# Rigid crosslinking of the CD3 complex leads to superior T cell stimulation

## TL;DR

A new study shows that rigid crosslinking of the CD3 complex on T cells leads to stronger stimulation and could improve the effectiveness of certain drugs.

## Contribution

The study reveals that rigid crosslinking of CD3 by Bi-Fab dimers enhances T cell stimulation compared to other formats.

## Key findings

- Anti-CD3 Bi-Fab causes more T cell fratricide than F(ab’)2 or mAb.
- Molecular simulations show Bi-Fab crosslinks CD3 more rigidly than F(ab’)2.
- Rigid crosslinking of CD3 may improve T cell effector function in drug design.

## Abstract

Functionally bivalent non-covalent Fab dimers (Bi-Fabs) specific for the TCR/CD3 complex promote CD3 signaling on T cells. While comparing functional responses to stimulation with Bi-Fab, F(ab’)2 or mAb specific for the same CD3 epitope, we observed fratricide requiring anti-CD3 bridging of adjacent T cells. Surprisingly, anti-CD3 Bi-Fab ranked first in fratricide potency, followed by anti-CD3 F(ab’)2 and anti-CD3 mAb. Low resolution structural studies revealed anti-CD3 Bi-Fabs and F(ab’)2 adopt similar global shapes with CD3-binding sites oriented outward. However, under molecular dynamic simulations, anti-CD3 Bi-Fabs crosslinked CD3 more rigidly than F(ab’)2. Furthermore, molecular modelling of Bi-Fab and F(ab’)2 binding to CD3 predicted crosslinking of T cell antigen receptors located in opposing plasma membrane domains, a feature fitting with T cell fratricide observed. Thus, increasing rigidity of Fab-CD3 crosslinking between opposing effector-target pairs may result in stronger T cell effector function. These findings could guide improving clinical performance of bi-specific anti-CD3 drugs.