Anti-CD3 Bi-Fab crosslinking of T cells enables their fratricide by activation induced cell death (AICD) and cytotoxicity

Abstract We showed in the past anti-CD3 Fabs can form stable bivalent non-covalent dimers we called Bi-Fabs, which appear stimulatory to T cells according to the up-regulation of the early activation marker CD69. Now we show that incubation of naïve T cells with anti-CD3 Bi-Fabs in the presence of proper co-stimulation results in their robust stimulation in comparison to mAb and F(ab’)2 species with same specificity. Nonetheless, anti-CD3 Bi-Fab treatment leads to abortive T cell proliferation with impaired accumulation of divided T cells. After detailed studies to dissect the mechanisms of T cell death enabled by Bi-Fab molecules in comparison with anti-CD3 mAb and F(ab′)2 recognizing the same epitope, we observed that anti-CD3 Bi-Fabs promote T cell fratricidal killing via activation induced cell death (AICD) and CD8 cytolytic activity (CTL) more efficiently than the mAb and F(ab′)2 counterparts. While low resolution structural studies indicate that Bi-Fab and F(ab’)2 adopt similar shapes, molecular dynamic simulation shows that F(ab’)2 is a highly flexible molecule, whereas Bi-Fabs are more rigid and of linear conformation, sampling a smaller range of Fab-Fab angles than F(ab’)2. We hypothesize anti-CD3 Bi-Fab is superior as T cell killer stimulatory modality over mAb and F(ab’)2 of equal specificity due to Bi-Fab’s greater rigidity when crosslinking in trans TCR/CD3 molecules located in neighboring T cells. These findings may be applicable into the design of Ig-based immunotherapeutic drugs targeting the TCR/CD3 complex. Supported by MU start-up funds