Rankin Alexander W, Pham-Danis Catherine, Novak Amanda J, Danis Etienne, Fry Terry J, Kohler M Eric
Division of Hematology, Oncology, and Blood and Marrow Transplant, Nationwide Children's Hospital, Columbus, Ohio, USA.
Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA.
J Immunother Cancer. 2025 Nov 13;13(11):e011971. doi: 10.1136/jitc-2025-011971.
Chimeric antigen receptor (CAR) T-cell therapy is effective in treating B-cell malignancies, however relapse due to lack of CAR persistence and antigen-modulated escape remains common. Multiple strategies to simultaneously target CD19 and CD22 have been able to reduce antigen-modulated escape but not completely eliminate relapse. A bicistronic CAR construct consisting of a CD19 CAR incorporating the CD28 costimulatory domain paired with a CD22 CAR incorporating a 4-1BB costimulatory domain (CD19xCD22) demonstrated superior preclinical activity compared with other configurations and is currently under clinical investigation (NCT05098613, NCT05442515, NCT06559189). We hypothesized that simultaneous activation of CD28-containing and 4-1BB-containing CAR molecules not only allows for targeting of both antigens but creates a unique signal which enhances CAR T-cell function and efficacy.
We tested CD19xCD22 CAR T-cells generated from primary human T-cells against NALM6 with wild-type expression of CD19 and CD22 (CD19+/CD22+) or CRISPR/Cas9 knockout of one or both antigens (CD19+/CD22-, CD19-/CD22+, CD19-/CD22-) to interrogate the effect of dual-CAR stimulation on T-cell function, signaling, and in vivo efficacy in xenograft models.
In vitro proliferation and cytokine production of CD19xCD22 CAR T-cells were primarily driven by activation of the CD19-28z CAR, however the CD22-BBz CAR drove equivalent cytotoxicity. Dual-CAR stimulation of CD19xCD22 CAR T-cells decreased leukemia relapse and improved survival in xenograft models. This increase in efficacy was associated with increased signaling through the phospholipase C-gamma 1 and nuclear factor of activated T-cells pathway after dual-CAR stimulation. Dual-CAR stimulation also led to decreased expression of markers associated with T-cell exhaustion in persistent CD19xCD22 CAR T-cells.
Stimulation of both CAR molecules in a CD19xCD22 bicistronic CAR construct impacts downstream signaling events within the CAR T-cell and subsequently drives a more efficacious in vivo response with evidence of decreased exhaustion in persisting cells. These data suggest that bicistronic CAR platforms have the potential to not only target two antigens to prevent antigen-modulated escape but can be engineered to improve multiple facets of CAR T-cell biology, such as mitigating exhaustion, thereby overcoming multiple mechanisms known to drive relapse in current CAR T-cell therapies.
嵌合抗原受体（CAR）T细胞疗法在治疗B细胞恶性肿瘤方面有效，然而，由于缺乏CAR持久性和抗原调节逃逸导致的复发仍然很常见。多种同时靶向CD19和CD22的策略能够减少抗原调节逃逸，但不能完全消除复发。一种双顺反子CAR构建体，由包含CD28共刺激结构域的CD19 CAR与包含4-1BB共刺激结构域的CD22 CAR配对（CD19xCD22），与其他配置相比，在临床前显示出卓越的活性，目前正在进行临床研究（NCT05098613、NCT05442515、NCT06559189）。我们假设同时激活含CD28和含4-1BB的CAR分子不仅能够靶向两种抗原，还能产生独特信号，增强CAR T细胞功能和疗效。
我们测试了从原代人T细胞产生的CD19xCD22 CAR T细胞，针对具有野生型CD19和CD22表达（CD19+/CD22+）或一种或两种抗原经CRISPR/Cas9敲除（CD19+/CD22-、CD19-/CD22+、CD19-/CD22-）的NALM6，以探究双CAR刺激对T细胞功能、信号传导及异种移植模型体内疗效的影响。
CD19xCD22 CAR T细胞的体外增殖和细胞因子产生主要由CD19-28z CAR的激活驱动，然而CD22-BBz CAR具有同等的细胞毒性。CD19xCD22 CAR T细胞的双CAR刺激降低了异种移植模型中的白血病复发率并改善了生存率。这种疗效的提高与双CAR刺激后通过磷脂酶C-γ1和活化T细胞核因子途径的信号增强有关。双CAR刺激还导致持续存在的CD19xCD22 CAR T细胞中与T细胞耗竭相关标志物的表达降低。
在CD19xCD22双顺反子CAR构建体中刺激两种CAR分子会影响CAR T细胞内的下游信号事件，随后驱动更有效的体内反应，且有证据表明持续存在的细胞中耗竭减少。这些数据表明，双顺反子CAR平台不仅有潜力靶向两种抗原以防止抗原调节逃逸，还可进行工程改造以改善CAR T细胞生物学的多个方面，如减轻耗竭，从而克服目前CAR T细胞疗法中已知的多种导致复发的机制。
