Biological Features and Therapeutic Potential of CD3 and MSLN

• Gene Information: CD3 is a multi-subunit T-cell receptor complex essential for T-cell signaling, while MSLN (Mesothelin) is a cell-surface tumor-associated antigen. Both are established targets for T-cell redirecting therapies.
• Protein Expression: CD3 is specifically expressed on T cells, whereas MSLN is normally expressed at low levels on mesothelial tissues and highly overexpressed in several solid tumors, including mesothelioma, pancreatic, and ovarian cancers.
• Signaling Pathway: CD3 engagement activates the TCR signaling cascade, leading to T-cell activation, proliferation, cytokine production, and cytotoxicity. MSLN binding provides tumor-cell targeting, enabling CD3-mediated T-cell redirection and tumor killing.
• Therapeutic Inhibition: CD3×MSLN bispecific antibodies recruit and activate T cells by simultaneously binding CD3 on T cells and MSLN on tumor cells, resulting in targeted killing of MSLN-positive cancer cells. Additional therapeutic strategies include monoclonal antibodies, CAR-T cells, antibody drug conjugates, and cancer vaccines targeting MSLN.

CD3EDG

• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hMSLN mice.

MSLN

• The mouse Msln gene sequences that encode the full length of coding region were replaced by human MSLN coding gene sequence in B-hCD3EDG/hMSLN mice.

Mouse and human CD3E expression analysis in splenocytes. Splenocytes and blood were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hMSLN mice (Female, 8-week-old, n = 1). CD3E expression on T cells was analyzed by flow cytometry using species-specific anti-CD3E antibodies (anti-human CD3E antibody, BD Horizon, 562426; anti-mouse CD3E antibody, Biolegend, 100312).

Major organs were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hMSLN mice (female, 8 weeks old), and analyzed by IHC using anti-mouse MSLN antibody (Abcam, ab213174) and anti-human MSLN antibody (Abcam, ab196235) . The arrow indicates tissue cells with positive MSLN staining (brown). "+" and "-" indicate positive and negative tissues, respectively.