Protein-Based TCR Knockdown for Off-Shell CAR T Cells

The patent replaces mechanical genome editing systems (ZFNs, TALENs, CRISPR/Cas) with a molecular biology-based approach using secreted blocking proteins. Instead of physically cutting and editing DNA sequences, the invention uses soluble proteins that bind to TCR components and prevent their surface expression through biochemical interference. This substitution eliminates the mechanical forces and enzymatic activities that cause off-target disruptions and translocations.

The patent introduces secreted blocking proteins as intermediaries between the CAR T-cell and the native TCR system. These proteins act as mediators that specifically bind to TCR components (such as CD3epsilon or zeta chains) and prevent their assembly or surface expression without directly contacting or modifying the T-cell genome. This intermediary mechanism achieves TCR knockdown while avoiding the harmful effects of direct genome editing.

The patent merges the TCR blocking function with the CAR expression system by co-delivering the secreted blocking protein construct alongside the CAR transgene. Both the CAR and the blocking protein are expressed from the same genetic modification event, ensuring that all CAR-expressing cells automatically produce the blocking protein that prevents native TCR surface expression. This merging eliminates the need for separate sorting steps to select for TCR-disrupted cells.

The patent implements preliminary action by having the secreted blocking protein continuously present in the culture medium during CAR T-cell production and activation. The blocking protein is secreted into the extracellular environment where it preemptively binds to TCR components before they can assemble or traffic to the cell surface, ensuring TCR knockdown is already in place before any sorting or clinical use is required.

The patent converts the harmful effect of HLA mismatch (which enables off-the-shelf production) into a benefit by using the resulting GVHD-risk scenario as the indication for TCR knockdown. The secreted blocking proteins specifically address the GVHD risk posed by HLA-mismatched donor cells while preserving the productivity benefits of off-the-shelf manufacturing. The HLA mismatch that would otherwise cause harm becomes the very condition that triggers the protective mechanism.

The patent extracts the harmful TCR-mediated GVHD activity from the CAR T-cell product while preserving the beneficial CAR-mediated anti-tumor function. By selectively blocking native TCR surface expression through secreted proteins while leaving CAR expression intact, the invention removes the harmful graft-versus-host component while maintaining the therapeutic anti-tumor component of the off-the-shelf CAR T-cell product.