vaccine antigens as well as therapeutic antibody agents for the treatment of meningococcal diseases.