Development of single chain antibodies for targeting Ewing sarcoma surface marker CD99

Area of Science:

• Oncology
• Immunology
• Biotechnology

Background:

• Recombinant antibodies are crucial therapeutics for cancer treatment.
• CD99 is a glycoprotein overexpressed in malignancies like Ewing sarcoma (ES).
• Current ES therapies have significant toxicity and late complications, necessitating novel treatments.

Purpose of the Study:

• To develop CD99-targeting single-chain antibodies (scFvs) for improved cancer therapy.
• To engineer and characterize two scFvs with different linker lengths for CD99 targeting.
• To evaluate the impact of linker length on scFv performance and specificity.

Main Methods:

• Isolated variable regions from a hybridoma-derived anti-CD99 antibody.
• Engineered two scFvs with distinct 15-mer and 18-mer linkers.
• Expressed and purified scFvs in Escherichia coli.
• Performed in vitro binding analyses to compare scFv affinity and specificity.

Main Results:

• Both engineered scFvs were successfully expressed and purified.
• The scFv with the shorter 15-mer linker showed significantly enhanced affinity and specificity.
• The 15-mer scFv demonstrated superior binding to CD99-overexpressing Ewing sarcoma cells compared to the 18-mer variant.
• Linker length was identified as a critical factor influencing scFv performance.

Conclusions:

• A rational design principle for optimizing CD99-targeting scFvs was established.
• The 15-mer scFv holds potential for CAR-T cell development in Ewing sarcoma.
• This scFv may also be applicable for targeting other CD99-overexpressing carcinomas.