Muthuvel Muthuganesh, Ganapathy Thamizhselvi, Spencer Trent, Raikar Sunil S, Thangavel Saravanabhavan, Srivastava Alok, Martin Sunil
Laboratory of Synthetic Immunology, Cancer Research Division, Biotechnology Research Innovation Council- Rajiv Gandhi Centre for Biotechnology (BRIC - RGCB), Department of Biotechnology, Thiruvananthapuram, India.
Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India.
Front Immunol. 2025 May 9;16:1545549. doi: 10.3389/fimmu.2025.1545549. eCollection 2025.
Despite the curative potential, high cost of manufacturing and the toxicities limits the wider access of Chimeric Antigen Receptor (CAR) T cell therapy in global medicine. CARs are modular synthetic antigen receptors integrating the single-chain variable fragment (scFv) of an immunoglobulin molecule to the TCR signaling. CARs allow HLA independent, T cell mediated destruction of tumor cells independent of tumor associated-HLA downregulation and survive within the patient as 'living drug.' Here we report a safer approach for engineering alpha beta T cells with anti- CD19-CD28ζ CAR using self-inactivating (SIN) lentiviral vectors for adoptive immunotherapy.
αβ T cells from the peripheral blood (PB) were lentivirally transduced with CAR construct containing hinge domain from CD8α, transmembrane and co-stimulatory domain from CD28 along with signaling domain from CD3ζ and driven by human UBC promoter. The cells were pre-stimulated through CD3/CD28 beads before lentiviral transduction. Transduction efficiency, fold expansion and phenotype were monitored for the CAR T cells expanded for 10-12 days. The antigen-specific tumor-killing capacity of CD19 CAR T cells was assessed against a standard CD19 expressing NALM6 cell lines with a flow cytometry-based assay optimized in the lab.
We have generated high titer lentiviral vectors of CAR with a titer of 9.85 ± 2.2×10 TU/ml (mean ± SEM; n=9) generating a transduction efficiency of 27.57 ± 2.4%. (n=7) at an MOI of 10 in total T cells. The product got higher CD8+ to CD4+ CAR T cell ratio with preponderance of an effector memory phenotype on day 07 and day 12. The CAR-T cells expanded (148.4 ± 29 fold; n=7) in serum free media with very high viability (87.8 ± 2.2%; n=7) on day 12. The antitumor functions of CD19 CAR T cells as gauged against percentage lysis of NALM6 cells at a 1:1 ratio is 27.68 ± 6.87% drawing up to the release criteria. CAR T cells produced IFNγ (11.23 ± 1.5%; n=6) and degranulation marker CD107α (34.82 ± 2.08%; n=5) in an antigen-specific manner. Furthermore, the sequences of WPRE, GFP, and P2A were removed from the CAR construct to enhance safety. These CAR T cells expanded up to 21.7 ± 5.53 fold with 82.7±5.43% viability (n=4).
We have generated, validated, and characterized a reproducible indigenous workflow for generating anti-CD19 CAR T cells This approach can be used for targeting cancer and autoimmune diseases in which CD19+ B lineage cells cause host damage.
尽管嵌合抗原受体（CAR）T细胞疗法具有治愈潜力，但高昂的制造成本和毒性限制了其在全球医学领域的广泛应用。CAR是一种模块化的合成抗原受体，它将免疫球蛋白分子的单链可变片段（scFv）整合到TCR信号传导中。CAR允许不依赖HLA，通过T细胞介导破坏肿瘤细胞，而不依赖肿瘤相关HLA的下调，并作为“活药物”在患者体内存活。在此，我们报告一种使用自失活（SIN）慢病毒载体构建抗CD19-CD28ζ CAR工程化αβ T细胞的更安全方法，用于过继性免疫治疗。
从外周血（PB）中分离出的αβ T细胞用含有来自CD8α的铰链区、来自CD28的跨膜区和共刺激结构域以及来自CD3ζ的信号结构域的CAR构建体进行慢病毒转导，并由人UBC启动子驱动。在慢病毒转导之前，通过CD3/CD28磁珠对细胞进行预刺激。对扩增10-12天的CAR T细胞监测其转导效率、扩增倍数和表型。使用实验室优化的基于流式细胞术的检测方法，评估CD19 CAR T细胞对表达标准CD19的NALM6细胞系的抗原特异性肿瘤杀伤能力。
我们已产生高滴度的CAR慢病毒载体，滴度为9.85±2.2×10 TU/ml（平均值±标准误；n = 9），在总T细胞中MOI为10时转导效率为27.57±2.4%（n = 7）。在第7天和第12天，该产物具有更高的CD8 +与CD4 + CAR T细胞比例，且效应记忆表型占优势。在无血清培养基中，CAR-T细胞在第12天扩增（148.4±29倍；n = 7），活力非常高（87.8±2.2%；n = 7）。以1:1比例针对NALM6细胞的裂解百分比衡量，CD19 CAR T细胞的抗肿瘤功能为27.68±6.87%，达到释放标准。CAR T细胞以抗原特异性方式产生IFNγ（11.23±1.5%；n = 6）和脱颗粒标记物CD107α（34.82±2.08%；n = 5）。此外，从CAR构建体中去除了WPRE、GFP和P2A的序列以提高安全性。这些CAR T细胞扩增至21.7±5.53倍，活力为82.7±5.43%（n = 4）。
我们已经建立、验证并表征了一种可重复的本土工作流程，用于生成抗CD19 CAR T细胞。这种方法可用于靶向CD19 + B淋巴细胞系细胞导致宿主损伤的癌症和自身免疫性疾病。
