Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells

Area of Science:

• Immunology
• Oncology
• Biotechnology

Background:

• Chimeric antigen receptor (CAR)-T cell therapy shows promise for cancer treatment.
• Targeting tumor-associated antigens (TAA) with CARs requires careful design to avoid on-target, off-tumor effects.
• Low-affinity CARs may improve tumor specificity but can compromise efficacy.

Purpose of the Study:

• To investigate the impact of intracellular signaling domains on the function of low-affinity CAR-T cells targeting CD38.
• To determine if low-affinity CARs can be engineered for both safety and optimal antitumor activity.

Main Methods:

• Generated CAR-T cells using single-chain variable fragments (scFv) with varying affinities for CD38.
• Incorporated three distinct CAR costimulatory designs (including CD28 and 4-1BB domains).
• Evaluated CAR-T cell functionality and toxicity in vitro and in vivo.

Main Results:

• Low-affinity CD38 CARs with a 4-1BB domain showed limited cytotoxicity, which was improved by using a CD28 costimulatory domain.
• The combination of CD28 and 4-1BB signaling enhanced CAR-T cell proliferation, memory phenotype, and in vivo antitumor function.
• These optimized CAR-T cells effectively discriminated target antigen density, reducing off-tumor effects.

Conclusions:

• Combinatorial costimulatory designs enable the development of safe and effective low-affinity CAR-T cells.
• Low-affinity scFvs, when combined with appropriate signaling elements, can enhance the clinical potential of TAA-targeting CAR therapies.