CTLA-4 tail fusion enhances CAR-T anti-tumor immunity

Abstract

Chimeric antigen receptor (CAR) T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here, utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CT), we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen, and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior anti-tumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

Dissecting the role of CAR signaling architectures on T cell activation and persistence using pooled screening and single-cell sequencing

“…CTLA4, recognized as an inhibitory receptor on T cells, when embedded in the CAR signaling architecture resulted in potent CARs capable of promoting a robust T cell response, particularly amongst the CD4 compartment, with a persistent cytotoxic phenotype. Despite the inhibitory effect of CTLA4 signaling ( 23 ), our findings align with recent research reporting that the addition of CTLA4 cytoplasmic tails to a 28z CAR led to increased cytotoxicity and reduced production of pro-inflammatory cytokines ( 7 ). Despite its previous association with enhancing T cell anti-tumor potential ( 21 ), the IL15RA cytoplasmic domain did not seem to provide impactful T cell co-stimulation amongst the variants.…”

“…In addition, we included the signaling domains of CD40 (TNFRSF5) and the cytokine receptor chain IL15RA, which in preclinical studies have demonstrated the ability to enhance the anti-tumor properties of CARs ( 6 , 20 , 21 ). Lastly, CTLA4 was chosen as an example of an inhibitory receptor on T cells that still may enhance anti-tumor responses when incorporated into CARs ( 7 ). As a negative control, a non-signaling CAR (NS-CAR) was designed that lacks any intracellular signaling domains and is therefore unable to initiate CAR-dependent T cell activation.…”

“…For instance, combining CD79A and CD40 signaling domains resulted in CARs exhibiting improved proliferation and superior in vivo antitumor activity compared to clinically approved designs ( 6 ). Furthermore, incorporation of CTLA4 cytoplasmic tails into a CD28-CD3ζ CAR increased its cytotoxic potential while delaying T cell activation and proinflammatory cytokine production, ultimately enhancing CAR-T efficacy in a murine model of leukemia ( 7 ).…”

Applications of CRISPR technology in cellular immunotherapy

“…To generate those cells, autologous or allogenic T cells are engineered with a TCR or CAR to recognize tumor‐specific antigen or tumor‐associated antigen and to active T cells for targeted lysis 63,64 . Traditionally, viral vectors such as lentivirus and retrovirus have been used to generate genetically engineered T‐cell products 65,66 . However, both lentivirus and retrovirus have limitations regarding their variable integration copy numbers, as well as the random genomic sites at which they integrate.…”

“…63,64 Traditionally, viral vectors such as lentivirus and retrovirus have been used to generate genetically engineered T-cell products. 65,66 However, both lentivirus and retrovirus have limitations regarding their variable integration copy numbers, as well as the random genomic sites at which they integrate. This can cause variability in transgene expression and cell phenotypes due to insertional mutagenesis.…”