Highly Specific and Effective Targeting of EGFRvIII-Positive Tumors with TandAb Antibodies

Area of Science:

• Oncology
• Immunotherapy
• Antibody Engineering

Background:

• Solid tumors often express unique antigens like EGFRvIII, presenting therapeutic targets.
• Harnessing T-cell-mediated cytotoxicity is a promising strategy for cancer treatment.
• Developing targeted therapies with minimal off-target effects is crucial for improving patient outcomes.

Purpose of the Study:

• To engineer novel tetravalent, bispecific tandem diabody (TandAb) antibodies for targeted immunotherapy of EGFRvIII-positive solid tumors.
• To assess the specificity, binding affinity, and cytotoxic potential of these TandAbs against EGFRvIII-expressing tumor cells.
• To evaluate the *in vivo* efficacy of EGFRvIII/CD3 TandAbs in a preclinical tumor model.

Main Methods:

• Phage display was used to identify and affinity-mature single-chain variable fragment (scFv) antibodies against EGFRvIII.
• Bispecific TandAbs were constructed by linking EGFRvIII-specific scFvs with CD3-specific scFvs.
• Antibody specificity was confirmed using Western Blot, SPR, ELISA, and FACS assays.
• Cytotoxicity was evaluated *in vitro* against EGFRvIII-expressing cell lines and *in vivo* in a xenograft tumor model.

Main Results:

• High-affinity, fully human scFvs targeting a unique EGFRvIII epitope were developed, achieving picomolar dissociation constants (KDs).
• EGFRvIII/CD3 TandAbs demonstrated exquisite specificity for EGFRvIII, with no binding to wild-type EGFR.
• Potent *in vitro* cytotoxicity was observed, with EC50 values in the picomolar range against EGFRvIII-expressing tumor cells.
• *In vivo* studies showed dose-dependent tumor growth control in an EGFRvIII-positive xenograft model.

Conclusions:

• EGFRvIII/CD3 TandAbs are highly specific and potent therapeutic candidates for selective immunotherapy of EGFRvIII-positive tumors.
• The tumor-exclusive expression of EGFRvIII and the target-dependent activation of these TandAbs minimize off-target effects and spare normal tissues.
• These novel antibodies offer a promising approach to reduce side effects associated with conventional anti-EGFR therapies and improve treatment outcomes.