Development of non-viral, ligand-dependent, EPHB4-specific chimeric antigen receptor T cells for treatment of rhabdomyosarcoma

Abstract

Ephrin type-B receptor 4 (EPHB4), expressed in tumors including rhabdomyosarcoma, is a suitable target for chimeric antigen receptor (CAR)-T cells. Ligand-independent activation of EPHB4 causes cell proliferation and malignant transformation in rhabdomyosarcoma, whereas ligand-dependent stimulation of EPHB4 induces apoptosis in rhabdomyosarcoma. Therefore, we hypothesized that ligand-based, EPHB4-specific CAR-T cells may kill rhabdomyosarcoma cells without stimulating downstream cell proliferation mechanisms. We developed novel CAR-T cells by targeting EPHB4 via EPHRIN B2, a natural ligand of EPHB4. The generation of EPHB4-CAR-T cells via piggyBac (PB) transposon-based gene transfer resulted in sufficient T cell expansion and CAR positivity (78.5% ± 5.9%). PB-EPHB4-CAR-T cells displayed a dominant stem cell memory fraction (59.4% ± 7.2%) as well as low PD-1 expression (0.60% ± 0.21%) after 14 days of expansion. The PB-EPHB4-CAR-T cells inhibited EPHB4-positive tumor cells without activating cell proliferation downstream of EPHB4, even after multiple tumor re-challenges and suppressed tumor growth in xenograft-bearing mice. Therefore, PB-EPHB4-CAR-T cells possess a memory-rich fraction without early T cell exhaustion and show potential as promising therapeutic agents for treating rhabdomyosarcoma and other EPHB4-positive tumors.

A lymphodepleted non‐human primate model for the assessment of acute on‐target and off‐tumor toxicity of human chimeric antigen receptor‐T cells

“…As previously reported, the EPHB4‐CAR‐T cells exhibited strong and durable potency against EPHB4 + RH30 cells, as demonstrated via xCELLigence ® Real‐Time Cell Analysis (Figure 3b). 7 These data were consistent with our previous data; therefore, we used these cells for conducting further NHP studies.…”

“…As this tendency was observed in the male macaque of the No Tx group and not observed in the male macaque of the CAR‐T group, it was considered an effect of the conditioning regimen. However, our previous study has revealed that EPHB4 is weakly expressed in renal tissue, 7 indicating the occurrence of possible renal toxicity. Nevertheless, the elevated BUN levels eventually returned to baseline levels; however, creatinine levels were not affected by the EPHB4‐CAR‐T cell infusion (Figure 2b).…”

“…piggyBac transposase plasmid (pCMV‐PB) and piggyBac (PB) transposon plasmids used for the stable expression of the EPHB4‐specific CAR transgene (pIRII‐EPHB4‐CAR‐28z) have been described previously, 7 as was the pIRII‐dEPHB4‐CD80‐CD137L plasmid, which contained an extracellular, transmembrane, 20‐amino acid sequence of the cytoplasmic portion fused to the full‐length CD80 and 4‐1BBL (CD137L) with self‐cleaving 2A sequence, for the production of feeder cells (Supplementary figure 3). For the overexpression of human or cynomolgus EPHB4 , the full length of human EPHB4 () or cynomolgus EPHB4 () gene fused to puromycin resistance gene and green fluorescent protein (GFP) gene was artificially synthesised (Fasmac Inc., Kanagawa, Japan) and cloned into pIRII transposon plasmids (pIRII‐humanEPHB4‐GFP or pIRII‐cynoEPHB4‐GFP, respectively) (Supplementary figure 3).…”

“…The CAR transgene was transduced into fresh, unstimulated PBMCs using the PB transposon system, as per methods described previously 7,26 (Supplementary figure 4). Briefly, the pCMV‐PB (7.5 μg) and pIRII‐EPHB4‐CAR‐28z (7.5 μg) were introduced into 4 × 10 7 PBMCs using the MaxCyte ATX electroporator (MaxCyte) with the optimised protocol for the introduction of DNA plasmid into resting T cells (Protocol; RTC 14‐3).…”

“…We have developed piggyBac transposon (PB)‐mediated CAR‐T cells redirected towards the Ephrin type‐B receptor 4 (EPHB4), which is a tumor‐associated antigen expressed not only on various tumors but also on certain normal tissues at low to moderate levels 7 . We have utilised a natural ligand of EPHB4, Ephrin B2, as an antigen recognition site of the CAR molecule, and these CAR‐T cells effectively recognised and eliminated EPHB4‐positive tumor cells 7 . EPHB4 and other immunomodulatory molecules are highly conserved between cynomolgus macaques and humans with more than 99% overlap of amino acid sequences (Supplementary figure 1).…”