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Abstract
Background CD19-targeted therapies, such as chimeric antigen receptor (CAR)-T or T-cell engagers (TCE), have been approved for the treatment of B cell malignancies. By depleting autoreactive B cells, CD19-targeted CAR-T have shown early but encouraging efficacy in treating systemic lupus erythematosus (SLE) patients. However, the clinical application of TCE continues to be greatly hindered by the unfavorable pharmacokinetics and toxicity associated with cytokine release syndrome. Here we developed a ‘2+1’ CD19 x CD3 TCE, ATG-201, which effectively depletes B cells with minimal risk of CRS. It demonstrates potent anti-disease in vivo efficacy in mouse models for lymphoma and autoimmune diseases.
Methods ATG-201 was constructed by introducing a high affinity, novel conformational epitope-targeted anti-CD3 single chain fragment variable (scFv) to the hinge region of one of the heavy chains of a CD19 monoclonal antibody. It was evaluated in a series of preclinical studies for binding affinity, T cell activation, T cell dependent cytotoxicity (TDCC), B cell depletion and cytokine release. The in vivo anti-lymphoma efficacy was evaluated in PBMC-humanized immunodeficient NDG mice engrafted with Raji lymphoma cells. The in vivo efficacy of ATG-201 in treating autoimmune diseases were investigated in a mouse MS model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE) and a spontaneous SLE mouse model using a mouse surrogate TCE with the same format.
Results ATG-201 bound to CD19+ Raji, NALM6 and primary B cells with an EC50 of 1.3 nM, 0.6 nM and 0.4 nM respectively. The CD3 binding site of ATG-201 is concealed by the CD19 Fab arm before binding to CD19, due to the steric hindrance. Therefore, ATG-201 demonstrated limited binding capability to CD3+ cells before CD19 crosslinking. It activated T cells only in the presence of CD19+ cells. In vitro, ATG-201 resulted in a dose-dependent, potent depletion of B cells in human PBMCs, with an IC50 of 76.1 pM. However, it induced significantly lower cytokine release than benchmarks (figure 1). ATG-201 showed potent anti-lymphoma activity in PBMC-humanized Raji xenograft model. The mice surrogated CD19xCD3 TCE demonstrated enhanced in vivo efficacy than CD20xCD3 bispecific antibody in MOG-EAE model and MRL-lpr mouse SLE model, with deeper B cell depletion observed.
Conclusions ATG-201 demonstrates CD19-dependent CD3 binding and activation with low risk of CRS. It effectively depletes B cells in vitro and in vivo, which provides potential in the treatment of B cell malignancies and B cell related autoimmune diseases.
ATG-201 effectively depleted B cells with low risk of CRS. A. Design of ATG-201. B. Binding of ATG-201 to Raji, NALM6 and primary B cells. C. Binding of ATG-201 and benchmark (BMK, a BITE-HLE CD19xCD3 TCE) to CD3+ Jurkat T cells. D. Raji or NALM6 cells were co-cultured with isolated human T cells at an E:T ratio of 5:1 in the presence of ATG-201 for 24 h. T cell dependent cellular cytotoxicity (TDCC) were assessed by flow cytometry E. PBMC isolated from healthy donors was incubated with ATG-201 at the indicated concentrations for 24 hours. Primary B cell depletion (cytotoxicity%) was assessed by flow cytometry. F. B cell depletion and concentration of the indicated cytokines (G) in the supernatant of isolated human PBMC incubated with ATG-201 or Benchmarks (20 nM) for 48 hours
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