Abstract
BackgroundChimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform 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 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.MethodsA specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) 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 CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium.ResultsELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining.ConclusionsThis fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.
Highlights
Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells
Using the antigen specificity of CAR as the determinant of a more specific reagent for the detection of the CD19-specific CAR, we developed a CD19/Fragment crystallizable region of an antibody (Fc) molecule that can be labeled for its use primarily as a reagent in flow cytometry studies
The fusion proteins containing the CD19 extracellular domain were designed in two versions, fusing exons 1–3 (CD19sIg1-3) and exons 1–4 (CD19sIg1-4), to human IgG1 fragment (Figure 1A); this strategy was chosen as previously published data suggested that the addition of exon 4 decreased protein secretion
Summary
Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. 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. Detection of CAR-bearing cells has usually been performed by flow cytometry, with the use of antibodies against the extracellular structure of the molecule, such as the hinge (using an anti-IgG Fc antibody or F(ab’) fragment) or the antigen-binding domains (as in the case of the use of an anti-idiotypic antibody). Fusion to the Fc domain has been used to allow secretion of peptide sequences, with enhanced solubility and stability, and a fusion protein of murine extracellular CD19 and Fc domain has been previously described [6]
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