Multivalent Tri-Functional T-cell Engagers by Chemically Induced Protein Self-Assembly | JoVE Visualize

Area of Science:

Biotechnology
Immunology
Nanotechnology

Background:

T-cell engagers (TCEs) show promise in cancer immunotherapy but face challenges in solid tumors, including heterogeneity, antigen escape, and limited T-cell infiltration.
Developing effective TCEs for solid tumors requires strategies to overcome these barriers and enhance T-cell activity.

Purpose of the Study:

To develop a modular platform using chemically self-assembled nanorings (CSANs) for creating potent multispecific TCEs.
To engineer a bifunctional fusion protein and demonstrate its self-assembly into CSANs for targeting EGFR-positive tumor cells and T-cells.
To explore the co-assembly of these CSANs with other targeting monomers to create trispecific TCEs for enhanced tumor cell targeting and cytotoxicity.

Main Methods:

Engineered a bifunctional fusion protein (E1-DHFR2-αCD3) with an EGFR-binding domain and an anti-CD3 scFv on a DHFR2 scaffold.
Utilized bis-methotrexate to induce self-assembly of monomers into multivalent cis-CSANs.
Performed co-culture assays to evaluate binding, internalization, and dose-dependent cytotoxicity of monomers and CSANs against EGFR+ tumor cells and T-cells.
Demonstrated co-assembly with additional DHFR2 monomers for creating trispecific CSANs targeting multiple antigens (e.g., GFP, EpCAM).

Main Results:

Both E1-DHFR2-αCD3 monomers and CSANs demonstrated binding to EGFR+ tumor cells and T-cells, followed by internalization.
CSANs induced dose-dependent, EGFR- and T-cell-dependent cytotoxicity in co-culture assays, showing reproducibility across T-cell donors.
Co-assembly enabled the formation of trispecific CSANs capable of binding multiple antigens and mediating enhanced cytotoxicity.
The CSAN platform demonstrated potential for developing multispecific TCEs to address solid tumor challenges.

Conclusions:

The modular CSAN platform provides a versatile strategy for engineering multispecific T-cell engagers.
This approach effectively addresses key challenges in solid tumor immunotherapy, such as antigen escape and heterogeneity.
Further optimization of antigen combinations within the CSAN platform holds promise for enhancing efficacy across diverse cancer subtypes, including breast cancer.