Figure 5. Very low-affinity CD38-CAR-T cells with 28z and 4–1BBL costimulation show better tumor control and persistence in vivo.

(A) Schematic of in vivo experimental set-up. Scaffolds consisting of three 2- to 3-mm³ triphasic calcium phosphate particles coated in vitro with human mesenchymal stromal cells were implanted subcutaneously in RAG^2−/−γc^−/− mice. 8–12 weeks later, mice were i.v. injected with 10×10⁶ cells of luciferase-transduced UM9 cells. One week after tumor injection mice were treated with i.v. injections of 5×10⁶ mock, CD38-CARB1 BBz, −28z or −28z-BBL T cells. (B) Representative bioluminescence images (BLI) are shown n=4 per group for week 1 and 7 (all BLI images in Figure S4). (C) Quantification of BLI measurements, normalized to week 1. Each group contained 4 mice and each mouse harbored 4 scaffolds (n=4). Indicated are the median values of normalized tumor load per group +/− range, * indicates p value <0.05, and ** <0.01 using Kruskal-Wallis analysis of variance. (D) Kaplan-Meier curve based on the set up of a maximum BLI measurement. In our model BLI measurement reaches a plateau after between 8 weeks without the tumor being lethal for the mice. Therefore we have chosen week 7 as the end point of our experiments. This plateau roughly corresponds with 500 cpm/m2. Thus we considered this BLI value as the potential human endpoint of the experiment to obtain a Kaplan-Meier curve. (E, F, G) Post-mortem analysis of scaffolds harboring UM9 tumor cells and infiltrated T cells. Scaffolds were dissociated and filtered through a 70μm filter. Single cells were stained for mouse and human CD45, counted and analyzed by flow cytometry. (E) Absolute numbers of MM cells (GFP⁺/CD138⁺). (F) Number of CAR-T cells (CD45⁺/CD3⁺ and LNGFR⁺ or dsRed⁺ or 4–1BBL⁺). Mock <10³ T cells. (G) Ratio between CAR T cells and MM cells. N=4, results are median cell number of 16 scaffolds (4 per mouse) +/− range. (H) Since tumor growth was similar for the mock treated group between experiments and tumor load between treatment groups was equal before CAR T cell injection, we normalized BLI measurements to mock control values and compared data to previous experiment¹⁵. Displayed is the median of relative tumor load (quantified BLI measurements) at week 7, relative to mock (set at 100%), n=4 mice. * indicates p value <0.05, ** <0.01, ***<0.001 using Kruskal-Wallis analysis of variance.