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Mono- and bispecific single-chain antibody fragments for cancer therapy
S Thirion1, K Motmans, H Heyligen
1Dr L Willems-Instituut, Diepenbeek, Belgium.
Abstract:
Especially when dealing with solid cancers, single-chain antibody fragments (scFvs) have a lot of advantages. Due to their small size (27 kDa), these proteins clear more rapidly from the blood, and penetrate faster and deeper into tissues, than whole antibodies. Furthermore, the lack of constant regions ensures that they are not retained in tissues such as the liver and kidney. This reduces possible toxic side-effects. Single-chain construction is normally done by polymerase chain reaction (PCR). To decrease the overall cost of oligonucleotide primer synthesis, time-consuming primer design, multiple PCR reactions and individual PCR optimization, we designed a universal single-step overlap extension PCR protocol using hybridoma cDNA as a template. To overcome the lack of effector function, bispecific scFvs, consisting of an scFv produced against a tumour-associated antigen fused to a T cell marker-specific scFv, are being created, starting from already assembled scFv, by means of two additional PCR reactions. In this paper we describe both PCR methods that were successfully used to create scFvs against the human transferrin receptor, the human interleukin-2 receptor, the human CD3 molecule, a breast tumour-associated antigen and an anti-transferrin-anti-CD3 bispecific scFv.
Insights
We developed a streamlined polymerase chain reaction (PCR) method for creating single-chain antibody fragments (scFvs). This approach simplifies scFv production, enabling faster development of targeted cancer therapies.
Area of Science:
- Biotechnology and Molecular Biology
- Immunology and Cancer Research
Background:
- Single-chain antibody fragments (scFvs) offer advantages over whole antibodies for solid cancer treatment due to their small size, rapid blood clearance, and enhanced tissue penetration.
- Traditional scFv construction via polymerase chain reaction (PCR) involves costly and time-consuming primer synthesis, design, and optimization steps.
Purpose of the Study:
- To design and validate a universal, single-step overlap extension PCR protocol for efficient scFv construction.
- To develop bispecific scFvs by fusing tumor-associated antigen scFvs with T cell marker-specific scFvs to overcome effector function limitations.
- To demonstrate the successful application of these PCR methods for generating scFvs against various targets, including tumor antigens and immune cell markers.
Main Methods:
- A novel, single-step overlap extension PCR protocol was developed using hybridoma cDNA as a template to streamline scFv production.
- Bispecific scFvs were generated by performing two additional PCR reactions on pre-assembled scFvs, fusing target-specific and T cell-specific fragments.
- The PCR methods were applied to create scFvs targeting the human transferrin receptor, human interleukin-2 receptor, human CD3 molecule, and a breast tumor-associated antigen.
Main Results:
- The developed single-step PCR protocol significantly reduces the cost and complexity associated with scFv synthesis and optimization.
- Successful generation of scFvs against multiple human receptors and a breast tumor antigen was achieved.
- An anti-transferrin-anti-CD3 bispecific scFv was successfully constructed, demonstrating the potential for enhanced therapeutic strategies.
Conclusions:
- The described universal single-step overlap extension PCR protocol offers an efficient and cost-effective method for scFv production.
- The ability to readily create bispecific scFvs opens new avenues for developing targeted immunotherapies, particularly for solid cancers.
- These optimized PCR methods facilitate the rapid generation of diverse scFvs for research and therapeutic applications.