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. Author manuscript; available in PMC: 2017 Mar 20.
Published in final edited form as: Immunity. 2016 Jun 21;44(6):1444–1454. doi: 10.1016/j.immuni.2016.05.014

Figure 1. 5E5 mAb and 5E5 CAR Specifically Recognize the Tn-MUC1, but Not the Peptide Alone.

Figure 1

(A) ELISA-peptide assay of glycopeptides OSM, aOSM, Muc1-9Tn, and unconjugated Muc1-60-mer with antibodies 3C9, 5F4, 3F1, and 5E5. The peptides were coated in the plate wells at concentrations of 2,000 ng/mL in bicarbonate buffer and halved in dilutions until 1 ng/mL. The bottom right panel used starting concentration of 2,000 ng/mL and diluted 8-fold until 233 fg/mL (noted with **). Statistical significance is calculated using an unpaired t test comparing 5F4 ELISA for Muc1-9Tn and Muc1-60-mer, 3F1 ELISA for OSM and aOSM, and 5E5 ELISA for Muc1-9Tn and Muc1-60-mer. p < 0.0001.

(B) Graphical representation of the chimeric antigen receptor developed using the 5E5 scFv, CD8α hinge, and transmembrane domain, 4-1BB and CD3zeta endodomain.

(C) Indirect ELISA assays quantifying the cytokine production in supernatant from 5E5 CAR and CD19 CAR T cells cultured on peptide-bound plates for 24 hr. Peptides were plated starting at concentrations of 2,000 ng/mL and diluted 256-fold until 1.82 ag/mL. 10 μg/mL of OKT3 mAb was used as a control T cell stimulant. Statistical significance of 5E5 CAR T cells on Muc1-9Tn versus Muc1-60-mer peptide is p = 0.0101 for IL-2 secretion and p = 0.0012 for IFN-γ secretion.