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: Zeng Eric , Zhan Tailan , Wu Yang , Chen Meili , Wang Junzheng , Zhang Yun , Tu Xiaojie , Yang Shuai , Zhang Wang , Nesheiwat Tonia , Liang Frank , Tadagavadi Raghu , Chai Kathy , Geng Dong , Wu Shu 
Published: June 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).