Extended Data Fig. 7: No obvious structural damage of CDH17CARTs to various normal mouse tissues.
(a) A/J mice were immunized three times with human CDH17 protein. Splenocytes from the immunized mice were fused into hybridoma cells. Single clones of fused hybridoma cells were generated in 96 well plates. Binding of the culture medium from fused hybridoma cells to coated human CDH17 protein were determined by ELISA and positive clones were further analyzed by flow cytometry. WT, human CDH17 or mouse Cdh17-expressing SKOV3 cells were incubated with culture medium from #3 and #29 clones of the positive clones and secondary anti-mouse-FITC antibody, followed by flow cytometry analysis. The results showed that #3 Ab only bound to human CDH17, not mouse Cdh17. The other clone, #29 bound both human (hCDH17) and mouse (mCdh17) CDH17. Representative of n = 2 independent experiments with similar results. (b-c) IHC staining of the colons from murine UTD T cell (mUTD) or murine VHH1-28BBz CART (mCAR) treated mice (Fig. 7) with anti-Foxp3 antibody. Scale Bar: 100 µM. Representative of n = 2 independent experiments with similar results. (d) Counting of the Foxp3+ Treg cells from (b-c) in the tumors (n = 6 mice/group). The data are presented as the mean ± SD (Two-tailed unpaired Student’s t-test). (e) Mouse small intestine, colon, pancreas, stomach, heart, liver and kidney were collected and fixed from control UTD T cell or the indicated VHH1-CART-treated NSG mice bearing NT-3 tumors at day 10 after the first T cell injection, followed by H&E staining. Experiments were performed two independent times with similar results. Scale Bar: 500 µM.
