Preclinical analysis of an autologous CD4-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy for relapsed or refractory peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma (CTCL).
Authors: Eric Zeng, Tailan Zhan, Yang Wu, Meili Chen, Junzheng Wang, Yun Zhang, Xiaojie Tu, Shuai Yang, Wang Zhang, Tonia Nesheiwat, Frank Liang, Raghu Tadagavadi, Kathy Chai, Dong Geng, Shu Wu
Journal: Journal of Clinical Oncology (2021)

e14510 Background: CD4 is highly and uniformly expressed T-cell lymphomas (TCL) including PTCL and CTCL, suggesting its potential as a surface target for CAR-T therapy. However, there is a significant risk of potential antigen masking by CAR introduced into tumor cells, which thereby leads to escape of tumor cells from recognition by CAR-T cells. For example, Ruella M et al ( Nat Med, 2018) reported that contaminating malignant B cells transduced with anti-CD19 CAR (called CAR-B cells) in the manufacturing process led to the CD19 antigen masking by CAR molecules. Hence, CAR-B tumor cells could not be recognized by CAR-T cells, which resulted in relapse of CAR expressing B lymphoma cells. A similar antigen masking effect might occur for the anti-CD4 CAR-T product. Methods: To identify single-chain variable fragment (scFv) without antigen-masking effect, large panels of fully human monoclonal antibodies were converted to format of CAR modality with the scFv-4-1BB-CD3z structure and introduced into CD4 + and CD8 + T cells. After lentiviral transduction, residual CD4 + T cells were quantified and the CARs with complete elimination of CD4 + T cells were selected for further validation. The selected CAR constructs were then introduced into CD4 + TCL cells HH (CAR-HH cells) to mimic the potential risk of introducing CAR into contaminating malignant T cells in the manufacturing process. Thereafter, CAR-HH cells were subject to in vitro killing assay by LB1901. Last, identified CAR constructs were further tested for in vitro and in vivo anti-tumor efficacy and off-target binding and killing. Results: Introduction of LB1901 CAR into CD4 + and CD8 + T cells led to complete elimination of CD4 + T cells, suggesting no masking effect of CAR on CD4 antigen. Furthermore, the introduction of CAR into CD4 + HH cells did not protect HH cells from being recognized and eliminated by LB1901, further confirming that the CAR modality of LB1901 does not mask CD4 antigen. An in vivo anti-tumor efficacy study showed that LB1901 exhibited dose-dependent anti-tumor activity without significant adverse effect. As low as 0.3 million CAR + cells completely suppressed tumor growth, suggesting the potent anti-tumor activity by LB1901. Immunohistochemical analysis of normal tissues with LB1901 scFV binder showed no off-target binding. Furthermore, no killing toward CD4 - cell lines and primary cells derived from vital organs or antigen-independent cytokine release was observed in vitro. Conclusions: Altogether, the in vitro and in vivo studies showed that LB1901 did not mask the CD4 antigen but exhibited potent anti-tumor activity without off-target effects. A phase 1 study of LB1901 CAR-T in patients with relapsed or refractory PTCL or CTCL is ongoing in the US to assess the safety and tolerability of LB1901 CAR-T (NCT04712864).