Screening human antibody libraries against carcinoma cells by affinity purification and polymerase chain reaction
Herald Reiersen1, Grete Berntsen, Marike Stassar
1Affitech AS, Oslo Research Park, Gaustadalleen 21, Oslo, Norway. herald@affitech.com
Abstract:
Bacterial scFv clones from a naïve antibody library have been isolated against cancer cell antigens with AffiSelect, a novel screening method that indirectly identifies candidate library members via an antigen reporter gene. The first step is the coating of carcinoma cell surface epitopes (antigen) with either mAbs, scFvs or phages (library members). Upon binding to a cell surface ligand, the library member generates a linking moiety. This facilitates magnetic affinity purification of the antibody-cancer cell complexes, detected by the polymerase chain reaction (PCR) using the beta-actin gene of the cancer cell as the target. Combining these well-known methods resulted in a higher resolution than a comparable cell-based ELISA method of detection. We have isolated human scFv antibodies against surface antigens of a lung carcinoma cell line. These were identified from a polyclonal mixture of phage display-enriched library clones comparing PCR patterns of the carcinoma cell line with the two negative cell types, HUVEC and peripheral blood cells (PBLs). The positive clones were sequenced and verified by FACS.
Insights
Researchers developed AffiSelect, a new method to find cancer-targeting antibodies. This technique uses magnetic purification and PCR to identify single-chain variable fragment (scFv) clones from antibody libraries, improving cancer antigen detection.
Area of Science:
- Immunotechnology
- Molecular Biology
- Oncology
Background:
- Identifying specific antibodies against cancer antigens is crucial for targeted therapies.
- Traditional screening methods like ELISA can lack the resolution for complex cell-surface antigen detection.
- Phage display libraries offer a rich source of potential antibody candidates.
Purpose of the Study:
- To introduce and validate AffiSelect, a novel screening method for isolating bacterial single-chain variable fragment (scFv) clones against cancer cell antigens.
- To compare the resolution of AffiSelect with existing cell-based ELISA methods.
- To isolate and characterize human scFv antibodies targeting surface antigens of a lung carcinoma cell line.
Main Methods:
- AffiSelect employs magnetic affinity purification of antibody-cancer cell complexes, facilitated by a linking moiety generated upon library member binding to cell surface epitopes.
- Detection of purified complexes is achieved via polymerase chain reaction (PCR) targeting the cancer cell's beta-actin gene.
- Phage display-enriched library clones were screened against a lung carcinoma cell line, with HUVEC and peripheral blood cells (PBLs) used as negative controls. Positive clones were sequenced and verified by Fluorescence-Activated Cell Sorting (FACS).
Main Results:
- The AffiSelect method demonstrated higher resolution compared to cell-based ELISA.
- Human scFv antibodies targeting surface antigens of a lung carcinoma cell line were successfully isolated.
- PCR pattern analysis of enriched library clones effectively distinguished positive clones from negative controls.
Conclusions:
- AffiSelect is an effective and high-resolution method for isolating specific antibody fragments against cancer cell surface antigens.
- The identified scFv antibodies represent potential candidates for lung carcinoma diagnostics or therapeutics.
- This approach offers a significant advancement in antibody library screening for cancer research.
