Extended Data Fig. 1: Covalently linked blocking-zipper inhibits extracellular leucine zipper pairing.
From: Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors
a, Zip-sorting system vector maps. b, Vector map and diagram for EE-Thy1.1-P2A–BFP vector. c, Flow cytometry analysis of C1498 cells co-transduced with FLAG–RR-CBR–GFP and EE-Thy1.1-P2A–BFP vectors. Arrows depict combined intracellular + extracellular pairing (orange) and extracellular pairing (blue). d, Comparison of FLAG–zipper staining on dual-transduced (EGFP+ BFP+) and capture-zipper single-transduced (EGFP− BFP+) C1498 cells from four independent biological replicate co-transductions with FLAG–RR-CBR–GFP and EE-Thy1.1-P2A–BFP vectors as in panel c. Data are mean ± SEM of replicate samples. e, Maps of non-blocked EE-Thy1.1-P2A–BFP and blocked RR-EE-Thy1.1-P2A–BFP capture-zipper vectors. f, Diagrams and flow cytometry analysis of co-culture of single-transduced FLAG–zipper-secreting FLAG–RR-CBR–GFP C1498 with C1498 cells single-transduced with either (top) non-blocked EE-Thy1.1-P2A–BFP or (bottom) blocked RR-EE-Thy1.1-P2A–BFP capture-zipper vectors at depicted cell ratios for 48 h. FLAG staining represents extracellular pairing of FLAG–RR zippers on single-transduced capture-zipper+ C1498 cells, which capture FLAG–RR zippers secreted into the media by single-transduced FLAG–RR-secreting C1498 cells. g, FLAG–RR zipper surface expression on C1498 cells expressing blocked or non-blocked capture zippers depicted in panel f. n = 1 transduction for each cell line and n = 3 co-culture experiments. Data are mean ± SEM of triplicate samples. Error bars were too small to depict. h, Flow cytometry contour plots and histograms of unsorted, mixed populations of C1498 cells co-transduced with FLAG–RR-CBR–GFP and RR-EE-Thy1.1-P2A–BFP vector variants with capture zippers containing different repulsive mutations in blocking-zippers (See Supplementary Table 1). “EE” blocking-zipper is engineered to be fully repulsive against EE capture-zipper and maximally attractive towards the FLAG–RR zipper. Remaining mutants contain varying numbers of repulsive mutations “2 or 3” in the N-terminal “N” or middle “M” regions of the blocking-zipper. Representative of n = 3 separate transductions. i, Diagram depicting predicted effect of repulsive amino acid substitution on zipper binding affinity. j, Maps for non-blocked and blocked EGFRt-based capture-zipper vectors. k, Flow cytometry analysis of C1498 cells dual-transduced with FLAG–RR-CBR–GFP and either (top) non-blocked EE-EGFRt-P2A–BFP or (bottom) blocked capture-zipper RR-EE-EGFRt-BFP. l, Zip-sort of FLAG–RR-CBR–GFP/RR-EE-EGFRt-P2A–BFP C1498 cells. Representative of n = 2 transductions and Zip-sorts.
