Human c-SRC kinase (CSK) overexpression makes T cells dummy.

Area of Science:

• Immunology
• Cancer Therapy
• Cellular Engineering

Background:

• Adoptive cell therapy using T-cell receptor (TCR)-engineered T cells shows promise for cancer treatment.
• TCRs target specific peptide-MHC (pMHC) complexes, but cross-reactivity and off-target effects pose significant safety risks.
• Developing strategies to validate TCR safety before clinical application is crucial.

Purpose of the Study:

• To investigate a novel method for validating the safety of TCR-engineered T cells.
• To determine if blocking intracellular signaling downstream of TCR engagement can prevent T-cell effector functions without compromising target recognition.

Main Methods:

• Overexpressed c-SRC kinase (CSK), a negative regulator of LCK, in T cells engineered with a therapeutic TCR.
• Assessed the impact of CSK overexpression on TCR proximal signaling, ERK phosphorylation, and downstream effector functions (IL-2 release, degranulation, target cell killing).
• Evaluated TCR expression, pMHC binding, and trogocytosis in CSK-overexpressing T cells.

Main Results:

• CSK overexpression inhibited anti-CD3/anti-CD28-induced ERK phosphorylation but did not affect TCR proximal signaling.
• CSK co-expression with a therapeutic TCR blocked pMHC-induced ERK phosphorylation and downstream effector functions, including target cell killing.
• TCR expression, pMHC recognition, and trogocytosis remained unaffected, indicating preserved target binding.

Conclusions:

• Co-expression of CSK with therapeutic TCRs creates
• dummy T cells
• that retain target recognition but are functionally inactivated.
• This approach offers a promising safety validation strategy for TCR-engineered T cells prior to clinical use, mitigating risks associated with off-target effects.