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Published on: October 15, 2016
Screening for antibodies against intact cancer cells with a naïve large phage antibody library
Yuanyuan Qiao1, Lijun Zhou, Yuxiao Wang
1Graduate School of Southern Medical University, Guangzhou, PR China.
Abstract:
Large phage antibody libraries are expected to be an efficient technology for obtaining humanized anti-tumor antibodies. However, few reports have been concerned about the selection strategies for intact cancer cells as targets. In this study, a 2x1011 large naïve scFv library from blood B lymphocytes of normal adult donors and neonatal cord blood was constructed using the LoxP-cre system. Three human esophageal cancer cell lines were equally mixed for use as the target. These intact cells were divided into two groups, PF and NF, according to the treatment method of the cells (fixed with 2% PFA or not, respectively). Positive phage antibodies were identified following 4 panning cycles of adhesion, elution and amplification. Using a cell ELISA assay, it was found that the additional procedure of directly infecting XL1-Blue bacteria with the cell pellet following washing with an acidic solution can effectively decrease the loss of positive phage, yielding a positive screening rate of 11.6% (61/525). Most of the phage antibodies displayed binding activity with all three esophageal cancer cell lines. Moreover, other phages (such as NFc53a and NFc70a) appeared to be specific for certain cell lines. Regarding the method used to treat the target cells, both the positive screening rate and the genetic diversity of the antibody variable region were significantly higher in the NF group (12.9 and 71.4%, respectively) than in the PF group (10.5 and 14.2%, respectively). Immunohistochemical analysis demonstrated that the scFv phages have good specificity for esophageal cancer. This technology is helpful for developing small molecular tracers and targeted therapies for malignant tumors.
Insights
This study developed a phage antibody selection strategy using intact esophageal cancer cells, yielding a high positive screening rate and diverse antibody candidates. The non-fixed cell method proved superior for identifying effective anti-cancer phage antibodies.
Area of Science:
- Immunotechnology
- Oncology
- Molecular Biology
Background:
- Phage antibody libraries offer a promising avenue for developing humanized anti-tumor antibodies.
- Effective selection strategies for intact cancer cells remain underexplored.
- Esophageal cancer requires novel therapeutic and diagnostic approaches.
Purpose of the Study:
- To construct a large naive single-chain variable fragment (scFv) phage library for identifying human esophageal cancer-targeting antibodies.
- To optimize selection strategies for intact cancer cells, comparing fixed and non-fixed cell targets.
- To evaluate the specificity and diversity of selected phage antibodies for esophageal cancer.
Main Methods:
- Construction of a 2x10^11 scFv phage library from normal adult donor and neonatal cord blood B lymphocytes.
- Panning against a mixture of three human esophageal cancer cell lines, treated either with paraformaldehyde (PF) or not (NF).
- Optimization of phage recovery using acidic solution washing and bacterial infection, followed by cell ELISA and immunohistochemical analysis.
Main Results:
- A positive screening rate of 11.6% was achieved with an optimized cell ELISA method.
- The non-fixed (NF) cell group demonstrated significantly higher positive screening rates (12.9%) and antibody variable region genetic diversity (71.4%) compared to the fixed (PF) group (10.5% and 14.2%, respectively).
- Selected scFv phages exhibited binding activity against multiple esophageal cancer cell lines, with some showing cell-specific binding and good specificity in immunohistochemical analyses.
Conclusions:
- The developed phage display selection strategy using intact, non-fixed esophageal cancer cells is efficient for isolating high-affinity, specific anti-cancer antibodies.
- This approach enhances antibody discovery yield and genetic diversity compared to using fixed cells.
- The identified scFv phages hold potential for developing targeted therapies and small molecular tracers for esophageal cancer.
