Highly sensitive and antigen-specific detection of CAR expression cells using novel CD19, BCMA, CD20 and TROP2 soluble fusion proteins by flow cytometry

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Conigen Bioscience research poster describing sensitive, antigen-specific detection of CAR-expressing cells using soluble CD19, BCMA, CD20, and TROP2 fusion proteins. Diagrams and flow cytometry results illustrate how these reagents bind CARs to identify cells displaying antigen-binding receptors.
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Abstract

Introduction: As CAR-T therapies advance through development, analytical methods must provide meaningful characterization of product identity and function. While FDA guidance emphasizes direct CAR detection for identity testing, anti-idiotype reagents are not always available, and surrogate methods remain common during early development. Recombinant bioactive target proteins offer attractive antigen-specific CAR-T detection, which enables direct flow cytometric detection of CAR expression while simultaneously confirming target-binding specificity and affinity in a single, biologically relevant measurement.

Methods: To further advance the anti-specific CAR-T evaluation, we have developed novel cell surface fusion proteins including Fc-free dimeric CD19 and TROP2, tetrameric BCMA, and scaffold CD20 extracellular loop 2 (ECL2) proteins for sensitive, and specific CAR detection by flow cytometry.

Results: The results demonstrated that the CD19-dimer could potently bind to the FDA approved therapeutic antibody clones including 1) Lisocabtagene maraleucel CAR-T therapeutics, FMC63; 2) Loncastuximab antibody conjugate drug (ADC); 3) Blinatumomab bispecific therapeutic antibody, clone HD37; and 4) Tafasitamab therapeutic monoclonal antibody, clone 4G7. It had excellent sensitivity and specificity for CD19-CAR mRNA transfected human CD8 and CD4 T cells with EC50 <2ug/ml and no binding to un-transfected T cells. The CD19-specific CAR T detection is more relevant to tumor-cell killing although the linker-detected CAR expression levels are very similar. Validation of CAR detection using the CD19-dimer method showed a strong positive correlation with a surrogate marker. Similar results are achieved using BCMA, TROP2 or CD20 fusion proteins for specific antibody binding and CAR detections on transfected HEK293 cells. 

Conclusions: The novel CD19, BCMA, CD20, and TROP2 soluble proteins preserve the desired conformations and epitopes binding to specific antibodies and CAR expressing cells.  Antigen-specific CAR binding is more relevant to CAR T function. These proteins can be readily incorporated into multicolor flow cytometry panels, enabling detailed phenotypic characterization of antigen-specific CAR-positive subpopulations through CAR design and development.

Conclusions

  • Novel Fc-free novel cell surface antigen-based fusion proteins are designed to mimic the natural structure and epitopes for specific antibody and ligand binding, and sensitive CAR recognition.
  • CD19 Fc-free homodimer shows strong binding to FDA approved therapeutic antibodies by ELISA; and specifically measures the CD19 CAR-T cells using flow cytometry.
  • CD19-specific CAR detection is more relevant to CD8+ CAR T cell function measured as tumor-cell killing
  • CD20-ECL2 scaffold protein can specifically and potently bind therapeutic antibody Rituximab and detect anti-CD20 CAR expression on transfected HEK293 cells.
  • BCMA-tetramer protein can specifically and potently bind its natural ligand BAFF, therapeutic antibody Belantamab and measure anti-BCMA CAR expression on transfected HEK293 cells.
  • Native-like TROP2 dimer antigen-specific detection reveals differences in differently designed CAR binding competence that linker staining alone cannot resolve.
  • Antigen-based CAR detections are specific and can reveals the differences in CAR binding competence of differently designed CAR Constructs that anti-linker staining alone cannot.
  • These novel fusion proteins provide a reliable, robust, and widely applicable method for detection of antigen-specific CAR recognition.