Optimizing efficacy and safety of T cell bispecific antibodies: the interdependence of CD3 and tumor antigen binder affinities in FOLR1 and CEACAM5 2 + 1 TCBs

T cell bispecific antibodies (TCBs) are an emerging class of cancer therapy that are typically designed for high binding affinity to CD3 and tumor antigen (TA). Using this approach, TCBs have demonstrated significant clinical efficacy, but they have also elicited cytokine release syndrome and off-target on-tumor toxicities. CD3 affinity-attenuation has recently been reported as an approach to maintain efficacy while reducing cytokine release, but the interdependence of CD3 affinity with other factors is often not systematically explored. For this purpose, we generated a series of TCBs comprising CD3 binders with varying affinities and TA binders with either high or low affinities, utilizing FOLR1 and CEACAM5 as tumor targets. The CD3 binders were classified into high, intermediate, low, and very low affine binders based on affinity measurements as well as functionality. Depending on the target, different combinations of binders showed the most advantageous profile of tumor-cell killing while coupled with lower cytokine secretion. For instance, within the FOLR1-TCBs series, CD3^intermed exhibited a favorable profile compared to CD3^high <i>in vitro</i> using cocultures and <i>in vivo</i> using humanized mice. For CEACAM5-TCBs, CD3^low, along with CD3^intermed, showed a favorable profile compared to CD3^high in both <i>in vitro</i> and <i>in vivo</i> settings. Additionally, CD3^low avoided on-target, off-tumor toxicity and reduced cytokine release in transgenic mice. Taken together, reducing cytokine release while maintaining adequate efficacy is possible through CD3 binder affinity attenuation, but optimizing cytokine release profiles by CD3 binder affinity-attenuation is dependent on additional parameters.