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Updated: Jul 26, 2026
Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting
Published on: March 8, 2018
Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers
A M Wu1, W Chen, A Raubitschek
1Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Background:
Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo.
Objectives:
To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model.
Study Design:
ScFv were constructed with either a 28 or 14 amino acid connecting peptide and expressed by secretion from E. coli. Following affinity purification, proteins were characterized by gel electrophoresis and mass spectrometry. Binding properties were assessed by size exclusion HPLC after incubation with antigen, and affinities determined by surface plasmon resonance. The shorter linker favored formation of dimers (and higher multimers) which showed unusual stability. ScFv were radiolabeled with 125I for tumor targeting and biodistribution studies of monomeric or dimeric forms were conducted in athymic mice bearing LS174T human colorectal carcinoma xenografts.
Results:
125I-scFv monomers and dimers targeted exhibited rapid clearance kinetics in tumor-bearing mice. Nevertheless, the anti-CEA scFvs targeted very well to xenografts, leading to high tumor: normal organ ratios (greater than 20:1 at 24 h) for both forms. Tumor localization of the non-covalent dimers was much higher than monomers, reaching 10-15% injected dose per gram at 1 h.
Conclusion:
Non-covalent dimers of scFv (also known as diabodies) are stable, easy to produce and show excellent targeting as compared to monomeric scFv, probably due to increased mass and valency.
Insights
Engineered antibody fragments called diabodies (non-covalent dimers of single-chain Fv) show superior tumor targeting compared to monomers. These stable diabodies offer improved properties for in vivo cancer therapies.
Area of Science:
- Biotechnology
- Immunology
- Molecular Biology
Background:
- Genetic engineering enables creation of novel antibody fragments for enhanced in vivo applications like tumor targeting.
- Antibody fragments offer potential for improved therapeutic efficacy and reduced immunogenicity.
Purpose of the Study:
- To produce stable monomeric and dimeric forms of a single-chain variable fragment (scFv) targeting carcinoembryonic antigen (CEA).
- To evaluate the in vivo tumor targeting and biodistribution of these engineered antibody fragments in a preclinical model.
Main Methods:
- Single-chain Fv (scFv) constructs with varying linker lengths were expressed and purified from E. coli.
- Proteins were characterized using gel electrophoresis, mass spectrometry, and surface plasmon resonance.
- Radiolabeled scFv monomers and dimers were assessed for tumor targeting and biodistribution in mice bearing colorectal carcinoma xenografts.
Main Results:
- Engineered scFv, particularly dimers (diabodies), demonstrated efficient targeting of colorectal carcinoma xenografts.
- High tumor-to-normal organ ratios (>20:1 at 24h) were achieved for both monomeric and dimeric forms.
- Non-covalent dimers exhibited significantly higher tumor localization compared to monomers.
Conclusions:
- Non-covalent dimers (diabodies) of anti-CEA scFv are stable and readily produced.
- Diabodies offer superior tumor targeting efficacy compared to monomeric scFv, likely due to increased mass and valency.
- These findings support the potential of diabodies for targeted cancer therapy.
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