Preclinical genO‑hCD3ε mouse model

Validation data

• CD3ε humanization does not alter the thymocytes’ distribution along the maturation process and its expression on T cells

  (A) Freshly isolated thymocytes from wild-type (WT) and humanized homozygous CD3ε (genO‑hCD3ε) mice were analyzed by flow cytometry and gated on single live CD4⁺CD8⁺ (DP), CD4⁺CD8^- (CD4 SP), CD4^-CD8⁺ (CD8 SP), CD4^-CD8^- (DN)

  (B) Freshly isolated splenocytes from wild-type (WT) and humanized homozygous CD3ε (genO‑hCD3ε) mice were analyzed by flow cytometry and T-cell subsets were gated on single live TCRβ⁺, TCRɣẟ⁺, CD4^-CD8⁺ (CD8), CD4⁺CD8^-FoxP3^- (Conv CD4), CD4⁺CD8^-FoxP3⁺ (Treg)

  

• Anti human-CD3 SP34 clone specifically binds to hCD3ε and induces T-cell activation in genO‑hCD3ε mice

  (A) Freshly isolated splenocytes from wild-type (WT) and humanized homozygous CD3ε (genO‑hCD3ε) mice were incubated with anti-human CD3 antibody (clone SP34) and were analyzed by flow cytometry and gated on single live CD4⁺CD8^-FoxP3^- (Conv CD4), CD4^-CD8⁺ (CD8), CD4⁺CD8^-FoxP3⁺ (Treg)

  (B) Isolated T cells were labelled with CTV and activated with various concentrations of coated anti human CD3 (SP34) for 72h

  (C) Isolated T cells were activated with various concentration of coated anti human CD3 (SP34) for 72h. IFN-γ level in supernatant was quantified by ELISA

  

• A T-cell engager targeting CD3 and a tumoral antigen, showed a dose-dependent anti-tumor effect in vivo

  T-cell engagers efficacy assessment. (A) Experimental design for in vivo experiments. Tumor growth curves in (B) wild-type and (C) genO‑hCD3ε mice.