CD3 humanized mouse models as validated tools to assess immune-related adverse events of T-cell engagers

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Perrine MARTIN-JEANTET, Gaëlle MARTIN, Angela PAPPALARDO, Florence RENART-DEPONTIEU, Patricia ISNARD-PETIT, Yacine CHERIFI, Fabiane SONEGO and Kader THIAM

T-cell engagers show high efficacy in B-cell malignancies. Immune-related adverse events (IrAEs), including cytokine release syndrome (CRS), are reported in patients due to on-target off-site effects of T-cell engagers (TCEs). Translational and predictive assays to assess IrAEs of TCEs are key to avoid pitfalls in clinical trials. Knockin humanized mouse models enable the assessment of human-target antibodies in a fully immunocompetent mouse. Indeed, immune responses in CD3 humanized model have been extensively evaluated, and humanization of CD3 has not impaired immune cell distribution. Furthermore, immunization studies showed that T–B cell cooperation is functional in these mice. CD3-TCR complex is also functional, as anti-human CD3 antibodies induce T-cell activation. Different classes of TCEs showed tumor cell killing ex vivo, as well as tumor growth inhibition in vivo. Here we report the use of CD3 humanized models to assess CRS ex vivo and in vivo.

Two CD3 humanized mice developed by Knockin were used for ex vivo and in vivo investigation of cytokine production. While one of the models is restricted to one clone of anti-human CD3, the other has been shown to bind several clones of anti-human CD3. Ex vivo, T cell-dependent cellular cytotoxicity assay was performed using different TCE concentrations on splenocytes from CD3 humanized mice. In vivo, CD3 humanized mice were treated with anti-human CD3, and cytokine release in blood, clinical monitoring, body weight and temperature were assessed.

Cytokine release in cytotoxicity assay showed that splenocytes from CD3 humanized mice produced IFN-γ, TNF-α, IL-6, IL-1β, IL-10 and IL-12p70 in a TCE concentration-dependent manner. In vivo, cytokine production was also observed in blood of CD3 humanized mice treated with anti-human CD3. Kinetics of secretion were dependent on the detected cytokine (IFN-γ, IFN-α, IL-6, IL-10) and chemokine (CXCL9, CXCL10, CCL3, CCL2, CCL4). An anti-CD3 dose effect on cytokine levels of secretion was demonstrated. In parallel, body temperature drops at early timepoints and an increase in late timepoints were observed upon treatment with anti-CD3.

Cytokine release and some clinical signs of CRS are reproduced in the two CD3 humanized models described here, validating their value to assess IrAEs of TCEs. Although the fully functional immune system is one of the main advantages of this model, assessment of mouse biology is a drawback. As a complementary approach, BRGSF-HIS mice, an immunodeficient mouse model reconstituted with human CD34⁺ cells, were shown to reproduce both clinical signs and cytokine production of human TCE-induced CRS.