Fig. 1. CD45 is a universal blood cancer antigen that can be targeted with CD45 knockout CAR-T cells.

(A) Schematic overview of the tested CAR45 constructs. The heavy and light chains of different anti-CD45 antibody clones were cloned into a second-generation CAR containing a 4–1BB costimulatory domain and truncated NGFR (tNGFR) as a transduction marker. (B) CD45^KO Jurkat reporter cells were transduced with CAR45 constructs and sorted for purity based on expression of tNGFR. Sorted cells were incubated with His-tagged recombinant CD45 followed by secondary staining with an anti-His antibody showing that CARs derived from clones 9.4, Gap8.3, and BC8 show highest expression of surface CAR. (C) CD45^KO CAR45 Jurkat reporter cells were incubated with wild type Jurkat cells (CD45+) for 4hrs and NFAT-mediated GFP expression was measured by flow cytometry. CARs derived from antibody clones Gap8.3, BC8, and 9.4 show significant upregulation of GFP compared to untransduced reporter cells (n=3, One-way ANOVA compared to UTD, ***p<0.001). Data are represented as the mean ± SD. (D) CD45^KO CAR45 Jurkat reporter cells were incubated with wild type Jurkat cells (CD45+) for 12hrs and NFAT-mediated GFP expression was measured by time lapse microscopy and the GFP positive area (normalized to t=0) was quantified. Data are represented as the mean ± SEM. (E) Population doublings of CD3/CD28 activated T cells transduced with either CAR19 or CAR45 after 13 days of ex vivo expansion. CAR45 transduced cells have significantly lower T population doublings due to fratricide (n=3 independent donors, one-ANOVA compared to CAR19, ***p<0.001; *p<0.05). Data are represented as the mean ± SD. (F) Human T cells were electroporated with SpCas9 protein and CD45 targeting gRNA (screening of CD45-specific gRNA not shown). Surface CD45 protein expression was assessed by flow cytometry (n=3 independent donors, unpaired t-test, ***p<0.001. Data are represented as the mean ± SD. (G) INDEL frequency in CD45^KO T cells were quantified by TIDE following 9 days of in vitro culture. (H) T cells were electroporated with SpCas9 protein pre-complexed with CD45 gRNA followed by CD3/CD28 activation and transduction with either CAR19 or CAR45 and expansion for 13 days. CD45 deletion enables the expansion of CAR45 transduced cells similarly to CART19 cells (n=3 independent donors, one-ANOVA compared to CAR19, n.s=p>0.05). Data are represented as the mean ± SD. (I) CD45^KO CART45 or CART19 cells were incubated with patient AML cells for 24hrs at a 1:4 E:T ratio and AML cells were quantified by flow cytometry. CART45 efficiently eliminates AML cells compared to CART19 (n=3 technical replicates, one way ANOVA compared to AML only, ***p<0.001; n.s=p>0.05). Data are represented as the mean ± SD. (J) CD45^KO CART45 or CART19 control cells were incubated with patient AML cells for 24hrs at a 1:1 E:T ratio and cytokines in the supernatant were quantified by cytometric bead array (n=3 technical replicates, one way ANOVA compared to AML only, ***p<0.001; n.s=p>0.05). Data are represented as the mean ± SD.