Department of Experimental Immunology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, Amsterdam, The Netherlands.
Department of Hematology, Cancer Center Amsterdam and Lymphoma and Myeloma Center Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands.
J Immunother Cancer. 2020 Jun;8(1). doi: 10.1136/jitc-2019-000218.
Bispecific antibodies are promising new therapeutics in B cell malignancies. Whether they lead to potent T cell activation despite described T cell dysfunction in chronic lymphocytic leukemia (CLL), and are able to effectively target high-risk or venetoclax-resistant samples, is currently unknown.
CD19 cell lines or primary (high-risk) CLL were cocultured in vitro with healthy donor (HD) or CLL-derived T cells in the presence of a CD3xCD19 dual affinity retargeting molecule (CD3xCD19 DART). Cell cytotoxicity, T cell activation, proliferation and effector molecule production were analyzed using flow cytometry.
Here, we report that a bispecific CD3xCD19 DART mediates efficient killing by HD T cells of CD19 cell-lines and primary CLL cells, regardless of immunoglobulin heavy chain variable region (IGHV) mutational status TP53 status or chemotherapy, ibrutinib or venetoclax sensitivity. Whereas TCR stimulation of CLL-derived T cells resulted in dysfunctional T cell activation and proliferation, treatment with CD3xCD19 DART led to a similar activation profile in CLL-derived and HD-derived T cells. Consistently, co-culture of CLL derived T cells with JeKo-1 or CLL cells in the presence of CD3xCD19 DART resulted in significant cytotoxicity by both CD4 and CD8 T cells. On stimulation of CLL cells with CD40L, CLL cells become resistant to the specific inhibitor of anti-apoptotic Bcl-2 protein venetoclax, due to upregulation of Bcl-2 family members such as Bcl-XL. Nevertheless, CD40L stimulated CLL cells were as efficiently lysed on CD3xCD19 DART treatment as unstimulated CLL cells. Further examination of the mechanism of CD3xCD19 DART mediated killing showed that lysis was dependent on granules, but was independent of BAX/BAK or caspase activity, indicating non-apoptotic cell death.
These data show that CD3xCD19 DART in CLL leads to robust T cell activation and lysis of high-risk venetoclax resistant CLL cells through a non-apoptotic mechanism.
双特异性抗体是 B 细胞恶性肿瘤中很有前途的新型治疗药物。尽管慢性淋巴细胞白血病（CLL）中描述了 T 细胞功能障碍，但它们是否能够导致有效的 T 细胞激活，并且能够有效靶向高危或 venetoclax 耐药样本，目前尚不清楚。
体外将 CD19 细胞系或原发性（高危）CLL 与健康供体（HD）或 CLL 衍生的 T 细胞共培养，同时存在 CD3xCD19 双特异性重定向分子（CD3xCD19 DART）。使用流式细胞术分析细胞毒性、T 细胞活化、增殖和效应分子产生。
在这里，我们报告称，双特异性 CD3xCD19 DART 介导 HD T 细胞有效杀伤 CD19 细胞系和原发性 CLL 细胞，无论免疫球蛋白重链可变区（IGHV）突变状态、TP53 状态、化疗、依鲁替尼或 venetoclax 敏感性如何。虽然 CLL 衍生 T 细胞的 TCR 刺激导致功能性 T 细胞活化和增殖受损，但 CD3xCD19 DART 治疗导致 CLL 衍生和 HD 衍生 T 细胞具有相似的活化谱。一致地，在 CD3xCD19 DART 存在下，共培养 CLL 衍生的 T 细胞和 JeKo-1 或 CLL 细胞导致 CD4 和 CD8 T 细胞产生显著的细胞毒性。在用 CD40L 刺激 CLL 细胞后，由于上调 Bcl-2 家族成员（如 Bcl-XL），CLL 细胞对 Bcl-2 蛋白抗凋亡抑制剂 venetoclax 变得耐药。然而，与未受刺激的 CLL 细胞一样，CD40L 刺激的 CLL 细胞在用 CD3xCD19 DART 处理时也被有效地溶解。进一步研究 CD3xCD19 DART 介导的杀伤的机制表明，裂解依赖于颗粒，但独立于 BAX/BAK 或半胱天冬酶活性，表明非凋亡细胞死亡。
这些数据表明，CD3xCD19 DART 在 CLL 中导致高危 venetoclax 耐药 CLL 细胞的强大 T 细胞活化和裂解通过非凋亡机制发生。
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