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Phage library-derived human anti-TETA and anti-DOTA ScFv for pretargeting RIT
S J DeNardo1, G L DeNardo, J Brush
1Department of Internal Medicine, University of California Davis Medical Center, Sacramento 95816, USA.
Abstract:
Pretargeting techniques have promise for radioimmunotherapy (RIT) in cancer because of the potential for markedly increasing the therapeutic ratio. Human antibody fragments can be retrieved from phage libraries and used to realize this potential. The library can be used to select the genetic material required to generate molecules with binding sites for both radiochelates and tumor antigens. In this study, human anti-chelate scFvs (single-chain fragments) for two metal chelates (Cu-TETA and Y-DOTA) were selected from a large, naive human scFv library. These anti-chelate scFvs were intended to serve as one arm of bispecific pretargeting molecules and to bind radiochelates given subsequently as Cu-67-TETA or Y-90-DOTA. Phage that displayed the anti-chelate scFv were selected by absorption to antibody (Lym-1) bound Cu-TETA or Y-DOTA. Enzyme-linked immunosorbent assays (ELISA) were performed to assess the intensity and specificity of phage binding to the specific chelate. Ninety-six clones demonstrating metal chelate binding seven times greater than to Lym-1 alone were chosen for diversity analysis. BstN I restriction digests were performed on DNA from these clones. Twenty-three and 43 different DNA fingerprint patterns were identified for anti-TETA and anti-DOTA clones, respectively. DNA sequencing of 39 anti-TETA clones for 23 different BstN I fingerprint patterns revealed 22 distinct sequences. Eleven of the anti-TETA clones were selected for further study. Five hundred to 1000 microg (100 to 320 microg per liter of culture) of purified scFv was produced from each of the 11 anti-TETA clones. Preliminary studies by BIAcore demonstrated evidence of 25- to 200-nM affinities. Comparable examination of the anti-DOTA clones is in progress. This study provides evidence that human scFv against unique synthetic targets can be readily selected from a large, naive human immunoglobulin phage library. Selections against metal chelated antibodies provided a wealth of scFvs with diverse binding affinities useful for engineering molecules for pretargeting RIT.
Insights
Researchers developed human antibody fragments to improve cancer radioimmunotherapy. These fragments target both radiochelates and tumor antigens, enhancing treatment effectiveness and safety.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Pretargeting techniques in radioimmunotherapy (RIT) offer potential for improved cancer treatment by increasing the therapeutic ratio.
- Human antibody fragments from phage libraries can be engineered for specific targeting applications.
Purpose of the Study:
- To select human anti-chelate single-chain fragments (scFvs) for radiometals (Cu-67-TETA and Y-90-DOTA) from a naive human scFv phage library.
- To generate bispecific molecules for pretargeting RIT by combining anti-chelate scFvs with tumor-targeting antibodies.
Main Methods:
- Selection of anti-chelate scFvs from a phage library by binding to antibody-bound metal chelates (Cu-TETA, Y-DOTA).
- Enzyme-linked immunosorbent assays (ELISA) to assess binding specificity and affinity.
- DNA fingerprinting (BstN I digests) and sequencing to analyze the diversity of selected scFv clones.
- BIAcore analysis to determine binding affinities of purified scFvs.
Main Results:
- Successfully selected human scFvs targeting Cu-TETA and Y-DOTA with high specificity.
- Identified significant diversity in anti-chelate scFv sequences, with 22 distinct sequences found among anti-TETA clones.
- Produced purified scFvs with preliminary binding affinities in the nanomolar range (25-200 nM).
Conclusions:
- Human scFvs against synthetic targets like metal chelates can be readily selected from large phage libraries.
- This approach yields diverse scFvs with varying affinities, suitable for engineering molecules for pretargeting RIT.
- The selected anti-chelate scFvs are valuable tools for developing advanced pretargeting strategies in cancer therapy.