CAR‐Aptamers Enable Traceless Enrichment and Monitoring of CAR‐Positive Cells and Overcome Tumor Immune Escape

Abstract

Chimeric antigen receptor (CAR)‐positive cell therapy, specifically with anti‐CD19 CAR‐T (CAR19‐T) cells, achieves a high complete response during tumor treatment for hematological malignancies. Large‐scale production and application of CAR‐T therapy can be achieved by developing efficient and low‐cost enrichment methods for CAR‐T cells, expansion monitoring in vivo, and overcoming tumor escape. Here, novel CAR‐specific binding aptamers (CAR‐ap) to traceless sort CAR‐positive cells and obtain a high positive rate of CAR19‐T cells is identified. Additionally, CAR‐ap‐enriched CAR19‐T cells exhibit similar antitumor capacity as CAR‐ab (anti‐CAR antibody)‐enriched CAR‐T cells. Moreover, CAR‐ap accurately monitors the expansion of CAR19‐T cells in vivo and predicts the prognosis of CAR‐T treatment. Essentially, a novel class of stable CAR‐ap‐based bispecific circular aptamers (CAR‐bc‐ap) is constructed by linking CAR‐ap with a tumor surface antigen (TSA): protein tyrosine kinase 7 (PTK7) binding aptamer Sgc8. These CAR‐bc‐aps significantly enhance antitumor cytotoxicity with a loss of target antigens by retargeting CAR‐T cells to the tumor in vitro and in vivo. Overall, novel CAR‐aptamers are screened for traceless enrichment, monitoring of CAR‐positive cells, and overcoming tumor cell immune escape. This provides a low‐cost and high‐throughput approach for CAR‐positive cell‐based immunotherapy.

Construction of Switch Modules for CAR-T Cell Treatment Using a Site-Specific Conjugation System

“…A previous study has shown that the CD8 + T cell-binding aptamer A3t combined with magnetic beads is an excellent choice for achieving the traceless enrichment of CD8 + T cells. Indeed, this approach not only reduces costs but also simplifies the purification process. , Additionally, our previous study identified a type of CAR-aptamers that could efficiently accomplish the traceless enrichment of CAR + T/NK cells. , In fact, the process of aptamer capture and release does not leave markers or reduce cell viability. We thus speculated that the combination of aptamers with magnetic beads is an economical and convenient method for enriching target cells such as CD8 + T cells.…”