Novel Soluble Fc-Free Dimeric Proteins of CD19 and Immune Checkpoints for CAR-T Detection, Receptor/Ligand Binding and Antibody Characterization Assays

CD19-directed chimeric antigen receptor T (CAR-T) cell therapy has produced remarkable outcomes in patients with B-cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B-cell non-Hodgkin lymphomas. Evaluating CAR expression on transfected T cells in vitro is essential to test the CAR construct design. Monitoring CAR-T cell kinetics in vivo is critical to elucidate the relationship between CAR-T cell expansion and persistence, as well as therapeutic responses and treatment-related toxicities. The assays rely on a recombinant human CD19 protein recognized by the single-chain variable fragment (scFv) of the CAR construct. CD19 is a type 1 integral membrane protein and the extracellular domain is the targeting region of CD19-specific scFv. In this study, we aimed to establish a robust, sensitive, and broadly applicable flow cytometry–based method for CD19 CAR-T cell detection. A novel soluble Fc-free CD19 cis-dimer protein with a His-Tag at C-terminus (CD19-dimer-His) was developed using the full-length extracellular domain (ECD). The CD19-dimer-His is designed to perform better CAR-T detection by increasing the binding avidity to scFv and reducing the aggregation of CD19-ECD monomer and the FcR binding noise caused by CD19-ECD Fc-fusion protein. CD19 CAR detection was performed using CD19-dimer-His followed by an APC-conjugated anti-His-Tag antibody by flow cytometry. The results demonstrated excellent sensitivity and specificity for CD19-CAR transfected human CD8 and CD4 T cells with EC50 <2ug/ml and no binding to un-transfected T cells. Validation of CAR detection using the CD19-dimer-His method showed a strong positive correlation with a surrogate marker. CD19-dimer-His also proved to be more cost-effective. CD19-dimer-His staining can be readily incorporated into multicolor flow cytometry panels, enabling detailed phenotypic characterization of CAR-positive subpopulations. Importantly, this approach is applicable across different anti-CD19 CAR products and sample types. Overall, our findings demonstrate that CD19-dimer-His based flow cytometry is a reliable, robust, and widely applicable method for detection of CD19 CAR-T cells. Many immune checkpoint proteins, as type 1 integral membrane proteins, have been reported to form dimers on cell membrane, including PD1, PD-L1, CTLA-4, CD28, CD80, TIGIT, CD155 and Nectin-4. Dimerization is important for their bioactivities. To better mimic the native dimer conformation and quaternary structure, we developed a panel of immune checkpoint proteins as soluble ECD Fc-free cis-dimer proteins. These dimer immune checkpoint proteins can bind to their specific antibodies very potently. More interestingly, these dimeric proteins dramatically enhanced the receptor ligand binding. For example, CD155 or Nectin-4 binding to their receptor TIGIT increased by >50 fold compared to the respective monomer proteins, as measured by ELISA and surface plasmon resonance (SPR). Additionally, very potent binding activities between dimeric CTLA-4 or CD28 and CD80, as well as dimeric PD1 and PD-L1 interactions were also demonstrated. These findings support that the engineered novel immune checkpoint cis-dimer proteins mimic native conformations and can be used to perform in vitro assays to study immune checkpoint receptor and ligand interactions, and antibody screening/characterization in immuno-oncology research and anti-tumor antibody discovery.