Chimeric Antigen Receptor Induced Antigen Loss Protects CD5 CART Cells from Fratricide Without Compromising On-Target Cytotoxicity
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Western Blot original images for Figures 1C, 1J, S1D, 2F,
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Figure 1C: To evaluate the presence of CD5 protein, non-transduced or CD19.CAR/CD5.CAR/∆CD5.CAR-expressing wild-type activated T-cells (ATCs) were probed using Abcam's anti-CD5 protein antibody (clone: EP2952) compared to CD5 knocked-out ATCs as negative control. Figure 1J: ∆CD19.CAR T-cells and ∆CD5.CAR T-cells expressing a dual-tagged myc.CD5.FLAG variant were probed for myc-tag or FLAG-tag to determine whether the entire CD5 protein was degraded. Myc-tag was on the N-terminus of CD5 while FLAG-tag was on the C-terminus. .: Construct #395c: Bi-cistronic expression of ∆CD5.CAR and dual-tagged myc.CD5.FLAG variant. Native CD5 was CRISPR knocked-out. .: Construct #396c: Bi-cistronic expression of ∆CD19.CAR and dual-tagged myc.CD5.FLAG variant. Native CD5 was CRISPR knocked-out. Figure S1D: ∆CD19.CAR T-cells and ∆CD5.CAR T-cells with transgenic over-expressing either full-length CD5 or truncated CD5 lacking its cytoplasmic signaling domains were probed for CD5 (Abcam clone: EP2952) to determine whether CD5 signaling was required for CD5.CAR-mediated loss of CD5 in cis. .: Construct #396: Bi-cistronic expression of ∆CD19.CAR and full-length CD5. Native CD5 was CRISPR knocked-out. .: Construct #395: Bi-cistronic expression of ∆CD5.CAR and full-length CD5. Native CD5 was CRISPR knocked-out. .: Construct #396b: Bi-cistronic expression of ∆CD19.CAR and truncated CD5 lacking all cytoplasmic signaling domains. Native CD5 was CRISPR knocked-out. .: Construct #395b: Bi-cistronic expression of ∆CD5.CAR and truncated CD5 lacking all cytoplasmic signaling domains. Native CD5 was CRISPR knocked-out. Figure 2F: ATCs were co-cultured with either CD19.CAR T-cells or ∆CD5.CAR T-cells to induce CD5.CART-mediated trans-removal of CD5 and subsequently analyzed for residual CD5 protein [Main Figure: Novus Biologicals (clone: H65); Additionally attached for reference: Abcam (clone EP2952)]. All data was acquired on an LICOR Odyssey cLX and probed using LICOR IR-Dyes 680/800 (Goat anti-Mouse IRDye 680RD Cat#925-68070, Goat anti-Rabbit IRDye 800CW Cat#925-32211).