CD19 Fc-Fusion Protein for Detection of Cells Expressing Anti-CD19 Chimeric Antigen Receptors.

Abstract 3756

Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain that is capable of activating T cells. CARs are displayed on the surface of transduced cells, performing non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of modified mature T lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. We developed a specific reagent for the detection of anti-CD19 CAR expressed on cells, a soluble fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (19sIg). Genes encoding 19sIg fusion proteins were constructed by fusing either Exons 1 to 3 (19sIg1-3) or Exons 1 to 4 (19sIg1-4) of the human CD19 cDNA to a human IgG₁Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The 19sIg proteins were produced from 293T cells by stable lentiviral vector transduction and purification from culture medium. ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule 19sIg1-4, but no binding to 19sIg1-3. Conjugation of the 19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays. Large-scale production and stable conjugation of this fusion molecule will provide a useful reagent to detect anti-CD19 CAR derived from any monoclonal antibody.