Protein catenation potentiates antitumor activity of malaria VAR2CSA navigation CAR-T cells in a mouse model of hematological malignancies.

BACKGROUND

While CD19-targeting chimeric antigen receptor T-cell (CAR-T) therapies have shown efficacy in B-cell hematological malignancies, treatment of antigen-negative or relapsed tumors remains challenging. The recombinant malaria protein VAR2CSA (rVAR2) binds specifically to placental-like chondroitin sulfate A (pl-CSA), which is expressed on many cancer types, enabling its use in various tumor-targeting platforms. However, rVAR2-based CAR-T strategies are limited by poor protein thermostability, short serum half-life, and safety concerns related to irreversible CAR activation. To address these limitations, we developed a navigable CAR-T cell system that uses an rVAR2-based navigator to direct CAR-T cells to pl-CSA+ tumors.

METHODS

We overexpressed and purified rVAR2 and three catenation proteins in Escherichia coli, then produced and epitope-mapped the anti-rVAR2 monoclonal antibody 5H4VAR2omab following murine immunization. Using its single-chain fragment variable, we constructed a third-generation CAR-T with CD28 and 4-1BB co-stimulatory domains. An interlocking rVAR2 catenane, AXVB, was identified via orthogonal screening and employed as a navigator for directing CAR-T cells to pl-CSA+ tumors. The antitumor efficacy of this navigable CAR-T cell system was evaluated in vitro and in hematologic cancer cell line-derived xenograft models.

RESULTS

We developed an orthogonal CAR-T cell system that employs a navigator protein (rVAR2 or AXVB) to target pl-CSA+ tumors. Through a reversible switch mechanism, this system enables indirect tumor recognition by CAR-T cells, thereby improving the safety profile of conventional CAR-T therapies. Compared with the rVAR2 monomer, the AXVB multimer exhibited enhanced thermostability and target affinity. Both in vitro and in vivo, AXVB-(navigation)-CAR T cells potently eliminated CD19-positive and CD19-negative pl-CSA+ tumor cells and significantly prolonged survival in tumor-bearing mice.

CONCLUSIONS

Protein catenation improved the thermostability and functional activity of rVAR2. In a murine model of hematologic malignancy, CAR-T cells utilizing the rVAR2 catenane (AXVB) as a targeting module exhibited superior antitumor activity.